Tag Archives: Howard Temin

To Resign over an Editorial Decision You Disagree With

What would you do if you were serving on the editorial board of a scientific journal which had just published a manuscript that you knew was seriously flawed. Moreover, you knew that publication of the manuscript might seriously undermine global public health? That was the circumstance of cell biologist Klaudia Brix, Professor of Cell Biology, Jacobs University Bremen, Germany, when, in 2011, the Italian Journal of Anatomy and Embryology (IJAE)—the official publication of the Italian Society of Anatomy and Histology—published a paper by infamous AIDS denialist, Peter Duesberg, which reiterated his already discredited argument that HIV (the human immunodeficiency virus) does not cause AIDS (1). Brix resigned in protest from the IJAE editorial board. But why is that noteworthy? Remarkably, she was, for a time, the only member of the journal’s 13-person editorial board to do so, despite other members having similar misgivings over the decision to publish the paper. Afterwards, Heather Young, an anatomy and neuroscience researcher at the University of Melbourne, likewise resigned from the IJAE editorial board. Here is the background to this state of affairs.

Peter Duesberg is not the only AIDS denialist. However, he has been the most infamous of the AIDS denialists. HIV is a retrovirus, and Duesberg is the only AIDS denialist who also happens to be an expert retrovirologist. In fact, Duesburg was at one time a highly esteemed retrovirologist. In 1985 he was elected to the U.S. National Academy of Sciences; mainly for his 1970 discovery, with Peter Vogt, of the first known retroviral oncogene—the Rous sarcoma virus v-src.

AIDS denialist, Peter Duesberg
AIDS denialist, Peter Duesberg

Duesberg first put forward his denialist view in a 1987 paper in Cancer Research (2), which asserted that AIDS results from drug abuse, parasitic infections, malnutrition, and antiretroviral drugs. In Duesberg’s assessment, HIV is just another opportunistic infection. He has maintained that view since then, despite overwhelming evidence to the contrary. Consequently, he is looked upon as a pariah by the scientific community.

Even though Duesberg’s denialist views have been rejected by AIDS experts, Duesberg’s standing as a retrovirologist enabled him to yet influence some public health officials. In 2000, Duesberg was serving on a panel advising Thabo Mbeki (President of South Africa after Nelson Mandela) on how to manage the South African AIDS outbreak. Although Mbeki was an able and intelligent leader, he accepted Duesberg’s denialist view that HIV was not the cause of the South African AIDS epidemic. Thus, Mbeki allowed the South African outbreak to get completely out of control (3). Two independent studies later concluded that over 300,000 South African AIDS deaths would not have occurred if the Mbeki government’s public health policy had not followed the denialist view. Many thousands of South African AIDS victims, including infants, would have been spared infection if the government had publicized that AIDS is an infectious disease, and if it had made antiretroviral drugs available, particularly to pregnant women (1).  See Asides 1 and 2.

[Aside 1: The reasons why Mbeki assented to Duesberg’s denialist view are not clear. One possibility is that Mbeki held strong anti-colonialist and anti-West sentiments—born of having come of age during South Africa’s apartheid era—which led him to see his country’s AIDS crisis as a means by which the West sought to exploit his nation. To that point, he may have doubted the efficacy of expensive antiretroviral drugs, which were available only from large Western pharmaceutical companies. Moreover, the cost of treating the 5 million or more HIV-infected South Africans with those drugs would have exceeded the annual health department budget of his poverty-stricken nation by a factor of ten. Mbeki did accept that AIDS is the consequence of a breakdown of the immune system. But he was inclined to believe (or at least claimed) that poverty, bad nourishment, and ill health, rather than a virus, led that breakdown; a stance that enabled him to justify treating poverty in general, rather than AIDS in particular. Duesberg defended Mbeki in his publications, denying that hundreds of thousands of lives were lost in South Africa because of the unavailability of anti-retroviral drugs. But in 2002, after Mbeki suffered political fallout from the consequences of having acceded to Duesberg’s views, he tried to distance himself from the AIDS denialists, and asked that they stop associating his name with theirs.]

[Aside 2: The 2000 International AIDS Conference was taking place in Durban (a city in the South African province of KwaZulu-Natal) at the same time that Mbeki’s AIDS panel was convening in Johannesburg. Consequently, the denialist views expressed by Mbeki’s panel were also being heard in Durban. This prompted the so-called “Durban Declaration,” signed by over 5,000 scientists and physicians, and published in Nature, which proclaimed that the evidence that HIV causes AIDS is “clear-cut, exhaustive and unambiguous”.]

Well before Duesberg submitted his paper to IJAE, the arguments put forward in the paper had already been appraised and rebuffed by the scientific community. Indeed, the paper had previously been rejected by several other journals. The first submission was to the Journal of Acquired Immune Deficiency Syndromes (JAIDS), a peer-reviewed medical journal covering all aspects of HIV/AIDS. The JAIDS editors found that Duesberg’s contentions in the paper were based on a selective reading of the scientific literature, in which he dismissed all the vast evidence that HIV is the etiologic agent of AIDS. Not surprisingly, JAIDS rejected the paper, with one peer reviewer even warning that Duesberg and co-authors could face criminal charges if the paper were published.

After JAIDS rejected the paper, Duesberg  submitted a revised version to Medical Hypotheses (4). Like the original paper sent to JAIDS (as well as the version accepted by IJAE), the paper submitted to Medical Hypotheses contained data cherry-picked to cast doubt on HIV as the cause of AIDS. Nonetheless, Medical Hypotheses accepted the paper. However, the paper never went to press. But first, what was the explanation for the seemingly bizarre decision to accept the paper?

The answer laid in the fact that Medical Hypotheses was the only journal of its parent publisher, Elsevier, that did not use peer review; instead relying on its editorial board to select papers for publication. In any case, before the accepted paper went to press, prominent AIDS researchers, including Nobel laureate Francoise Barre-Sinoussi (co-discoverer that HIV is the cause of AIDS, 5), complained to Elsevier that the paper would have a negative impact on global healthcare, and requested that the paper be withdrawn.

Elsevier responded to these protests by asking the editors of another of its journals, The Lancet, to oversee a peer review of the paper. The Lancet editor sent the paper to five external reviewers, each of whom found that it contained numerous errors and misinterpretations, and that it could threaten global public health if it were published. Elsevier then permanently withdrew the paper.  Elsevier also instituted a peer-review policy at Medical Hypotheses (and fired the journal’s editor, who resisted the change).

The Medical Hypotheses incident resulted in more notoriety for Duesberg when the University of California, Berkley, where Duesberg is still a professor of molecular and cell biology, bought charges of misconduct against him for making false scientific claims in the paper, and for a conflict-of-interest issue. Apropos the latter, Duesberg did not reveal that co-author David Rasnick had earlier worked for Matthias Rath, a German doctor and vitamin entrepreneur, who sold vitamin pills as a therapy for AIDS. Duesberg was later cleared of both charges. But the next iteration of paper, to IJAE, did not respond to these allegations.

Duesberg regarded Elsevier’s actions as another example of “censorship” imposed by the “AIDS establishment.” Undeterred however, he submitted a revision of the paper to IJAE, which that journal then accepted, prompting Klaudia Brix and Heather Young to resign from that journal’s editorial board. The IJAE paper contained the same cherry-picked data and discredited assertions that were rejected earlier by JAIDS and Elsevier.  Moreover, publication of the paper still posed a threat to global public health. What then was behind the IJAE decision to publish?

Here is what happened. The paper was “peer-reviewed” by IJAE, but by only two reviewers; one of whom was Paolo Romagnoli, the IAJE editor-in-chief, who is neither a virologist or an epidemiologist but, instead, a cell anatomist. Consequently, the paper underwent only one external review, and there is no information regarding whether the lone external reviewer was an AIDS expert. One board member (who did not resign) later commented: “Only one [external] reviewer in my mind is not enough for manuscripts of a sensitive nature… (6)” [But this comment too is a bit troubling. Bearing in mind that the paper contained numerous errors and misinterpretations, would those have been okay if the paper were not of a “sensitive nature”?]

One also might ask why a journal that specialized in anatomy and embryology would consider a paper about the cause of AIDS. To that point; Klaudia Beix gave, as a reason for her resignation from the IJAE board, her belief that a journal should function within its scientific “scope” (6). So how did Romagnoli, the IJAE editor-in-chief, justify his decision to consider the paper?  He did so by asserting that it dealt with “issues related to the biology of pregnancy and prenatal development and with the tissues of the immune system (6).” But despite Romagnoli’s contention, the only mention of embryology in the paper was a short comment in the abstract: “We like to draw the attention of scientists, who work in basic and clinical medical fields, including embryologists, to the need of rethinking the risk-and-benefit balance of antiretroviral drugs for pregnant women, and newborn babies (1).”

As for Romagnoli’s reliance on only two reviewers, he justified that stance on the fact that the reviewers had concurring opinions. Moreover, he claimed that his criteria for selecting reviewers—apparently irrespective of their expertise—was to choose individuals (himself included) who he believed would not reject a paper merely because it challenged prevailing opinion.

But is there any possibility that Duesberg might be right? The answer is virtually none whatsoever. An earlier post noted: “…the evidence that HIV causes AIDS is, without exaggeration, overwhelming. Consider just the following. Data from matched groups of homosexual men and hemophiliacs show that only those who are infected with HIV ever develop AIDS. Moreover, in every known instance where an AIDS patient was examined for HIV infection, there was evidence for the presence of the virus. These data have been available for years, and Duesberg should have been aware of them. What is more, there has been the enormous success of antiretroviral therapy in changing AIDS from a nearly invariably fatal disease, into a manageable one, for many HIV-infected individuals (3).”

Even so, Duesberg is not regarded as a pariah by AIDS experts merely because his views concerning the connection between HIV and AIDS challenge accepted wisdom.  Instead, as asserted by Harvard University AIDS epidemiologist, Max Essex, Duesberg has sustained a “dangerous track of distraction that has persuaded some people to avoid treatment or prevention of HIV infection (6)”.

A scientist mounting a long-time challenge to the “establishment,” and being ridiculed for his views, before eventually being vindicated, makes for a very good story. However, such instances are very rare. Exceptions include Howard Temin (7) who hypothesized reverse transcription, and Stanley Pruisner (8) who hypothesized prions—infectious agents that contain no nucleic acid genome. Both researchers had to endure widespread ridicule for several years. But, and importantly, irrefutable evidence eventually accumulated to support their hypotheses. And, finally, both were awarded Nobel Prizes. But Duesberg has not been vindicated and, almost certainly, he  never will be.

References:

1. Duesberg PH, et al., 2011. AIDS since 1984: no evidence for a new, viral epidemic – not even in Africa. Italian Journal of Anatatomy and Embryololgy 116:73–92. http://fupress.net/index.php/ijae/article/view/10336/9525

2. Duesberg P, 1987. Retroviruses as carcinogens and pathogens: expectations and reality. Cancer Res. 47:1199–220. PMID3028606.

3. Thabo Mbeki and the South African AIDS Epidemic, Posted on the blog July 3, 2014.

4. Duesberg PH, et al., 2009. WITHDRAWN: HIV-AIDS hypothesis out of touch with South African AIDS – A new perspective. Medical Hypotheses. doi:10.1016/j.mehy.2009.06.024. PMID19619953.

5. Who Discovered HIV, Posted on the blog, January 24, 2014.

6.  Corbyn Z. 2012. Paper denying HIV–AIDS link sparks resignation: Member of editorial board quits as editor defends publication. Nature doi:10.1038/nature.2012.9926.

7. Howard Temin: “In from the Cold, Posted on the blog December 14, 2013.

8. Stanley Pruisner and the Discovery of Prions: Infectious Agents Comprised Entirely of Protein, Posted on the blog December 15, 2016.

 

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Stanley Pruisner and the Discovery of Prions: Infectious Agents Comprised Entirely of Protein

Stanley Prusiner (1942) received the 1997 Nobel Prize in Physiology or Medicine for discovering the agents responsible for the transmissible spongiform encephalopathies—diseases so named because the brains of afflicted subjects contain numerous holes or vacuoles, which give them a spongy (“spongiform”) appearance under the microscope. These diseases include scrapie in sheep, bovine spongiform encephalopathy (“mad cow disease”) in cattle, and Creutzfeldt-Jacob disease (CJD) and kuru in humans. Each of these diseases is invariably fatal.

Stanley B. Pruisner (left) and His Majesty the King, at the Nobel Prize Ceremony
Stanley B. Pruisner (left) and His Majesty the King, at the Nobel Prize Ceremony

Pruisner’s discovery was iconoclastic in the extreme because the etiologic agents of these diseases, which can multiply and kill, are comprised entirely of protein! Since they can replicate, despite carrying no genetic information, they defy the very foundation of biology. Pruisner dubbed them “prions”—an acronym he derived from “proteinaceous infectious particle.” The spongiform encephalopathies are now more commonly called “prion diseases.”

How might an infectious agent, which contains no genetic information, replicate? Pruisner provided the answer. Prion proteins (PrPSc) (Sc for scrapie, the prototype prion disease) are misfolded forms of corresponding normal cellular proteins (PrPC), which are generally present in all vertebrates, and which are particularly plentiful in the brain. The PrPSc isoforms act as templates that cause the normally configured proteins to refold into the PrPSc configuration. Once underway, this conversion process might escalate exponentially. In that way PrPSc isoforms “replicate,” and their accumulation in the brain leads to the characteristic prion disease pathology. See Aside 1.

[Aside 1: Prion diseases were once referred to as “slow virus diseases,” where “slow” referred to the course of the disease, rather than the agent. All the prion diseases have a clinically unapparent incubation period that may last for as long as 50 years. But once symptoms emerge, the duration of the clinical stage is only a matter of months, and invariably ends in death. The length of the incubation period appears to be inversely correlated with the level of PrPC. The actual cause of cell death in prion diseases is not known.]

The medical relevance of Pruisner’s discovery of prions, and of their mode of replication, may be much more significant than merely their association with the relatively rare infectious prion diseases. That is so because similar aggregates of misfolded proteins have since been observed in the much more widespread Alzheimer’s and Parkinson’s diseases, as well as Lou Gehrig’s (ALS) and Huntington’s diseases. Misfolded PrP-like proteins associated with Alzheimer’s disease include amyloid-β and tau. In Parkinson’s disease, these aggregates are comprised of α-synuclein. [The entire family of the PrP-like misfolded proteins are referred to as amyloids.] Thus, Pruisner’s discovery may have significant implications for the diagnosis and treatment of much more prevalent neurodegenerative diseases and dementias. See Aside 2.

[Aside 2: Potential therapies include the targeting of toxic species of PrP with monoclonal antibodies or with other ligands that bind to amyloid aggregates. Apropos that, in November 2016, the drug solanezumab, which targets amyloid, failed in a clinical trial to determine whether it might help people with mild dementia. Critics of the “amyloid hypothesis” (who still remain; see below) cited the failed trial as evidence against the amyloid premise. For a review of evidence in support of the amyloid hypothesis, and for analyses of the meaning of the failed solanezumab trial, see references 1 and 2. For a current review of the field, see reference 3.]

If Alzheimer’s and Parkinson’s diseases, as well as other neurodegenerative disorders such as ALS, indeed resemble the transmissible prion diseases, as disorders of protein conformation, then they also suggest new disease paradigms. Some of these diseases are infectious, while others are sporadic, or genetic. However, and importantly, some of these diseases may be transmitted by several of these various ways that determine the frequency and distribution of a disease in a population. For instance, Pruisner’s group demonstrated that CJD can be an infectious as well as a familial disease (see below). In the latter instance, it results from a particular mutation in the cellular PrP gene. Indeed, more than 20 mutations in PrP are now known which underlie inherited prion diseases. See Asides 3 and 4.

[Aside 3: It is not surprising that Pruisner’s proposal of an entirely new type of infectious agent—one comprised entirely of protein—met with considerable skepticism. Reflecting on the rather vicious ridicule that some of the naysayers subjected him to, Pruisner wondered “how the course of scientific investigation might have proceeded had transmission studies not been performed until after the molecular genetic lesion had been identified (4).”]

[Aside 4: Might Alzheimer’s disease be transmissible? There is no epidemiological evidence to suggest that possibility. However, bearing in mind that infectious prion diseases, such as CJD, can be transmitted during medical or surgical procedures (e.g., corneal transplant), it is reasonable to suggest that Alzheimer’s too might be transmitted by a physician’s or surgeon’s treatment. Other known routes of iatrogenic CJD transmission include injection of pituitary hormones obtained from cadavers, and intracerebral exposure to contaminated neurosurgical instruments. Apropos the latter possibility, amyloid-β adheres stubbornly to metal surfaces, and prions are highly resistant to sterilizations that would inactivate a true virus. Yet, because of the already high prevalence of Alzheimer’s disease in the population, and a possible decade-long non-clinical incubation period, the risk of iatrogenic transmission in that instance is not yet known. New (and expensive) methods have been developed for removing amyloids from surgical instruments, but they are not widely used because of the uncertainty of the danger of iatrogenic transmission.]

Here now is the story of Pruisner’s discovery of prions, with a nod toward how he persevered in the face of the widespread disbelief and scorn that his discovery engendered.

Pruisner first became interested in neurological diseases as a third-year medical student at Penn, during his rotation on the neurology service (5). However, that exposure to neurological diseases did not immediately affect his career goals. Instead, in his fourth year, he satisfied his desire to do research by investigating oxidative metabolism of brown fat cells. Nonetheless, his research on fat cells excited him enough to envision a career as a physician-scientist. “I was astonished that people actually got paid to solve puzzles every day—what a fantastic way to make a living (5).”

Pruisner continued his medical training as an intern at the University of California, San Francisco (UCSF). Providentially perhaps, he found his internship to be demanding enough to dissuade him of any thoughts of a career practicing clinical medicine. So, Pruisner spent the next three years at the NIH researching enzyme regulation in bacteria; an experience that he found gratifying enough to decide that a career in medical research would be his goal.

Next, Pruisner had to choose a research area. He was still interested in neurological diseases. “With its billions of neurons, its ability to affect all aspects of human activity and its endless mysteries, the brain seemed a perfect subject for research… (5).” To gain the background he would need for his new calling, Pruisner decided to carry out an “abbreviated residency” in neurology at UCSF.

The next step in Pruisner’s path would be choosing a solvable research problem. Here now is an example of one of those fortuitous happenings that can make a scientific career and, with a bit of luck, lead to a singularly important scientific breakthrough. “It was during my residency at UCSF that I encountered a patient with a rare progressively debilitating illness called Creutzfeldt-Jacob disease (or CJD), and the mysteries surrounding this illness launched my scientific studies for the next four decades (5).”

Pruisner was intrigued as well as perplexed by his CJD patient. She had suddenly developed severe intellectual and memory deficits, and myoclonus (jerky movements in her muscles). But more puzzling: “She exhibited no signs of an infectious disease…she did not have a fever, and she had no increase in white blood cells in either her blood or cerebrospinal fluid (5).” Yet CJD is a transmissible disease, as shown earlier by Carleton Gajdusek’s finding that the illness could be passed to a chimpanzee by injecting it with a brain extract from a dead human CJD patient. See Aside 5.

[Aside 5: Gajdusek also experimentally transmitted kuru, via homogenates of human patient biopsies, to chimpanzees. Moreover, his epidemiological studies showed that kuru was transmitted among the Fore people of New Guinea via ritual cannibalism. For more on this, and for an account of how Bill Hadlow first suggested that scrapie and kuru might have a similar underlying basis, see reference 6.]

Gajdusek also found that CJD symptoms did not emerge in injected chimpanzees until months after the inoculation; a finding that agreed with the prevailing view in the scientific community that the scrapie-like diseases are caused by “slow viruses”—a term originally coined by Bjorn Sigurdsson in 1954 while he was working on scrapie in Icelandic sheep. Gajdusek referred to the scrapie agents as “unconventional viruses,” although he had no knowledge of how they might differ from “conventional viruses.”

Pruisner became fascinated by the prospect of once-and-for-all defining the nature of the agents responsible for the transmissible spongiform encephalopathies. However, more experienced, and cautious colleagues at UCSF saw the scrapie project as fraught with too many pitfalls, and tried to steer Pruisner away from it. But he would not be deterred.

One of Pruisner’s UCSF colleagues alerted him to papers by radiation biologist Tikvah Alper, who incidentally trained with Lisa Meitner (7). Alper noted other bizarre properties of the scrapie agents. In brief, she found them to be extremely resistant to UV light and X-rays, which should have inactivated any “conventional” virus by damaging its DNA or RNA genome. And: “Since a single X-ray photon should be sufficient to kill a single scrapie agent, Alper was able to calculate the minimal size of the agent. She estimated that it was less than one-hundredth the size of a typical virus (5).”

Alper’s findings clearly suggested the provocative idea that the scrapie agent does not contain a nucleic acid genome. Yet Pruisner, like everyone else, held to the belief that some sort of “novel” virus was responsible for her unusual results. “What else could the “scrapie agent” be? There was nothing else (5).”

Nonetheless, in the back of Pruisner’s mind, he did not completely dismiss “…the most startling interpretation: All the data might be pointing to an infectious particle devoid of nucleic acid and thus with no apparent way to replicate (5).” Pruisner indeed was intrigued by this radical possibility. And, notwithstanding the advice of more experienced colleagues, Pruisner, a former chemistry major, believed that the scrapie problem would be easy. “It’s just a problem in protein chemistry (5).” And, if it were true that the scrapie agents contain no genetic information, “then it would be worth an enormous effort to decipher the structure (5).”

Pruisner’s first step would be to isolate the scrapie agent from brain homogenates; a feat not accomplished by Alper, nor by anyone else. To monitor his progress towards purification, Pruisner planned to assay his fractions by means of a biological assay, making use of the 1961 finding by British scientist, Richard Chandler, that scrapie disease could be transmitted from one mouse to another via injection of brain homogenates. Pruisner would employ the so-called “endpoint dilution” procedure, in which the titer of a sample is the last dilution (e.g., 1/2, 1/4, 1/8, etc.) able to induce scrapie infection.

But, as predicted by others, complications soon materialized: “…so little was known about the physical nature of the mysterious scrapie agent that hundreds of fractions would have to undergo titration measurements (5).”  Additionally, Pruisner’s assay would require 60 mice to measure the titer of each sample. What’s more, since the scrapie incubation period could be a year or longer, some titrations might very well require that long as well. Consequently, Pruisner might have to maintain thousands of mice during this entire time. And, in the end, the assay might not be sensitive enough to show small increases in purity.

The above issues alone may explain why no one before Pruisner tried to systematically investigate the scrapie agent’s molecular nature. But, there is more. Even if Pruisner’s assays were sufficiently sensitive, the critical experiments would need to be repeated before they might be published. And, his findings would still need to be confirmed by others before they might be accepted. Furthermore, before Pruisner could make headway on this hugely expensive project, he would need to acquire a grant to support it. The NIH—the usual source for large biomedical research grants—appeared unlikely to provide that support, since its Virology Study Section held the view that a slow virus causes scrapie and that the issue should be approached as a virological problem, rather than by Pruisner’s chemical approach. Nonetheless, Pruisner was undeterred, “The hubris of youth was all that propelled me forward (5).”

The NIH indeed rejected Pruisner’s application for support of his scrapie project. However, Pruisner did obtain modest funding at UCSF from the Howard Hughes Medical Institute, which enabled him to set out on his project. Meanwhile, William Hallow and Carl Eklund, at the Rocky Mountain Laboratory in Hamilton, Montana, had been studying scrapie pathogenesis in sheep and goats, and had made some failed “hit-or-miss” attempts to define the molecular nature of the agent. When they met Pruisner, his more systematic approach impressed them, and they then taught him “an immense amount” about scrapie.  Importantly, they helped him to characterize the scrapie agent’s sedimentation behavior—a key step towards purifying it.

Pruisner then began to produce his first experimental results, which were curious in the least: “I had anticipated that the purified scrapie agent would turn out to be a small virus and was puzzled when the data kept telling me that our preparations contained protein but not nucleic acid (8).”

But while Pruisner’s findings raised the possibility that he might be on to something new and exciting, not all was going well for him. He lost his funding at UCSF from the Howard Hughes Medical Institute. Worse yet, UCSF told him that he would not be promoted to tenure. But, the tenure decision was reversed, and because he had by now developed a starting point for his studies, and because his early results suggested that the project might yield intriguing new findings, he was awarded modest support from the NIH, as well as more substantial funding from the R. J. Reynolds Company (really).

Pruisner’s rate of progress was significantly enhanced when he found that he could shorten the length of time needed for his assays by moving from mice to hamsters, in which 70 days were required, rather than the 360 days needed in mice. And, he also, redesigned his measurement method. By 1982, in addition to the results of his biochemical analysis (which implied that the scrapie agent was comprised entirely of protein), he also found that scrapie infectivity could be reduced by treatments that alter proteins, but not by treatments that alter nucleic acids. Believing that he now had sufficient data to support his premise that the scrapie agent is comprised only of protein, he published his findings in a paper in Science (9).

Pruisner introduced the term “prion” for the first time in the 1982 Science paper (9). But in doing so, he set off a “firestorm (8).” Most virologists were skeptical of his findings, and some competitors, who had been working on scrapie and CJD, were incensed by his claims. “At times the press became involved since the media provided the naysayers with a means to vent their frustration at not being able to find the cherished nucleic acid that they were so sure must exist. Since the press was usually unable to understand the scientific arguments and they are usually keen to write about any controversy, the personal attacks of the naysayers at times became very vicious (8).” Nonetheless, Pruisner was confident that he was right: “Despite the strong convictions of many, no nucleic acid was found; in fact, it is probably fair to state that Detlev Riesner (Aside 6) and I looked more vigorously for the nucleic acid than anyone else (8).”

[Aside 6: Detlev Riesner had been studying viroids when he met Pruisner. These agents, which mimic viruses, are small, naked, single-stranded, circular RNAs, that infect plants. They were discovered by Theodore O. Diener, a Swiss plant pathologist. Since the small size of viroids is consistent with Alper’s X-ray data, which showed that the scrapie agent too is extremely small, Pruisner sought out Riesner to inquire whether viroids might be the causative agents of CJD. The meeting between the two researchers led to a long-time collaboration, and both contributed to the discovery that the scrapie agents do not contain nucleic acids.]

By the next year Pruisner had isolated the scrapie prion protein, and the following year Leroy Hood (who helped found the human genome program) determined a portion of its amino acid sequence. Meanwhile, skeptics kept searching for a nucleic acid-containing scrapie agent. And while they never succeeded in their efforts to overturn the wealth of evidence Pruisner was accumulating in support of the prion hypothesis, the mystery of how prions might replicate still remained to be solved. Toward that end, Pruisner collaborated with Charles Weissmann to clone the cellular gene encoding the prion protein.

“Once cDNA probes for PrP became available, the PrP gene was found to be constitutively expressed in adult uninfected brain. This finding eliminated the possibility that PrPSc stimulated the production of more of itself by initiating transcription of the PrP gene… (8).” Moreover, with the isolated PrP proteins in hand, it was clear that PrPSc was not the translational product of an alternatively spliced mRNA. [“The entire open reading frame of all known mammalian and avian PrP genes resides within a single exon (8).”] Furthermore, PrPSc was not the result of a posttranslational modification of PrPC.

Pruisner and coworkers next considered the possibility that PrPC and PrPSc differed only in their conformations. However, bear in mind that the molecular biology dogma of the day held that the amino acid sequence of a protein specifies only one biologically active conformation of the protein. Consequently, the idea that PrPC and PrPSc differed only in their conformations “was no less heretical than that of an infectious protein (8).” Nonetheless, the results of structural studies indeed bore out the conformation premise. See Aside 7.

[Aside 7: For aficionados: “Fourier transform infrared (FTIR) and circular dichroism (CD) studies showed that PrPC contains about 40% α-helix and little β-sheet, while PrPSc is composed of about 30% α-helix and 45% β-sheet. Nevertheless, these two proteins have the same amino acid sequence!” The PrPSc structure enables amyloids to form their characteristic tightly interacting, many stranded and repetitive intermolecular β-sheets. Readers interested in these, and additional structural studies might begin with Pruisner’s Nobel lecture (4).]

Pruisner and coworkers then carried out a series of telling experiments that began to unlock the mystery of prion replication. The first step was to generate mice that lacked both copies of the mouse PrPC gene. Importantly, this treatment rendered these mice completely resistant to mouse PrPSc. However, when hamster PrPC genes were incorporated into the genomes of these mice, and were expressed in them,  these transgenic mice then were susceptible to hamster PrPSc. However, the mice remained resistant to mouse PrPSc. Thus, in mice, the hamster PrPC transgene product was required to promote the replication of hamster scrapie prions, whereas the mouse PrPC protein was required to promote the replication of the mouse scrapie prions.

These results show that the scrapie PrP isoform and the normal cellular PrP protein each  play crucial roles in the transmission and pathogenesis of prion disease. Importantly, these results are completely consistent with the “misfolding hypothesis,” in which the scrapie isoform catalyzes the conversion of the normal cellular PrP protein into the scrapie conformation.

The finding that expression of the hamster PrPC promotes (and indeed is required for) replication of the hamster PrPSc, but does not promote replication of the mouse PrPSc, is an example of the “species barrier” to prion infection—in which the passage of prions between species is generally restricted. Differences in the amino acid sequence homology between the PrPSc of one species and the PrPC of another species, which might impair their interaction, readily explain the species barrier. See Aside 8.

[Aside 8: Interestingly, despite prions not having genomes, prion “mutation” can occur, in the sense that prions encoded by the same PrP gene may assume different conformations, thereby giving rise to a kind of prion “polymorphism,” which may enable prions to cross species barriers by a process of conformational selection.]

In 1990, in another series of key experiments, Pruisner’s research group discovered a mutation of the human PrP gene (a leucine substitution at codon 102), which appeared to be linked to Gerstman-Straussler-Scheinker syndrome (a very rare, exclusively inheritable, progressive spongiform encephalopathy in humans). Next, they generated a recombinant mouse PrP gene that encoded the leucine substitution at codon 102. Importantly, transgenic mice, which expressed the recombinant PrP gene, developed a scrapie-like disease with many of the pathological features of Gerstman-Straussler-Scheinker syndrome. What’s more, inoculates, which contained brain extracts from those mice, transmitted the disease to inoculated mice. Thus, Pruisner’s group demonstrated that a prion protein, containing a single amino acid substitution, can be the cause of a human familial prion disease.

Only a portion of Pruisner’s contributions up until he received his 1997 Nobel award were noted in the above narrative. For a more complete review of his work until then, see his 1997 Nobel lecture (4). Pruisner is still investigating neurodegenerative and dementing diseases at the UCSF School of Medicine, where he also serves as the director of its Institute for Neurodegenerative Diseases.

As already noted, Pruisner’s career is also remarkable for his having persevered in authenticating his iconoclastic protein-only prion hypothesis, despite the continuing and widespread skepticism and ridicule from colleagues in the scientific community. In that regard, his story is reminiscent of Howard Temin’s after announcing his discovery of reverse transcription by the RNA tumor viruses; which eventually would be re-designated “retroviruses” (10).

Pruisner himself was unprepared for the level of resistance to his discovery of prions: “…this created a rather harrowing and arduous journey for more than a decade…Many argued that I was spewing heresy and I had to be wrong (5).” What’s more, the ferocity of some of the personal attacks against Pruisner, particularly those in the media, were affecting his family.

But what might have explained the extent of the enmity on the part of some naysayers? Could it have simply been that the prion hypothesis conflicted with molecular biology dogma of the day? Or could it have been, as Pruiner suggested, that some critics perhaps were reacting to their frustration at not being able to find the nucleic acid, which they were sure had to be there?

Resigned to the criticism, Pruisner stated: “When there is a really new idea in science, most of the time it’s wrong, so for scientists to be skeptical is perfectly reasonable.” And, stoically, Pruisner’s answer to his critics was to keep producing data. “The incredulity of my colleagues only strengthened my conviction that scientists have a responsibility to convince their skeptics of the validity and importance of discoveries that run counter to prevailing opinions, and they can do so only by performing experiments that challenge their own hypotheses. Sometimes the road of testing and retesting is long and arduous—such was the case for me (5).” Moreover, and to Pruisner’s credit as a scientist, he also attributed his tenacity to his fascination with prions.

By the late 1980s, enough scientific data (particularly the knockout mouse studies) had emerged to begin garnering a measure of acceptance for the protein-only prion hypothesis. Then, in 1996, with the appearance in Britain of the first human cases of mad cow disease (incidentally identified as such using techniques originally developed by Pruisner), prions were suddenly a hot topic in the media. And, the very next year Pruisner was awarded the Nobel Prize.

Did the spotlight on mad cow disease and prions influence the Nobel committee’s decision? Pruisner conceded that “It didn’t hurt.” And, he graciously admits to the part that luck may have played in his exceptional career. “Extremely intelligent men and women can toil for years in the vineyards of science and never be fortunate enough to make a great discovery. And then there are a few people who are recipients of mammoth doses of good luck. The infectious pathogen that we now call a prion might well have turned out to be an atypical virus—not nearly as interesting as an infectious protein…or another group instead of mine might have discovered prions; that sort of preemption happens all the time in science (5)”

Yet some of Pruisner’s critics remained skeptical of the prion hypothesis even after he was awarded the Nobel Prize. For example, consider the following excerpts from a 1998 Science paper by Bruce Chesebro (11): “Although the notion that “protein only” can account for the infectious agent has received considerable publicity as a result of the Nobel prize award to S. Prusiner for the discovery of prions, the fact remains that there are no definitive data on the nature of prions… There are arguments both for and against the hypothesis that abnormal PrP itself is the transmissible agent, but on either side of this controversy no argument is as yet completely convincing … Clearly, we are in the very early stages of exploration of this subject. It would be tragic if the recent Nobel Prize award were to lead to complacency regarding the obstacles still remaining. It is not mere detail, but rather the central core of the problem, that remains to be solved.”

References:

1. Abbott A. 2016. The red-hot debate about transmissible Alzheimer’s. Nature 531: 294–297 doi:10.1038/531294a

2. Abbott A and Dolgin E. 2016. Failed Alzheimer’s trial does not kill leading theory of disease. Nature  doi:10.1038/nature.2016.21045

3. Collinge J. 2016. Mammalian prions and their wider relevance in neurodegenerative diseases. Nature 539:217–226.

4. Pruisner SB, Prions, Nobel Lecture, December 8, 1997.

5. Pruisner S. Madness and Memory: The Discovery of Prions-A New Biological Principle of Disease, Yale University         Press, 2014.

6. Carlton Gajdusek, Kuru, and Cannibalism, Posted on the blog April 6, 2015.

7. Max Delbruck, Lisa Meitner, Niels Bohr, and the Nazis, Posted on the blog November 12, 2013.

8. Stanley B. Prusiner – Biographical”. Nobelprize.org. Nobel Media AB 2014. Web. 24 Nov 2016. <http://www.nobelprize.org/nobel_prizes/medicine/laureates/1997/prusiner-bio.html&gt;

9. Prusiner, SB. 1982. Novel proteinaceous infectious particles cause scrapie. Science 216:136–144.

10. Howard Temin: “In from the Cold, Posted on the blog December 14, 2013.

11. Chesebro B. 1998. BSE and Prions: Uncertainties About the Agent. Science 279:42-43.
DOI: 10.1126/science.279.5347.42

Harold Varmus: From English Literature Major to Nobel Prize-Winning Cancer Researcher

Harold Varmus and J. Michael Bishop changed cancer research in a fundamental way in the 1970s, when they discovered proto-oncogenes at the University of California at San Francisco (UCSF). Proto-oncogenes are cellular genes that normally play an important role in controlling cell division and differentiation. However, Varmus and Bishop found that proto-oncogenes can be altered by mutation, to become oncogenes that contribute to cancer. When Varmus and Bishop first began their collaboration in 1970, cancer research was, for the most part, focused on epidemiology (e.g., studies linking smoking to lung cancer) and empirical approaches to therapy (e.g., radiation and chemotherapy).

Harold Varmus, Cancer Researcher, Nobel Laureate, Director of the NIH
Harold Varmus, Cancer Researcher, Nobel Laureate, Director of the NIH

The discovery of proto-oncogenes is a pertinent topic for our Virology blog because it depended crucially on Varmus and Bishop’s earlier finding that retroviral oncogenes are mutated versions of cellular genes that retroviruses “captured” from their host cells. Varmus and Bishop hypothesized and then demonstrated that since retroviral oncogenes are versions of genes that actually are part of a normal cell’s genetic makeup, mutations in those genes, or their inappropriate expression, can lead to cancer. The v-src gene of Rous sarcoma virus was the first retroviral oncogene that Varmus and Bishop showed is derived from a cellular genome (1).

Varmus and Bishop continued searching for proto-oncogenes in the 1980s. Varmus also began investigating HIV (also a retrovirus and the cause of AIDS). In 1989 Varmus and Bishop were awarded the Nobel Prize in Physiology or Medicine for their discovery of proto-oncogenes.

In the early 1990s Varmus stepped out from his role as a research scientist to take up the cause of public funding for biomedical research. In 1993 President Bill Clinton acknowledged Varmus’ efforts in that regard, as well as his stature as a scientist, by appointing him to serve as Director of the National Institutes of Health (NIH). Thus, Varmus became the first Nobel laureate to head the NIH.

In 2000 Varmus left the NIH to accept the presidency of the Memorial Sloan-Kettering Cancer Center in New York. In 2010 Varmus returned to the NIH, this time appointed by President Barak Obama to serve as director of the National Cancer Institute (NCI). In 2015 Varmus was back again in New York where he is the Lewis Thomas University Professor of Medicine at Weill-Cornell Medical College.

Varmus was featured in two earlier blog postings. The first of these described how he mediated the dispute between Robert Gallo and Luc Montagnier over the right to name the AIDS virus (2). The second posting covered some of the political and social dilemmas Varmus faced during his days leading the NIH (3).

Here, we relate first how Varmus opted for a career in biomedical science and, second, how his collaboration with Bishop came about. This is an interesting tale because Varmus’ remarkable career as a science researcher, administrator, and spokesperson happened despite his initial intention to become a teacher of English literature. Indeed, his career in science did not begin until after he earned an M.A. degree in English from Harvard University, and then spent four years in medical school preparing for a career in clinical medicine.

We begin our story in 1950 as Varmus recounts how, as a ten-year-old, he witnessed his physician father receive a call that conveyed shocking news: “one of my mother’s favorite cousins, a robust man in the middle of his life, had just been diagnosed with leukemia. Of course, I did not know very much about leukemia, but I did know immediately from my parents’ expressions–and within a few weeks, from our cousin’s death—that his disease was a veritable tidal wave.” [All quotations are from Varmus’ book The Art and Politics of Science (4), in which he reflects back on his entire career.]

His cousin’s leukemia actually resulted from a mutation in one of the genes that Varmus would discover more than two decades later. And Varmus notes just how far the science in general had progressed during that 25-year interim: “…when my father heard about our cousin’s leukemia, biologists were not even sure that genes were made of DNA, had no idea how genetic information could be encoded in genes, and, of course, had no way of knowing that cancers are driven by mutations.”

Varmus was urged by his father to prepare for a career in medicine. Nonetheless, when Varmus enrolled as a freshman at Amherst College he strongly favored studying the humanities. Thus, he “toyed with the idea of majoring in philosophy (ultimately too abstract), physics (ultimately too hard), and English literature (ultimately selected).”

Throughout his undergraduate days, Varmus envisioned preparing for an academic career teaching literature. Still, he dutifully fulfilled premed requirements to keep open the possibility of obliging his father’s wishes that he become a medical doctor. Yet he never considered majoring in biology. “I couldn’t understand how some of my close friends (among them, some now distinguished scientists) could spend long afternoons and evenings incarcerated in a laboratory, when they could be reading books in a soft library chair or reciting poetry on Amherst’s green hills.”

Varmus began having doubts about his career choice when his Amherst College classmate Art Landy (later a well-known molecular biologist at Brown University) won an Amherst biology prize that allowed him to attend a 1961 international biochemistry meeting in Moscow. Importantly, Landy invited Varmus to accompany him to the Moscow meeting, where Varmus learned that Marshal Nirenberg had deciphered the genetic code. “Even though I did not understand its meaning or its importance at the time, I was not oblivious to the excitement around me…Scientists seemed likely to discover new, deep, and useful things about the world, and other scientists would be excited by these discoveries and eager to build on them. Would this be true of literary critics and teachers?”

Notwithstanding these misgivings, Varmus continued on his path to a career in English literature after graduating from Amherst College in 1961, earning an M.A. in English from Harvard in 1962 (his focus was on Anglo-Saxon poetry). But his uncertainties about his future only grew stronger. “Despite outward signs that I had chosen a life of studying and teaching literature, soon after starting my graduate work at Harvard I began to suffer some further internal doubts about abandoning medicine. The graduate curriculum in English literature was not especially onerous, but it felt like a prolongation of college. Most of my courses were heavily populated with Harvard and Radcliff undergraduates.” Varmus leaves the impression that he looked upon much of his course work at Harvard as a tiresome chore.

Varmus was also aware of the enthusiasm of former Amherst College classmates who were then studying at Harvard Medical School. “Occasionally, on Saturday mornings, I traveled across the Charles River to join some Amherst classmates at Harvard Medical School, while they sat in the Ether Dome at Massachusetts General Hospital, entranced by diagnostic dilemmas discussed at the weekly pathology conference. These stories struck me as far more interesting than those I was reading, and my medical school friends expressed general excitement about their work. They also seemed to have formed a community of scholars, with shared interests in the human body and its diseases and common expectations that they would soon be able to do something about those diseases…These Saturday excursions probably account for an influential dream I had one night about my continuing indecision. In that dream, my future literature students were relieved when I didn’t turn up to teach a class, but my future patients were disappointed when I didn’t appear. It seemed I wanted to be wanted.”

So, Varmus finally came to grips with his qualms about a career teaching English literature, hastily preparing an application to Harvard Medical School and biking across the frozen Charles River to deliver it just in time to meet the deadline. But it was to no avail, since the dean of admissions thought Varmus was “too inconstant and immature” for medical school.

Varmus next sent off an application to Columbia University’s College of Physicians and Surgeons (P&S). His interviewer at Columbia was an esteemed physician named David Seegal, who also happened to be rather literate. Seegal asked Varmus if he might translate the Anglo Saxon phrase Ich ne wat. “This was easy; it simply means ‘I don’t know.’” Seegal used his question as a lead-in to discuss why a physician might admit fallibility to a patient. In any case: “By the fall of 1962, I was happily enrolled at P&S, helped for the first, but not the last, time by someone’s exaggerated appreciation of my competence in two cultures.”

Now ensconced at P&S, Varmus thought he might become a psychiatrist; an ambition stoked by an interest in Freud and by his winning of an essay prize at P&S in psychiatry. But, he found his “first hour alone in a room with a psychotic patient to be more difficult and less interesting than an hour reading Freud.” So, Varmus’ interests in medical school turned from the “elusive mind” to the physical brain and then, more generally, to diseases that might be explained by known physiology and biochemistry.

When graduation from medical school was impending, Varmus had to consider his career options more deliberately than he had in the past. A key factor was the Vietnam War, which was in progress, and which he and many others of that era vehemently opposed. “I was determined not to serve in it. Medical graduates were subject to the draft; however, we did have the more palatable option of two years training at one of the agencies of the Public Health Service. For most of my classmates with academic ambitions similar to my own, the NIH was the favored choice. As the largest biomedical research campus in the world, it offered unequaled opportunities to learn virtually any form of biomedical research…”

Varmus confesses that he had a “woeful lack of laboratory credentials.” Nonetheless, he entered the competition for one of the coveted research slots at the NIH. But, because of his lack of research experience, he was not encouraged by most of the NIH laboratory chiefs who interviewed him. However, one of them, endocrinologist Jack Robbins, suggested to Varmus that he speak to Ira Pastan; a young endocrinologist who, at that time, was interested in the production of thyroid hormones.

As Varmus relates, “The recommendation proved to be wise and fateful. My schooling in literature turned out to be more important than my interest in endocrinology, Ira’s field, because Ira’s wife Linda, a poet, had often complained that Ira’s colleagues seldom talked about books…When matches were announced I was told I would become Ira’s first clinical associate, having been passed over by the more senior investigators. This outcome could not have been more fortunate.”

But, before Varmus could take up his position at the NIH, he received a “shocking phone call” from Pastan, to the effect that he (Pastan) was giving up his work on thyroid hormones because he and colleague Bob Perlman “had made a shocking discovery about gene regulation in bacteria.” Pastan and Perlman found that cyclic AMP is a major regulator of bacterial gene activity, and that it plays a similar role in animal cells—findings which led Pastan to pioneer the field of receptor biology in animal cells.

The discovery by Pastan and Perlman had important consequences for Varmus. First, it immediately forced him to give up his plan to train in endocrinology. Instead, Pastan assigned Varmus to find out whether cyclic AMP augments bacterial gene expression by increasing transcription of mRNA. Second, as explained below, Pastan’s new research direction led to Varmus’ introduction to and fascination with virology.

So, Varmus was now a budding molecular biologist. But, since he had no prior research experience, his first days in the Pastan lab were a near disaster, leading Pastan to half jokingly ask, “Now remind me why I took you into the lab.”

In any case, Varmus worked closely with Pastan to develop a molecular hybridization assay to measure transcription of E. coli lac mRNA. [Their specific the goal was determine whether the mechanism by which cyclic AMP reverses catabolite repression of the E. coli lac operon is by enhancing transcription of lac mRNA.] And, they used an E. coli phage, which had incorporated the lac operon into its genome, as their source of isolated lac operon DNA. Thus, Varmus was introduced to virology. [Aficionados, note, “These experiments with the lac operon proved to be analogous in several ways to experiments that revealed the first proto-oncogene a few years later.”]

The satisfaction that Varmus derived from his research in Pastan’s lab caused him to reconsider his aspirations for a career in clinical medicine, and instead to think about a future in biomedical research. He thought he might next try his hand at cancer research, motivated in part, by his mother’s breast cancer, first diagnosed in 1968, to which she succumbed two years later. But there were other factors at work as well. In particular, Varmus’ use of the E. coli phage in Pastan’s lab led to his fascination with virology. And his interest in virology was relevant to his new plans because the DNA and RNA tumor viruses held immense potential for cancer research. 1970s technology could not identify which one of the tens of thousands of cellular genes might have mutated to result in a cancer. However, that technology was potentially able to identify which of the handful of a tumor virus’ genes might underlie its ability to transform a normal cell into a tumor cell.

That line of thought led Varmus to apply for a research position in Renato Dulbecco’s lab at the Salk Institute. [Dulbecco would win a share of the 1975 Nobel Prize in Physiology or Medicine for his pioneering studies of the DNA tumor viruses (5).] However, reminiscent of Varmus’ unsuccessful application to Harvard Medical School, he was “rebuffed by not one but two letters from his (Dulbecco’s) secretary.”

While the rejection from Dulbecco was a disappointment, it would be another of the seemingly providential happenings in Varmus’ career. In the summer of 1969 he chanced to visit Harry Rubin, an eminent Rous sarcoma virus researcher at U Cal Berkeley. Rubin, who had earlier introduced Howard Temin to virology (another auspicious happening; see reference 6), told Varmus about a new group at UCSF that had begun to study retroviruses. Importantly, the goal of the UCSF group was to discover cancer-causing genes. Thus, Varmus stopped over at UCSF, where he met members of the group, including a smart young virologist named Mike Bishop. Varmus reports, “we recognized from the first moments that we were destined to work together.”

Varmus came to Bishop’s lab in 1970 as a postdoctoral fellow. However, their relationship quickly evolved to one of equals, and they made all of their major discoveries in the 1970s and 1980s as a team, and they rose together through the UCSF academic ranks. Bishop relates that their bond formed not just by a shared fascination with cancer viruses but “by our mutual love of words and language.” Varmus, for his part, notes that “after many years of ambivalence and indecision…I appeared to be headed in a clear direction, even if not towards medicine or literature.”

References:

1. Stehelin D, Varmus HE, Bishop JM, Vogt PK., 1976. DNA related to the transforming gene(s) of avian sarcoma viruses is present in normal avian DNA. Nature 260:170-173.

2. How the Human Immunodeficiency Deficiency Virus (HIV) Got Its Name, posted on the blog February 4, 2014.

3. The Politics of Science: Vignettes Featuring Nobel Laureate Harold Varmus during his Tenure as Director of the NIH, posted on the blog June 2, 2014.

4. Varmus, H. 2009. The Art and Politics of Science, (W. W. Norton & Company).

5. Renato Dulbecco and the Beginnings of Quantitative Animal Virology, posted on the blog December 3, 2013.

6. Howard Temin: “In from the Cold”, posted on the blog December 14, 2013.

“The Upright Thinkers”

The Upright Thinkers by Leonard Mlodinow—former professor of Physics at Caltech and author of several other best selling books on science—tells his version of “the human journey from living in trees to understanding the cosmos.” The story is epochal. We begin by noting several of Mlodinow’s general themes.

upright thinkers

Mlodinow asserts that our odyssey of discovery has been driven by our inborn and virtually insatiable curiosity; our propensity to ask “why?” “Human children all around the world ask their first questions at an early age, while they are still babbling and don’t yet speak grammatical language…Chimpanzees and bonobos, on the other hand, can learn to use rudimentary signing to communicate with their trainers, and even answer questions, but they never ask them. They are physically powerful, but they are not thinkers.”

We also are reminded that our astonishing progress was facilitated by a unique characteristic of our species—we add to knowledge already in existence. Recall the famous quote of Isaac Newton, “”If I have seen further than others, it is by standing upon the shoulders of giants.”

Related to the above, there is archaeological evidence that schools existed as early as 2,500 B.C. “…the idea that society should create a profession devoted to passing on knowledge, and that students should spend years acquiring it, was something entirely new—an epiphany for our species.”

Another of Mlodinow’s themes is that the major players in our quest to understand the cosmos (e.g., Newton, Mendeleev, Darwin, Einstein, Bohr, and Heisenberg) were not merely brilliant. They were also stubbornly persistent individuals. Contrary to popular belief, the groundbreaking discoveries they made did not result from sudden “aha moments” but, instead, came about only after years of dogged hard work. There was no apocryphal apple that serendipitously landed on Newton’s head.

Mlodinow also reminds us that scientists, as well as laypeople, do not easily to let go of the conventional beliefs of their day, even when faced with strong evidence to the contrary. Consequently, new scientific paradigms, even when supported by incontrovertible evidence, are often initially rejected by the establishment. And since the revolutionary breakthroughs that Mlodinow highlights in his tale indeed challenged the established wisdom of their times, the scientists who put them forward had to posses more than a little courage and even audacity to complement their doggedness. [Howard Temin provides a recent example of a virologist who persevered in the face of scorn and ridicule from his colleagues. See Howard Temin: “In From the Cold, posted on the blog December 16, 2013.]

Mlidinow allocates several pages to the origin and acceptance of the concept of universally applicable natural laws. The idea of natural laws already existed in ancient Greece. However, Greek natural laws were each specific to a particular situation. Our modern concept of universally applicable natural law came into being only in the early seventeenth century, as prompted by the discoveries of Kepler, Newton, and their contemporaries (see below).

Why might it have taken mankind so long to recognize that nature acts according to certain regularities? The answer may lie in the fact that when primitive humans were faced with seemingly inexplicable droughts, floods, plagues, earthquakes, and so forth, it was difficult for them to view the world as anything but chaotic. Remarkably, even today there still are those who reject the idea of a universe ruled by natural laws. [Consider this. Mlodinow relates that Einstein was astonished by the fact that nature has order. Einstein wrote: “one should expect a chaotic world, which cannot be grasped by the mind in any way… the most incomprehensible thing about the universe is that it is comprehensible.”]

Returning to the theme of our difficulty transitioning from judging the truth of a statement according to how well it fits convention or religious belief, to how well it is supported by empirical evidence, note that for nearly two millennia scientific progress was impeded by the conventions of ancient Greek science. Contrary to popular belief, the Greeks did not invent the scientific method (2). Nor were their theories developed with the intent of experimental verification. Aristotle’s science held that nature did what pure logic suggested it should do. For example, Aristotle said that heavier objects fall faster than lighter objects, because it is their purpose to do so. Neither Aristotle nor his contemporaries actually looked to see if heavier objects indeed fall faster than lighter ones. In fact, the first individual known to have actually tested this premise was Galileo (1564-1642). Only afterwards did observation and experimentation become the basis for western science.

Despite the fact that Aristotle’s non-quantitative search for purpose impeded scientific progress for nearly two thousand years, Mlodinow credits him with applying reason in the pursuit of understanding and, also, for the idea that nature acts according to certain regularities. Mlodinow also notes that Aristotle was hamstrung by the technology of his day. In particular, since Aristotle did not have a stopwatch, concepts such as velocity and acceleration—keystones of Newtonian physics—may have been beyond his reach. But, “More important was the fact that Aristotle was, like everyone else, simply not interested in quantitative description.”

Notwithstanding the above, why did the leap from Aristotelian physics to the breakthroughs of Galileo and Newton take 2,000 years? According to Mlodinow, the conquest of Greece by Rome was a key reason. The Romans were superb engineers, but they had little interest in the pursuit of knowledge for its own sake. Thus, the Roman conquest of the Greeks resulted in the fading out of the Greek scientific heritage from Western culture.

At the end of the eleventh century, a Benedictine monk, Constantinus Africanus, translated ancient Greek medical texts (preserved for centuries by Muslim court scholars) from Arabic to Latin. This was a first step in the revival of the Greek scientific heritage in Europe. Moreover, it was a step towards the eventual resumption of Western science (see Aside 1). Additionally, the development of European universities, such as Oxford (by 1250), facilitated scientific progress in the West, since—then as now—universities are a place that brings people together, where they might interact with and stimulate one another. [Most scientific progress still emanates from universities.] The emergence of moveable-type printing at around 1450 was another key development, since it enabled the widespread dissemination of new ideas.

[Aside 1: Intellectual progress in the Islamic world and in China often surpassed that in Europe during the Middle Ages. Why then did modern science not emerge in the Islamic world, or in China, instead of in Europe? For an answer, I turn briefly to Why the West Rules—For Now by Ian Morris (3). Morris tells us that the resurgence of science in Western Europe was largely driven by Europe’s “new frontier” across the oceans, and the need it created for the precise measurement of time and space. Moreover, “…by the point that two-handed clocks had become the norm Europeans would have to have been positively obtuse not to wonder whether nature itself was not a mechanism.” Additionally, the industrial revolution in the West, which was largely driven by the Atlantic economy, stimulated further mechanical invention and scientific development.]

Galileo was one of the most influential scientists who ever lived and a key figure in the resurgence of European science. One reason for his fame was his experiment at the Leaning Tower of Pisa, which showed that light objects fall as rapidly as heavy ones. Yet while he was disenchanted by the lack of experimentation in Aristotelian physics, he did not invent experimental physics per se. However, his quantitative approach to experimentation was indeed revolutionary. Moreover, “when he got a result that surprised him or went against conventional thinking, he didn’t reject it—he questioned convention and his own thinking.”

One of Galileo’s most insightful and important contentions was that objects in uniform motion tend to remain in that state of motion. That is, an object does not require the continuous application of a force to remain in motion; an assertion that was contrary to the prevailing Aristotelian view that objects require a continuing reason for their motion. If Galileo’s assertion sounds oddly familiar, it is because Newton later adapted it as his first law of motion. [“A few pages after stating the law, Newton adds that it was Galileo who discovered it—a rare instance of Newton giving credit to someone else.”]

Galileo is also famous for his conflict with the Catholic Church over his claim that the Earth is not at the center of the universe. But the idea of a heliocentric (i.e., sun-centered) universe did not originate with Galileo. In fact, it existed as early as the third century B.C. in Greece, and a “modern” European version can be attributed to Copernicus (1473-1543). But Galileo was the first to offer irrefutable proof that the Earth is not at the center of the universes. What’s more, Galileo made his case compelling only after he perfected the telescope, which enabled him to achieve unprecedented levels of power..

Isaac Newton was born in 1642; the year that Galileo died. He is commonly said to have invented the calculus, and to have discovered his laws of motion and universal gravitation, all in the single year, 1666. The last discovery supposedly followed his serendipitous encounter with a falling apple. The reality was a bit different. First, the calculus was concurrently and independently invented by Leibnitz. Second, Newton labored continuously for a period of three years—1664 through 1666—in order to discover his laws of motion. Yet even after accomplishing that feat, Newton was still not a Newtonian. “He still thought of uniform motion as arising from something internal to the body, and by the term ‘gravity’ he meant some inherent property arising from the material an object is made of , rather than an external force exerted by the earth.”

After discovering his laws of universal motion, Newton spent additional years acquiring information, largely compiled by others, regarding planetary orbits. Kepler’s finding—that planetary orbits are ellipses, with the sun at one of the foci—would be especially significant, as follows.

Edmond Halley (the astronomer who computed the orbit of the comet bearing his name), Robert Hooke (the discoverer of cells and perhaps the greatest experimentalist of the seventeenth century), and Christopher Wren (best known as an architect, but also an accomplished astronomer) gleaned from Kepler’s data a premise of singular importance; that the pulling force of the sun diminishes in proportion to the square of the planet’s distance from it. Yet Halley, Hooke, and Wren were unable to prove their conjecture.

Halley, happening to be in Cambridge, took the opportunity to visit Newton. During his visit with Newton, Halley informed his host of the conjecture concerning the pulling force of the sun. Imagine Halley’s surprise when Newton claimed that he had already discovered the premise and, indeed, had already proven it. Newton then set about looking for his proof. But when he found it, he discovered that it was in error. He then set about reworking his proof and, in so doing, he confirmed that Kepler’s planetary orbits indeed are explained by an inverse square law of attraction.

Newton’s proof of the inverse square law of gravitational attraction was followed by more hard work that resulted in “what is perhaps the most significant intellectual discovery that had ever been made”—Newton’s universal theory of motion, which showed that free fall and orbital motion are instances of the same laws of force and motion. Newton set down his laws of motion, and his law of universal gravitation, in his famous Principia; three books published in 1687, which are regarded as the foundation of classical mechanics and, thus, as one of the most important works in the history of science. Halley personally paid the cost of publishing the Principia.

Newton died in 1726. “Newton’s life and Galileo’s had together spanned more than 160 years, and together they witnessed—and in most respects accounted for—most of what is called the scientific revolution.”

A revolution in chemistry was occurring concurrent with the ones in physics and astronomy. The key breakthrough in chemistry would be the discovery that some substances—the elements—are fundamental, and that all else is made from them. Actually, the concept of elements dates back to Aristotle and the ancient Greeks. However, Aristotle’s chemistry had but four “elements”—earth, air, fire, and water.

The experimental results of Robert Boyle (1627-1691) were the first to free chemistry from Aristotelian convention. Mlodinow relates how in 1642, the 15-year-old Boyle was taken with science after reading Galileo’s book on the Copernican system. Afterwards, the adult Boyle (incidentally assisted by Robert Hooke) investigated respiration and combustion. His findings led him to conclude that air is not an element but, instead, is made up of different substances. Moreover, Boyle’s quantitative approach set in place a new convention in chemistry of careful experimental analysis.

Boyle’s experiments were followed by those of Joseph Priestly (1733-1804) and of Antoine Lavoisier (1743-1794). The relationship between these two men makes for an interesting tale, one that is too long to review here. But, together, their researches showed that respiration and combustion removed something from the air. Priestly may have discovered it first, but Lavoisier gave it its name, oxygen. Lavoisier went on to reveal one of chemistry’s major principles—the conservation of mass. That is, the total mass of the products produced in a chemical reaction must be the same as the combined mass of the initial reactants.

Lavoisier had been a tax collector in Paris in the days preceding the French Revolution. His day job provided financial support for his experiments, but it did not endear him to the revolutionaries who overthrew the French monarchy. He was arrested in 1793 during the Reign of Terror and sentenced to death. The presiding judge is said to have told him, “The Republic has no need of scientists.” “By the time of his execution, Lavoisier had identified thirty-three known substances as elements. He was correct about all but ten.”

The next important advancements would involve the nature of atoms. Although Lavoisier identified the elements as the reactants in chemical processes, he chose not to think in terms of atoms. His reason was simply that he did not know of any way by which something as tiny as an atom could be observed or experimented on. John Dalton (1766-1844) took the first step towards solving that problem when he discovered how to determine atomic weights by applying Lavoisier’s law of conservation of mass. [Mlodinow details Dalton’s approach.]

Dmitry Mendeleev (1834-1907) made a crucial breakthrough that would lead to an understanding of the relationship between the weight of an atom and its chemical properties. [The relationship of course involves atomic numbers, rather than atomic weights. Please be patient.] How that development came to pass was one of my favorite anecdotes in Mlodinow’s book. It was as follows. In 1866, while Mendeleev was a chemistry professor in St. Petersburg, he could not find a first-rate, up-to-date chemistry book, written in Russian, that he might use in his teaching. So, he decided to write his own. Mendeleev’s book was a labor of love that he worked on for years. Early on, he wrestled with the question of how to organize the book. His solution was to arrange the elements and their compounds into groups, as defined by their properties. Then, after much arranging and rearranging of the elements, he discovered a pattern that we know today as the “periodic table;” a discovery of singular importance that Mlodinow calls “chemistry’s version of Newton’s laws.”

Much important work remained to be done. Because there were as yet undiscovered elements, there were gaps in the Mendeleev’s table. Moreover, some of the assigned atomic weights were wrong and, crucially important, the chemical properties of an element are determined by the as yet undiscovered atomic numbers, rather than by atomic weights. Notwithstanding that, to Mendeleev’s credit, he accurately predicted the existence and chemical properties of the elements which corresponded to the gaps in his table.

The twentieth-century revolution in physics led to profound insights into the nature of space and time, and to a deeper “understanding” of the atom. Mlodinow tells that story only after he reviews several key breakthroughs in biology. Keeping with his format, he notes the early influence of Aristotelian convention on biology. Two Aristotelian beliefs were especially important. One was the belief that a divine intelligence designed all living beings. The other was the belief in spontaneous generation.

Readers of the blog are likely familiar with Louis Pasteur’s late nineteenth-century experiments, which laid spontaneous generation to rest once and for all (4). Many readers may also know that Francesco Redi experimentally challenged spontaneous generation more than two centuries earlier. In 1668, Redi’s microscopic observations of minute organisms showed that they are much more complex than had been imagined. In particular, these organisms had reproductive organs, which called into question one of Aristotle’s arguments for spontaneous generation; that lower organisms are too simple to reproduce.

The notion, that a divine intelligence was necessary to design all living beings, was a foundation stone of nineteenth-century thinking. Under that circumstance, Charles Darwin (1809-1882) gave the world a scientific theory—evolution by natural selection— that explained how species came to have their particular characteristics. Darwin’s theory directly challenged the biblical account of creation, which still has a very significant number of adherents. Thus, it is all the more remarkable that his grave occupies hallowed ground under Westminster Abbey.

Legend has it that Darwin had an “aha moment” while observing the beaks of finches on different islands of the Galapagos. The reality is that Darwin only gradually came to believe that species are not unchanging forms designed by God but, instead, evolved over time. And Darwin devoted many more years to discovering the mechanism behind evolution—natural selection. Moreover, knowing that his findings would expose him to ridicule and attack, he spent more years amassing incontrovertible evidence in support of his theory.

Darwin did not know of genes and mutations. Consequently, he was unaware of how genetic variability might provide the raw material for adaptation. His contemporary, Gregor Mendel (1822-1884), made the breakthrough in genetics. Mendel, carried out his experiments while a monk at a monastery in what is now the Czech Republic, perhaps explaining why Darwin never knew of his work.

One way or another, serendipity is usually a factor in all great scientific discoveries. Apropos the theory of evolution, Darwin likely would have remained an unknown English churchman were it not for the unlikely and unexpected letter inviting him, at twenty-two years of age, to sail around the world as a naturalist on the Beagle. The position on the Beagle was beforehand offered to a number of others, none of whom were willing to spend two years at sea.

Mlodinow devotes the last 100 or so pages of his book to the twentieth-century revolution in physics. Considering the stunning discoveries that were about to unfold, it is more than a bit ironic that the prevailing belief at the start of the century was that everything in physics was already known and that nothing else remained to be done.

Key individuals who fill Mlodinow’s final pages include Max Planck (see Aside 2), Albert Einstein (see Aside 3), Ernest Rutherford (see Aside 4), Niels Bohr (see Aside 5), and Werner Heisenberg and Erwin Schrodinger (see Aside 6).

[Aside 2: Max Planck was a German physicist who deduced the relationship between the frequency of radiation and its energy. Moreover, he proposed that electromagnetic energy could only take on discrete values, or quanta. In so doing, he originated quantum theory.

Mlodinow tells the following quip: “Since my name is so hard to spell and pronounce, when I make a restaurant reservation, I often do it under the name Max Planck. It’s very rare that the name is recognized, but one time when it was, I was asked if I was related to the “guy who invented quantum theory.” I said, “I am that man.” The maitre d’, in his early twenties didn’t believe me. He said I was too young. “Quantum theory was invented around 1960,” he said. “It was during World War II, as part of the Manhattan Project.”]

[Aside 3: In the single year 1905, while Einstein was employed as a third-class clerk at the Swiss patent office in Zurich, he published three singularly important, ground-breaking papers. The first paper explained Brownian motion, the second the photoelectric effect (in which light impinging on a metal causes it to emit electrons), and the third, his discovery of special relativity. Although each of the three papers dealt with a different topic, astonishingly, each was important enough to merit a Nobel-Prize. And, since Einstein is best known for his theories of relativity—which revolutionized our concepts of space and time—it may surprise some that his Nobel Prize was actually awarded for his explanation of the photoelectric effect. Einstein’s key insight was to treat light as a quantum particle, rather than as a wave. Consequently; his analysis of the photoelectric effect would significantly advance further developments in quantum theory; which Einstein eventually came to abhor (see below). His explanation of Brownian motion provided the most convincing evidence of the day for the existence of atoms. After years more of work, Einstein put forth his theory of general relativity, which incorporated gravity into relativity theory.]

[Aside 4: Rutherford discovered of the structure of the atom, with its positive charge concentrated in the nucleus. Interestingly, this key advancement  began fortuitously, as a “small research project,” for a young undergraduate student named Ernest Marsden; for him “to get his feet wet.”]

[Aside 5: Bohr applied quantum mechanics to the whole atom, asking what it might mean if the atom, like light quanta, could have only certain energies. His premise, that electrons occupy only certain allowable orbits, provided an explanation for why electrons don’t spiral into the atom’s nucleus. And his proposal that the atom’s outer orbits determine its chemical properties, gave new insights into why Mendeleev’s heretofore mysterious periodic table worked.]

[Aside 6: Heisenberg and Schrodinger proposed dissimilar quantum theories, each of which left behind Newton’s conventional description of reality. In Newton’s physics, position and velocity are independently measured quantities, one event causes another, and the world exists independently of our observation of it. In quantum theory, reality is based on probabilities and uncertainties, and reality does not exist independently of our observations. At a scientific conference, when Einstein famously attacked the indeterminacy and probabilistic nature of quantum physics, saying “God does not play dice with the universe,” Bohr famously replied, “Einstein, stop telling God what to do (5).” Yet even Schrodinger eventually turned against quantum theory, offering up his famous fable of the cat that was neither alive nor dead, to point up the seeming absurdity of the theory. Still, quantum theory remains as the most predictive of all scientific theories.]

The Nazis came to power in Germany in 1933. They hated the new physics for being surreal and abstract but, more importantly, because they considered it to be the work of scientists of Jewish heritage (e.g., Einstein, Born, Bohr, Pauli). [Incidentally, the new physics was also condemned in the Soviet Union in the early 1920s; in that instance for not conforming to the principles of Marxist-Leninist ideology. The Soviets similarly banned Mendelian genetics for the same reason.]

Einstein was still living in Germany when the Nazis took control. But, fortunately, he was visiting Cal Tech on the very day in January 1933 when Hitler became Chancellor. He never returned to Germany. His property in Germany was confiscated, his notes on relativity were burned, and a five-thousand-dollar bounty was put on his head.

Heisenberg tried to accommodate himself to the Nazis, but he was nevertheless harassed by them for having worked on “Jewish physics” with Jewish physicists, and for trying to have Max Born (incidentally Max Delbruck’s mentor) exempted from the Nazi non-Aryan work prohibition. Under pressure, Heisenberg disavowed the Jewish physicists and “their” science.

Max Born, with the help of Pauli’s refugee organization, escaped to Cambridge. Schrödinger, an Austrian who had been living in Berlin when Hitler came to power, was an outspoken anti-Nazi. He left Berlin to take a position at Oxford. Bohr’s real-life exploits during the Nazi era are more fascinating than fiction, and are covered in some detail in reference (5).

In 1933, Max Planck met face-to-face with Hitler to dissuade him from carrying out the anti-Jewish policies that were causing top Jewish scientists to leave Germany. The meeting came to nothing, and Planck quietly went on with his work. However, Planck’s youngest son was a member of the failed plot to assassinate Hitler on July 20, 1944. He was arrested by the Gestapo and was executed along with the other conspirators. Earlier, in 1907, when women were nearly entirely barred from receiving advanced education, Planck invited Lisa Meitner (only the second woman to receive a physics doctorate from the University of Vienna) to carry out postdoctoral studies under his guidance (5). Mlodinow includes Meitner among the short list of physicists who he believes deserved, but did not receive the Nobel Prize. See Aside 6. [Reference 6 notes the wartime experiences of Andre Lwoff, Francois Jacob, Jacques Monod, and Elie Wollman. Reference 7 describes a wartime experience of Renato Dulbecco.]

[Aside 7: By 1938, Lisa Meitner’s situation in Nazi Germany had become desperate. So she fled to Holland, aided by Dutch physicists who persuaded their government to admit her on her Austrian passport, which was no longer valid after the Anschluss (5). She next moved to Sweden, where Niels Bohr found for her a laboratory where she could continue her work. While in Sweden, she received a correspondence from her former collaborator in Germany, chemist Otto Hahn, from which she and her nephew Otto Frisch calculated that Hahn had unknowingly witnessed nuclear fission (5). Meitner passed that information on to Niels Bohr, which Bohr took to America and made public at a January 1939 conference at George Washington University. Leading proponents of the new physics immediately realized that a nuclear bomb was now possible. The famous Einstein letter of August 1939, which warned Franklin Roosevelt of that possibility, led to the American Manhattan Project to develop an atomic bomb. German scientists, led by Heisenberg, likewise began work on nuclear energy. In September 1939, Germany invaded Poland and World War II was underway. By September, 1941, Britain was fighting alone against Germany in the west, and the Soviets were reeling under the German onslaught in the East. Those were the circumstances under which Heisenberg attended a German-sponsored conference in Nazi-occupied Copenhagen. Bohr boycotted the conference in protest against the Nazis. However, Heisenberg sought out his former mentor in Copenhagen for a private meeting. While it is known that Bohr and Heisenberg discussed the nuclear weapon issue, the two men were never able to agree on exactly what was said, or even where their conversation actually took place, and to this day their meeting remains shrouded in mystery. Perhaps because Bohr was potentially a huge scientific asset to the Germans, or perhaps because his life was in danger because of his Jewish heritage, the British in 1943 carried out a harrowing rescue mission to smuggle him out of occupied Denmark. Details of the rescue are recounted in reference 5. Bohr then came to America, where he worked on the Manhattan Project and he later became an outspoken arms control advocate. Although it is not entirely clear, there are reasons to believe that Heisenberg may have surreptitiously impeded the German nuclear effort. Bohr and Heisenberg reestablished their relationship after the war, but their shaky friendship was sustained only by their mutual understanding not to revisit their 1941 meeting.]

Mlodinow asserts that “quantum theory was created by a concentration of brain power in Central Europe that surpassed or at least rivaled that of any of the intellectual constellations we’ve encountered in our journey through the ages.” Moreover, “It was a magical time in Europe, with burst after burst of imagination lighting the sky, until the outline of a new realm of nature began to appear.”

A photo in The Upright Thinkers, taken at the famed 1927 Fifth Solvay International Conference on Electrons and Photons, shows Erwin Schrodinger, Wolfgang Pauli, Werner Heisenberg, Paul Dirac, Max Born, Niels Bohr, and Albert Einstein among others; thus bearing out Mlodinow’s contention that it was a very special time in the history of science.

The “golden age of molecular biology” of the 1950s and 1960s is not discussed in The Upright Thinkers. Thus, I feel an urge to proclaim before closing that it too was a very special time in the history of science, when fundamental discoveries were made in biology that were comparable in significance to those made earlier in the century in physics . As a graduate student in the 1960’s I was privileged to experience the thrill of attending, and speaking at the summer conferences at Cold Spring Harbor, which were simultaneously graced by James Watson, Francis Crick, Max Delbruck, Sidney Brenner, Francois Jacob, Jacques Monod, Seymour Benzer, and Norton Zinder (all of whom are discussed elsewhere on the blog), as well as by the likes of Erwin Chargaff, Matthew Meselson, Frank Stahl, Sol Spiegelman, John Beckwith, and so forth.

This piece at last concludes with an item from The Upright Thinkers, which I found to be especially intriguing. Recent DNA analysis shows that around 140,000 years ago, a catastrophic event—possibly related to climate change—caused the entire human population to plummet to a mere several hundred individuals. We truly were an endangered species. “Isaac Newton, Albert Einstein, and everyone else you’ve ever heard of, and the billions of us who live in the world today, are all descendants of those mere hundreds who survived.”

References:

(1) Leonard Mlodinow, The Upright Thinkers, Pantheon Books, 2015.

(2) Thucydides and the Plague of Athens, posted on the blog September 30, 2014.

(3) Ian Morris, Why the West Rules—For Now, Farrar, Straus and Giroux, 2010.

(4) Louis Pasteur: One Step Away from Discovering Viruses, posted on the blog January 7, 2015.

(5) Max Delbruck, Lisa Meitner, Niels Bohr, and the Nazis, posted on the blog November12.

(6) Genealogies and a Selective History of Lysogeny: Featuring Friedrich Loeffler, Emile Roux, Andre Lwoff, Elie Wollman, and Francois Jacob, posted on the blog January 28, 2015.

(7) Renato Dulbecco and the Beginnings of Quantitative Animal Virology, posted on the blog December 3, 2013.

Norman McAlister Gregg and the Discovery of Congenital Rubella Syndrome

Our last posting (1) reviewed key facts about mumps and rubella; the other two viruses targeted by the trivalent MMR vaccine. The current posting tells how, in 1941, Australian ophthalmologist, Norman McAlister Gregg (1892-1966), discovered a link between rubella infection of a woman during pregnancy and her baby suffering from severe birth defects. Gregg’s finding was astonishing at the time because rubella, which is characterized by a rash and swollen glands, was regarded as nothing more than a mild childhood illness; a mere nuisance.

Norman Mcalister Gregg
Norman McAlister Gregg

Rubella is also known as German measles, since it was initially recognized in Germany (in 1814), and since it was at first thought to be a variant of measles. It was suspected of having a viral etiology as early as 1914, but rubella virus per se was not isolated until 1961. Norman Gregg’s story and his remarkable 1941 discovery are as follows.

Gregg received his medical degree in Australia in 1915. World War I was underway at the time, and Gregg immediately joined the British Expeditionary Force, which was then fighting in France. While Gregg was serving in France, as a captain in the Royal Army Medical Corps, he was wounded and was awarded the British Military Cross for gallantry in the field.

Gregg’s Military Cross citation read: “For conspicuous gallantry and devotion to duty during a raid. He untiringly attended to the wounded under heavy enemy fire until the last man was cleared, and showed great coolness and devotion to duty. He worked persistently throughout the raid in the open, and searched for any wounded that might have been overlooked. He behaved splendidly.” [See Aside 1]

[Aside 1: For more on the wartime experiences and heroism of several other important individuals in the history of virology see references 2, 3, and 4.]

Incidentally, Gregg was an outstanding athlete, who excelled at several sports. And, if it were not for the occurrence of World War I, he likely would have played on the Australian Davis Cup team. Note that Australian tennis players dominated international tennis tournaments until the 1960s.

When World War I ended, Gregg returned to Australia, where he served as a resident at the Royal Prince Alfred Hospital in Sydney. He then went to England in 1922 for further training in ophthalmology, and returned to Australia the following year to practice ophthalmic surgery. By 1941, Gregg established himself as senior ophthalmic surgeon at both the Royal Prince Alfred Hospital and the Royal Alexandra Hospital for Children. That very same year he published his landmark paper linking rubella infection during pregnancy to congenital birth defects.

The story of Gregg’s discovery began in 1940, during the Second World War. Australia was then in the midst of a severe rubella epidemic that began in 1939 under the crowded conditions in Australia’s wartime army camps. The illness was spread to the general population by infected soldiers, and it is very likely that some of these young soldiers transmitted the virus to their young wives; some of whom were likely pregnant. There had not been a rubella epidemic in Australia for many years prior to the 1939 outbreak.

In 1940, Gregg, in his role as an ophthalmologist in Sydney, noticed an unusually high incidence of infants born with cataracts. Gregg’s concern over the frequency of these infant cataract cases led him to write to other doctors in Australia to inquire into whether they might likewise have noticed an unusually high incidence of newborns with cataracts. After Gregg’s colleagues reported back, he knew of a total of 78 infant cataract cases, 44 of whom also had heart defects.

Several aspects of these of fetal cataract cases led the perceptive Gregg to suspect that they might be caused by an infectious agent, rather than by a solely developmental or genetic abnormality. First, from his perspective as an ophthalmologist, Gregg noted that the cataracts were atypical in that only the innermost layers of the lens, which form early in development, were affected. Second, many of the children also had heart defects and stunted development, suggesting to Gregg that a more systemic factor caused the syndrome . And, third, there was the uncommonly high frequency and widespread distribution of cases.

To determine whether these cases of congenital birth defects indeed might have a common thread, Gregg carried out a retrospective study (also called a case-control study), in which one tries to identify possibly relevant factors or conditions that existed before the outbreak of the disease. Gregg interviewed the mothers of the 78 affected infants. Remarkably, from these interrogations he learned that 68 of these mothers had contracted rubella early in their pregnancies. [Note that rubella can be so mild that some mothers, who did not report having been infected, may unknowingly have been infected.]

To inquire further into whether there might be a causal relationship between maternal rubella infection and congenital fetal deformities, Gregg next carried out a prospective study (also called a cohort study), in which one tracks a sample of the population that was exposed to the putative etiologic agent before the onset of disease. Thus, Gregg identified a cohort of pregnant women, who had experienced a rash-like illness during their current pregnancies, and then monitored those women to see if their babies displayed congenital defects. A comparison of this group of babies, with those born of mothers who had not experienced a rash-like illness, corroborated the relationship between congenital defects and maternal exposure to rubella virus.

Gregg was bold to assert that rubella during pregnancy could cause congenital malformations. First, the prevailing view at the time was that the placenta provides an impenetrable barrier to infections in utero. Second, the established belief among medical doctors was that all congenital abnormalities are inherited. Third, doctors found it difficult to accept that rubella, which was thought of as a mild disease of childhood, could be connected to severe birth defects. Fourth, there was not yet a laboratory test for rubella. Thus, Gregg’s proposition was based entirely on clinical assessments. For these reasons, Gregg’s work was initially met with skepticism by the medical community.

Nonetheless, Gregg’s findings aroused enough interest that the Australian National Health and Research Council sponsored a follow-up study by medical researcher, Charles Swan, which, when published in 1943, completely substantiated Gregg’s findings. Incidentally, it was Swan who added deafness to the symptoms of congenital rubella syndrome. Mental retardation was noted later by others.

Despite Swan’s corroborating findings, several more years would pass before Gregg’s determinations were widely accepted. [Curiously, the British medical journal The Lancet, which later published Andrew Wakefield’s discredited paper claiming a link between autism and the measles vaccine (5), stated in 1944 that Gregg had failed to prove his case.] The key that led to Gregg’s findings being finally accepted was the analysis by Oliver Lancaster, a statistician and epidemiologist at the University of Sydney, who concluded that Gregg’s data, which related severe birth defects to rubella infection during pregnancy, was statistically significant. Once accepted, Gregg’s pioneering study would greatly stimulate further research into birth defects and their causes. See Aside 2.

[Aside 2: The history of science contains many examples of correct hypotheses that were initially viewed as too radical to be accepted by the scientific community. Howard Temin’s provirus hypothesis is a particularly apt case in point (6).]

Gregg received numerous prestigious awards for his discovery from the governments and scientific societies of Australia, Canada, Great Britain, and New South Wales. But despite his many honors, Gregg is said to have remained an exceptionally humble, friendly, and caring man, who was “held in great respect and affection by all.” When he was notified that there was interest in nominating him for the 1958 Nobel Prize in physiology or medicine, he modestly stated: “I must confess that it comes as a great surprise and rather a shock that my name should even be considered . . . I feel it only fair to you to inform you that I have really no serious publications except those on Rubella as I have found very little time or inclination for writing during a very busy life.”

As noted above, rubella virus was isolated in 1961, and a live attenuated rubella vaccine was developed by 1969. The vaccine, usually given as a component of the trivalent MMR (measles, mumps, and rubella) vaccine, has vastly decreased the incidence of congenital rubella syndrome in regions where it has been used (1).

References:

(1) Andrew Wakefield and the MMR Vaccine Controversy: What about Mumps and Rubella?, Posted on the blog February 18, 2015.

(2) Max Delbruck, Lisa Meitner, Niels Bohr, and the Nazis, Posted on the blog October 30, 2014.

(3) Renato Dulbecco and the Beginnings of Quantitative Animal Virology, Posted on the blog November 19, 2014.

(4) Genealogies and a Selective History of Lysogeny: Featuring Friedrich Loeffler, Emile Roux, Andre Lwoff, Elie Wollman, and Francois Jacob, Posted on the blog January 28, 2015.

(5) Andrew Wakefield and the Measles Vaccine Controversy, Posted on the blog February 9, 2015.

(6) Howard Temin: “In from the Cold”, Posted on the blog November 25, 2014.

Genealogies and a Selective History of Lysogeny: Featuring Friedrich Loeffler, Emile Roux, Andre Lwoff, Elie Wollman, and Francois Jacob

I am intrigued by the genealogies of our leading scientists, since their mentors too were often preeminent scientists. Earlier postings noted the example of Jonas Salk, who did postgraduate studies under Thomas Francis; one of the great pioneers of medical virology, perhaps best known for developing the first influenza vaccine (1, 2). James Watson, who did his doctoral studies in Salvatore Luria’s laboratory, and Renato Dulbecco, who trained under both Luria and Max Delbruck (3), are other examples. In fact, Watson and Dulbecco shared a lab bench in Luria’s lab. Howard Temin did his doctoral (and postdoctoral studies too) in Dulbecco’s lab (4). And Delbruck, who hugely influenced the new science of molecular biology, did his doctoral studies under Max Born, the 1954 Nobel Laureate in physics. Moreover, Delbruck later served as an assistant to Lisa Meitner (5).

Important research paths were undertaken, and major contributions were made, which resulted from less formal interactions between budding young scientists and top scientists of the day. Howard Temin’s chance encounter with Harry Rubin, while on a mission to Dulbecco’s lab, is a case in point (4).

Our last posting told how Louis Pasteur came within a whisker of adding the discovery of viruses to his list of extraordinary achievements (6). Robert Koch played a part in that story for developing his famous postulates, which provided the standard for demonstrating that a particular microbe causes a particular disease.

The Pasteur article also noted that in 1898 Friedrich Loeffler and Paul Frosch isolated the foot and mouth disease virus; the first virus isolated from animals. However, the piece did not point up that Loeffler had trained under Robert Koch. Also, it did not underscore the special significance of what Loeffler and Frosch achieved. In brief, by the 1890s Dmitry Ivanovsky and Martinus Beijerinck had independently discovered that the agent responsible for tobacco mosaic disease passes through bacterium-proof filters. Nevertheless, neither Ivanovsy nor Beijerinck appreciated the implication of their observation. Ivanovsky believed his filters might be defective, while Beijerinck thought the disease was caused by a “living liquid.” In contrast, Loeffler and Frosch, in addition to isolating the first virus that is pathogenic in animals, also carefully considered all possible explanations for their experimental findings, and then were the first to conclude the existence of a kind of microbe too small to be retained by bacterium-proof filters, and too small to be seen under a microscope, and that will not grow on laboratory culture media. They also correctly predicted that smallpox, cowpox, cattle plague, and measles are similarly caused by a “filterable virus.”

Loeffler made another major discovery, fourteen years earlier, in 1884, when he used his mentor’s postulates to identify the bacterium that causes diphtheria, Corynebacterium diphtheriae. Importantly, Loeffler also discovered that when he injected C. diphtheriae into animals, the microbe did not need to spread to the tissues it damaged. This observation led Loeffler to propose the bacteria were secreting a poison or toxin that spread to the remote sites and caused disease there.

Loeffler’s idea of a toxin was a new concept that subsequently was confirmed by Emile Roux, who had been Louis Pasteur’s assistant (6). Using bacterium-proof filters developed by Charles Chamberland in Pasteur’s lab, Roux showed that injecting animals with sterile filtrates of C. diphtheriae cultures caused death with a pathology characteristic of actual diphtheria. Roux was also a co-founder of the Pasteur Institute, where he was responsible for the production of diphtheria anti-toxin; the first effective diphtheria therapy. See Aside 1.

[Aside 1: Earlier, Roux suggested the approach Pasteur used to generate attenuated rabies virus for the Pasteur rabies vaccine (aging spinal cords from rabbits that succumbed to experimental rabies infections of their spinal cords). Roux later withdrew from the rabies project because of a disagreement with Pasteur over whether the rabies vaccine might be safe for use in humans (6).]

So, Loeffler and Roux trained under Koch and Pasteur, respectively. But why might toxin production by C. diphtheriae interest virologists. Well, in 1951, Victor Freeman at the University of Washington showed that the lethal toxins produced by C. diphtheriae (and by Clostridium botulinum as well) are the products of lysogenic bacteriophage carried by the bacteria. This was shown by the finding that avirulent strains of these bacteria became virulent when infected with phages that could be induced from virulent strains. So, are diphtheria and botulism due to bacteria or to viruses? Our chain of genealogies continues with a selective history of lysogeny.

Almost from the beginning of phage research (bacteriophage were discovered independently by Frederick Twort in Great Britain in 1915 and by Félix d’Hérelle in France in 1917), some seemingly normal bacterial cultures were observed to generate phage. Initially, this phenomenon was thought to be a sign of a smoldering, steady state kind of persistent phage infection. Then, during the 1920s and 1930s, the French bacteriologists, Eugene Wollman and his wife Elizabeth, working together on Bacillus megatherium at the Pasteur Institute, provided evidence that instead of a steady state infection, the phage actually enter into a latent form in their host cells; a form in which they might be harmlessly passed from one cell generation to the next. [Considering the state of knowledge back then, note the insightfulness of Eugene Wollman’s 1928 comment, “the two notions of heredity and infection which seemed so completely distinct and in some ways incompatible, . . . almost merge under certain conditions.”] See Aside 2.

[Aside 2: Since some bacterial strains would, on occasion, spontaneously undergo lysis and release bacteriophage, the cryptic bacteriophage they carried were called “lysogenic.” Thus, it is a bit odd that “lysogeny” eventually came to refer to the temperate relationship between these phages and their host cells.]

In the late 1930s, the Wollmans developed a close friendship with Andre Lwoff, their new colleague at the Pasteur Institute. The Wollmans introduced Lwoff to their ideas about lysogeny, but, as Lwoff confesses, he was not then impressed by bacteriophage (7).

The Nazi occupation of Paris during the Second World War began in 1940. From then on, the Jewish Wollmans were prevented from publishing their research findings. Nevertheless, they continued their research at the Pasteur Institute until 1943, when they were seized by the Nazis and sent to Auschwitz. They never were heard from again. Their friend, Lwoff, grieved their loss and became active in the French resistance, gathering intelligence for the Allies, while also hiding downed American airmen in his apartment.

After the war, Lwoff received several honors from the French government for his efforts against the Nazis. He also returned to his research at the Pasteur Institute, studying the genetics of Moraxella; a bacterial pathogen of the human respiratory tract. Because of his work as a microbial geneticist, he was invited to the 1946 Cold Spring Harbor Symposium, where he met Max Delbruck. And as happened to others, meeting Delbruck resulted in Lwoff being seduced by bacteriophage.

Andre Lwoff
Andre Lwoff

Back in Paris, Lwoff’s passionate interest in phages was heightened further by discussions with Jacques Monod, a friend of Max Delbruck, and Lwoff’s neighbor in the attic of the Pasteur Institute. Although Monod was Lwoff’s junior colleague (in fact, it was Lwoff who first stirred Monod’s interest in microbiology), Lwoff’s conversations with the future Nobel Laureate resulted in Lwoff becoming intensely fascinated by lysogeny, which he began to study in 1949 (7).

Because of Lwoff’s earlier friendship with the Wollmans, he chose to study a lysogenic strain of B. megatarium. And, making use of techniques he learned from Renato Dulbecco during a brief stint at Cal Tech, he was able to follow a single lysogenic bacterium, which enabled him to observe that a bacterium could go through multiple rounds of replication without liberating virus. What’s more, he discovered that the phages are released in a burst when the cell lyses, thereby dispelling the still current notion that phages are liberated continuously by lysogenic bacteria. Furthermore, Lwoff showed that lysogenic bacteria usually do not contain phage particles, since none are detected when the cells are experimentally lysed with lysozyme; confirming the earlier (1937) findings of the Wollmans.

Lwoff went on to show that temperate phage genomes are maintained in a previously unknown integrated state in their host cell, and he gave the integrated phage genomes a name, “prophage.” He also discovered, unexpectedly, that irradiating lysogenic bacteria with ultraviolet light could induce the temperate phages to emerge from their latent state, and then replicate in, and lyse their host cells. And, he discovered that the phages lyse their host bacterial cells by producing enzymes that destroy bacterial cell walls.

Prophage
Prophage

Lwoff’s elucidation of the fundamental nature of lysogeny in bacteria would later provide a paradigm for the DNA tumor viruses, the herpesviruses, the oncogenic retroviruses, and HIV. He was awarded a share of the 1965 Nobel Prize for physiology or medicine for his lysogeny research. He shared the award with his fellow Pasteur Institute scientists, François Jacob and Jacques Monod, who received their awards for their pioneering studies of gene regulation in E. Coli.

A rather intriguing aspect of this story is that Lwoff was joined in his research on lysogeny at the Pasteur Institute by Elie Wollman; the son of Eugene and Elizabeth. Elie, born in 1917, escaped from the Nazis in Paris in 1940 and worked in the French resistance as a physician. In 1946, after the war, he came to the Pasteur Institute, where he took its microbiology course and then became Lwoff’s research assistant. Then, in 1947, Elie too happened to meet Max Delbruck (in Paris in this instance) and was invited to join the Cal Tech phage group, where he spent the next two years. See Aside 3.

Elie Wollman
Elie Wollman

[Aside 3: By the early 1940s, the then young Cal Tech “phage group,” headed by Max Delbruck, was on its way to becoming the World’s great center for phage research (5). However, the American group had little interest in lysogeny, since Delbrück neither believed in it, nor saw its importance. Instead, Delbruck was totally committed to the study of lytic phages. Then, during the late 1940s, Delbruck began to lose interest in molecular biology and looked for new research directions. When he thought of turning his attention to brain function, he asked his group to put together a series of seminars based on papers written by prominent neuroscientists of the day. Elie Wollman was the only member of the Cal Tech group who declined to participate in that endeavor, since he was totally committed to bacteriophage. Moreover, Elie was the one who finally convinced Delbruck that “such a thing as lysogeny does exist (7).”

Elie himself tells us that when he looked into a bibliographical index at Cal Tech, he came across an index card referring to his parent’s 1937 paper, which reported their finding that lysogenic cells contain a non-infectious form of the phage (8). “Delbruck’s comment on the card was “Nonsense.”]

After Eli’s two-year stint with Delbruck in Pasadena, he returned to the Pasteur Institute. Meanwhile, Francois Jacob had come to the Institute in the hope of beginning a research career in genetics under the tutelage of either Lwoff or Monod. Before that, in 1940, Jacob, who also was Jewish, left medical school in occupied France to join Free French Forces in London. He then served as a medical officer in North Africa, where he was wounded, and was later severely wounded at Normandy in August 1944, ending his dream of becoming a surgeon.

Francois Jacob
Francois Jacob

Initially, Jacob was spurned by both Lwoff and Monod, but was finally taken on by Lwoff, who suggested that he, Jacob, start work on “the induction of the prophage.” Jacob confesses he had no idea what that meant, but he accepted the project. Thus it came to pass that Francois Jacob and Elie Wollman established a particularly close and friendly collaboration, in which they turned their attention to the lambda prophage of E. coli. Their initial goal was to clarify the events of bacterial conjugation so that they might then understand the phenomenon whereby a temperate phage carried by a lysogenic bacterium is activated to undergo vegetative replication when that bacterium conjugates with, and transfers its integrated phage genome to a non-lysogenic bacterium.

To accomplish their goal, Wollman and Jacob began with experiments to locate the lambda genome on the chromosome of the lysogenic cell, and to follow its transfer during conjugation into a non-lysogenic recipient cell. A key feature of their experimental approach was conceived by Wollman (8). It was simply to interrupt conjugation between a lysogenic donor (Hfr) cell and a non-lysogenic recipient (F-minus) cell, at various times, by using a kitchen blender to break the mating cells apart. Using the blender to interrupt conjugation, and also using bacterial strains in which the recipient bacteria contained a set of mutations, and plating the mating mixture on selective media, Wollman and Jacob were able to measure the length of time required for each of the corresponding wild-type genes to be transferred from the Hfr donor cells to the F-minus recipient cells. Indeed, the time intervals between the appearances of each wild type gene in the recipient cells directly correlated with the distances between the genes, as independently determined by recombination frequencies. Thus, the interrupted mating approach gave Wollman and Jacob a new means to construct a genetic map of the bacterium, while also enabling them to locate the integrated phage genome on that map. Their experimental approach also allowed Wollman and Jacob to establish that, during conjugation, the donor cell’s genome is transferred linearly to the recipient cell. [The designation “Hfr” was coined by William Hayes because Hfr strains yielded a high frequency of recombinants when crossed with female strains.]

Importantly, Wollman and Jacob’s study of the activation of a lambda prophage when it enters a non-lysogenic F-minus recipient (a phenomenon they called “zygotic induction”), showed that the temperate state of the lambda prophage is maintained by some regulatory factor present in the cytoplasm of a lysogenic bacterium, but which is absent from a non-lysogenic one. It led to the discovery of a “genetic switch” that regulates the activation of the lysogenic bacteriophage, and of a phage-encoded repressor that controls the switch. These findings are among the first examples of gene regulation, and are credited with generating concepts such as the repressor/operator, which were firmed up by Jacob and Monod in their Nobel Prize-winning studies of the E. coli lac operon. See Aside 4.

[Aside 4: At the time of Wollman and Jacob’s interrupted mating experiments, kitchen blenders had not yet made their way to European stores. Eli was aware of these appliances only because of his earlier stint at Cal Tech. He bought a blender for his wife before returning to France, and then “borrowed” it for these experiments.]

Wollman and Jacob went on to demonstrate that the fertility or F factor, which confers maleness on the donor bacteria, can exist either in an integrated or an autonomous state. Indeed, this was the first description of such a genetic element, for which they coined the term “episome;” a term now largely replaced by “plasmid.”

Wollman and Jacob also determined that the E. coli chromosome is actually a closed circle. The background was as follows. Only one F factor is integrated into the chromosome of each Hfr strain, and that integration occurs at random. And, since the integrated F factor is the origin of the gene transfer process from the Hfr cell to the F-minus cell, interrupted mating experiments with different Hfr strains gave rise to maps with different times of entry for each gene. However, when these time-of-entry maps were taken together, their overlapping regions gave rise to a consistent circular map. The discovery of the circular E. coli chromosome was most intriguing, because all previously known genetic maps were linear. See Aside 5.

[Aside 5: The bacterial strain used by Wollman and Jacob in their study of zygotic induction was, in fact, the original laboratory strain of E. coli (i.e. E. coli K12) that was isolated in1922 from a patient with an intestinal disorder. In 1951, Esther Lederberg discovered that K12 is lysogenic. The discovery happened when she accidentally isolated non-lysogenic or “cured” derivatives of E. coli K12 that could be infected by samples of culture fluid from the parental K12 strain, which sporadically produced low levels of phage. Esther gave the lysogenic phage its name, lambda.

Esther was the wife of Joshua Lederberg, who received a Nobel Prize in 1958 for discovering sexual conjugation in bacteria, and the genetic recombination that might then ensue. Prior to Lederberg’s discoveries, genetic exchange and recombination were not believed to occur in bacteria. Lederberg’s Nobel award was shared with George Beadle and Edward Tatum (the latter was Lederberg’s postdoctoral mentor) for their work in genetics.

Joshua Lederberg, working with Norton Zinder (9), also discovered transduction, whereby a bacterial gene can be transferred from one bacterium to another by means of a bacteriophage vector. And, working together with Esther, Joshua discovered specialized transduction, whereby lambda phage transduces only those bacterial gene sequences in the vicinity of its integration site on its host chromosome. Esther and Joshua also worked together to develop the technique of replica plating, which enabled the selection of bacterial mutants from among hundreds of bacterial colonies on a plate and, more importantly perhaps, to provide direct proof of the spontaneous origin of mutants that have a selective advantage.]

In 1954 Elie Wollman was appointed a laboratory head in his own right at the Pasteur Institute. He retired from research in 1966 to become vice-director of the Institute, which he then rescued from a severe financial crisis. He continued to serve in that role for the next 20 years, while garnering numerous prestigious awards for his research and service.

Francois Jacob earned his doctorate in 1954 for his lysogeny studies. Then, realizing that he and Jacques Monod, his senior neighbor in the Pasteur Institute attic, were actually studying the same phenomenon, gene repression, he entered into a hugely productive collaboration with Monod that led to the elucidation of the genetic switch that regulates beta-galactosidase synthesis in E. coli (9). Their collaboration established the concepts of regulator genes, operons, and messenger RNA, for which they shared in the 1965 Nobel Prize for physiology or medicine, as noted above. See Asides 6 and 7.

Jacques Monod
Jacques Monod

[Aside 6: One of Jacob and Monod’s first experiments was the famous 1957 PaJaMa experiment, carried out in collaboration with Arthur Pardee, who was then on sabbatical at the Pasteur Institute. In brief (for aficionados), a Lac-positive, Hfr strain was grown in an inducer-free media, and then mated, still in an inducer-free media, with a Lac-minus, F-minus strain. (Note that the deletion in the Lac-minus, F-minus strain included the LacI gene, which encodes the yet to be discovered lac repressor.) As expected, in the absence of inducer, no beta-galactosidase is detected initially. But, after the donor DNA sequence, which bears the normal Lac genes (including LacI), is transferred to the Lac-minus recipient, it initially finds no repressor in the recipient cell and begins to synthesize beta-galactosidase. Then, as the donor cell’s lac repressor gene begins to be expressed in the recipient cell, in the inducer-free media, expression of the donor cell’s beta-galactosidase gene ceases. The PaJaMa experiment thus showed that the genetic regulation of enzymatic induction depends on a previously unknown regulatory molecule, the repressor.

Notice the similarity between the rationale for the PaJaMa experiment and that of the earlier Wollman and Jacob experiment on zygotic induction. In each instance, a process regulated by a repressor is suddenly in the repressor-free environment of a recipient cell.]

[Aside 7: In June of 1960, Francois Jacob, Matt Meslson, and Sidney Brenner came together in Max Delbruck’s Cal Tech lab to carry out an experiment that confirmed the existence of messenger RNA. The key to the experiment was their ability to distinguish ribosomes present in the cell before infection from ribosomes that might have been made after infection. They cleverly did that by incorporating heavy isotopes into ribosomes before infection, so that they might be separated in a density gradient from ribosome made after infection. Then, they showed that RNA produced by T2 phage in E. Coli associates with ribosomes that were synthesized by the cell entirely before infection. Furthermore, the new phage-specific RNA directs the synthesis of phage-specific proteins on those “old” ribosomes. I vote for this experiment as the most elegant in the entire history of molecular biology (11).]

Incidentally, during the Nazi occupation of Paris, Monod too was active in the French Resistance, eventually becoming chief of staff of the French Forces of the Interior. In that capacity, he helped to prepare for the Allied landings in Normandy. Monod and Jacob each received France’s highest honors for their wartime service.See Aside 7.

[Aside 7: I am singularly intrigued by the experiences of Andre Lwoff, Elie Wollman, Francois Jacob, and Jacques Monod during the Second World War. References 3 and 5 recount the wartime experiences of Renato Dulbecco and of Max Delbruck, and of other great scientists of the time. Other posts on the blog give accounts of virologists courageously placing themselves in harm’s way under different circumstances. Examples include pieces featuring Ciro de Quadros, Carlo Urbani, Peter Piot, and Walter Reed.]

References:

(1) Jonas Salk and Albert Sabin: One of the Great Rivalries of Medical Science, Posted on the blog March 27, 2014.

(2) Ernest Goodpasture and the Egg in the Flu Vaccine, Posted on the blog November 25, 2014.

(3) Renato Dulbecco and the Beginnings of Quantitative Animal Virology, Posted on the blog, December 4, 2013.

(4) Howard Temin: “In from the Cold,” Posted on the blog December 16, 2013
(5) Max Delbruck, Lisa Meitner, Niels Bohr, and the Nazis, Posted on the blog November 12, 2013.

(6) Louis Pasteur: One Step Away from Discovering Viruses, Posted on the blog January 7, 2015.

(7) Lwoff, Andre, The Prophage and I, pp. 88-99, in Phage and the Origins of Molecular Biology, J. Cairns, G.S. Stent, and J.D. Watson eds., Cold Spring Harbor Laboratory Press, 1966.

(8) Wollman, Elie L, Bacterial Conjugation, pp. 216-225, in Phage and the Origins of Molecular Biology, J. Cairns, G.S. Stent, and J.D. Watson eds., Cold Spring Harbor Laboratory Press, 1966.

(9) “The Phage in the Letter,” Posted on the blog November 4, 2013.

(9) Francois Jacob, Nobel Lecture, December 11, 1965.

(11) Norkin, Leonard C., Virology: Molecular Biology and Pathogenesis, ASM Press, 2010.

Andre Lwoff

Ernest Goodpasture and the Egg in the Flu Vaccine

There is a cautionary note on the info sheet accompanying the influenza vaccine, which advises individuals who are allergic to eggs to speak with their doctors before receiving the vaccine. As most readers know, the reason for the warning is that the usual flu vaccine is grown in embryonated chicken eggs.

[Aside 1: The current trivalent influenza vaccine is prepared by inoculating separate batches of fertile chicken eggs; each with one of the three influenza strains (representing an H1N1, an H3N2, and a B strain) recommended by the WHO for the upcoming winter flu season. The monovalent viral yields are then combined to make the trivalent vaccine.]

But, why chicken eggs, and how did this state of affairs come to be? The backdrop to this tale is that until the third decade of the twentieth century, virologists were still searching for fruitful means to cultivate viruses outside of a live laboratory animal. This was so despite the fact that, as early as 1907, researchers had been developing procedures for maintaining viable tissues in culture. And, soon afterwards, virologists began to adapt tissue cultures as substrates for propagating viruses.

Yet as late as 1930, there were still only two antiviral vaccines—the smallpox vaccine developed by Edward Jenner in 1798 (1) and the rabies vaccine developed by Louis Pasteur in 1885. Bearing in mind that Jenner’s vaccine preceded the germ-theory of disease by a half century, and that Pasteur’s vaccine came 15 years before the actual discovery of viruses (as microbial agents that are distinct from bacteria), the development of these first two viral vaccines was fortunate indeed (2).

The principal factor holding up the development of new viral vaccines was that viruses, unlike bacteria, could not be propagated in pure culture. Instead, for reasons not yet understood, viruses could replicate only within a suitable host. And, notwithstanding early attempts to propagate viruses in tissue culture (reviewed below), developments had not yet reached a stage where that approach was fruitful enough to generate a vaccine. How then were Jenner and Pasteur able to produce their vaccines? See Aside 2 for the answers.

[Aside 2: Jenner, without any awareness of the existence of infectious microbes, obtained his initial inoculate by using a lance to pierce a cowpox postule on the wrist of a young milkmaid, Sarah Nelmes. Jenner then propagated the vaccine, while also transmitting immunity, by direct person-to-person transfer. (The rationale underlying Jenner’s vaccine, and his story, is told in detail in reference 1.)

Jenner’s live cowpox vaccine protected against smallpox because cowpox, which produces a relatively benign infection in humans, is immunologically cross-reactive with smallpox. Thus, inoculating humans with cowpox induces immunity that is active against cowpox and against smallpox as well. Jenner’s discovery of the smallpox vaccine, while not entirely fortuitous, was still providential, since immunity per se, as well as microbes, were unknown in Jenner’s day.

Following a successful worldwide vaccination program, smallpox was officially declared to be eradicated in 1977. The smallpox vaccine currently stockpiled in the United States contains live vaccinia; a virus that is immunologically related to cowpox and smallpox. Like cowpox, vaccinia causes a mild infection in humans.

The existing smallpox vaccine was grown in the skin of calves. It is now more than 40 years old and has not been used for years, but it is still believed to be effective.

Pasteur (probably the greatest and most famous microbiologist) was a pioneer of the germ theory of disease. Yet he developed his rabies vaccine more than a decade before the discovery of viruses. He did so by applying the same principle that he used earlier to produce a vaccine against cholera. That is, he “attenuated” the rabies agent. He began with virus that was contained in an extract from a rabid dog. Pasteur attenuated the virus for humans by successively passing extracts in the spinal cords of live rabbits, and then aging the last extracts in the series. Modern rabies vaccines are generally killed virus vaccines, prepared by chemically inactivating tissue culture lysates.]

In the years following the pioneering 19th century contributions of Pasteur, Koch, and Lister, and with the widespread acceptance of the germ theory of disease, microbiologists (that is, bacteriologists) appreciated the importance of working with “pure cultures” that could be grown in a sterilized medium. Yet this was proving to be impossible in the case of viruses. Moreover, as late as the 1930s, it was not understood why that should be so

At the very least, virologists would have liked to be able to cultivate viruses outside of a living animal host. The possibility of achieving that goal began to emerge when Ross G. Harrison, working at Johns Hopkins in 1907, became the first researcher to maintain bits of viable tissue outside of an animal. Harrison maintained frog neuroblasts in hanging drops of lymph medium. What’s more, under those conditions, the neuroblasts gave rise to outgrowths of nerve fibers.

In 1913, Edna Steinhardt became the first researcher to cultivate (or at least maintain) a virus (cowpox) in a tissue culture. Steinhardt did this by infecting hanging-drop cultures with corneal extracts from the eyes of cowpox-infected rabbits and guinea pigs. However, there was no methodology at the time for Steinhardt to determine whether the virus might have replicated in her tissue cultures.

In 1912, Alexis Carrel, working at the Rockefeller Institute, began a two-decade-long experiment that significantly increased interest in tissue culture. Carrel maintained tissue fragments from an embryonic chicken heart in a closed flask, which he regularly supplied with fresh nutrients. Later, he claimed that he maintained the viability of the culture for more than 20 years; well beyond the normal lifespan of a chicken. See Aside 3.

[Aside 3: Carrel’s experimental results could never be reproduced. In fact, in the 1960s, Leonard Hayflick and Paul Moorhead made the important discovery that differentiated cells can undergo only a limited number of divisions in culture before undergoing senescence and dying. It is not known how Carrel obtained his anomalous results. But, Carrel was an honored, if controversial scientist, having been awarded the 1912 Nobel Prize in Physiology or Medicine for pioneering vascular suturing techniques. In the 1930s Carrel developed an intriguing and close friendship with Charles Lindbergh, which began when Lindbergh sought out Carrel to see if Carrel might help Lindbergh’s sister, whose heart was damaged by rheumatic fever. Carrel could not help Lindbergh’s sister, but Lindbergh helped Carrel build the first perfusion pump, which laid the groundwork for open heart surgery and organ transplants. Carrel and Lindbergh also co-authored a book, The Culture of Organs. In the 1930s, Carrel, promoted enforced eugenics. During the Second World War, Carrel, who was French by birth, helped the Vichy French government put eugenics policies into practice. Moreover, he praised the eugenics policies of the Third Reich, leading to inconclusive investigations into whether he collaborated with the Nazis. Carrel died in November, 1944.]

In 1925 Frederic Parker and Robert Nye, at the Boston City Hospital, provided the first conclusive evidence for viral growth in a tissue culture. The virus was a strain of herpes simplex, which Parker and Nye received in the form of an extract from Ernest Goodpasture; soon to be the major character in our story. Parker and Nye established their first culture from the brain of a rabbit that was inoculated intracerebrally with an extract from an infected rabbit brain. The animal was sacrificed when in a convulsive state, and its brain was then removed aseptically. Small pieces of normal rabbit testes were added to pieces of brain in the cultures, to provide another potential host cell for the virus. Virus multiplication was demonstrated by inoculating diluents of subculture extracts into laboratory animals. A 1:50,000 diluent was able to transmit the infection.

At this point in our chronology, the pathologist Ernest Goodpasture, and the husband-wife team of Alice and Eugene Woodruff, enters our story. Goodpasture’s principal interest was then, as always, in pathology. He became interested in viruses while he was serving as a Navy doctor during World War I. But his focus was on the pathology of the 1918 influenza pandemic, which he studied in the first sailors stricken by the infection (3). He was later interested in herpetic encephalitis, and in how rabies virus made its way to the central nervous system, but always from the perspective of a pathologist.

Ernest Goodpasture. (I was unable to find a picture of Alice Woodruff.)
Ernest Goodpasture. (I was unable to find a picture of Alice Woodruff.)

In 1927, Eugene Woodruff was a newly graduated physician who joined Goodpasture in the Pathology Department at Vanderbilt University for training as a pathologist. Eugene’s wife, Alice, a Ph.D., came to the Vanderbilt Pathology Department a year later, as a research fellow in Goodpasture’s laboratory.

Goodpasture set Eugene Woodruff to work on fowlpox; a relative of smallpox, which, unlike cowpox, can not infect humans. Goodpasture was interested in the cellular pathology of fowlpox infection; specifically, in the nature of the inclusion bodies seen in fowlpox-infected cells. Using a micropipette, Woodruff was able to pick single inclusion bodies from infected chicken cells, and to then determine that inclusion bodies are intracellular crystalline arrays of the virus.

More apropos to our story, in the late 1920s, virologists still could not generate large amounts of virus that were free of bacteria and contaminating tissue elements. For that reason, Goodpasture believed that future important advancements in virology would require the development of methods to grow large amounts of virus in pure culture; an impossible goal. In any case, Goodpasture delegated Alice Woodruff to develop a method for growing fowlpox outside of a live chicken.

Goodpasture had already adapted Carrel’s tissue culture methods, which he used to maintain chick kidney tissue in culture. So, Alice’s first experiments were attempts to get fowlpox to propagate in cultures of chick kidney tissue. However, the virus stubbornly declined to grow in the tissue cultures. Goodpasture then suggested to Alice that she try to grow the virus in embryonated chicken eggs. But why did Goodpasture make that suggestion?

The answer isn’t clear. But, back in 1910, Peyton Rous and colleague James Murphy, at the Rockefeller Institute, fruitfully made use of fertile chick eggs to cultivate a virus, as described in Aside 4. However, Rous’ accomplishments, which eventually would be recognized as huge, were largely ignored for the next 50 or so years. (The reasons are discussed in reference 4.) Goodpasture may well have been unaware of Rous’ earlier work when he suggested to Alice that she try to cultivate fowlpox in chicken eggs. If so, then his suggestion to Alice may have been an original idea on his part, perhaps inspired by his thinking of the chick embryo as a sterile substrate that is enclosed in a naturally sterile container. On the other hand, he and Alice did note the earlier work of Rous and Murphy in the 1931 report of their own work. (In that paper, they state: “The production of experimental infection in the chorio-allantoic membrane has, however, been done only in the one instance where Rous and Murphy grew the virus of the Rous sarcoma.”). In any case, the chick embryo method for growing viruses had lain dormant for twenty years.

[Aside 4: Rous and Murphy cut a small window into the shells of six-to-sixteen-day-old embryonated chicken eggs, and then placed a bit of a filtered, cell-free extract from a chicken sarcoma into each. By one week’s time there was a tumor mass growing in each of the inoculated embryos. These studies led to Rous’ 1911 report of a filterable, infectious agent, eventually named the Rous sarcoma virus, which causes sarcomas in chickens. The Rous sarcoma virus was the first virus known to cause solid tumors and, moreover, it was the prototype of a virus family that eventually would be known as the retroviruses (4).]

Alice Woodruff’s procedure for infecting the chicken eggs began with her making a small window in the egg shell, at the site of the air sac. (An egg cup served as the operating table, and the window was cut with a dentist’s drill.) She then inoculated the viral extract into the outermost layer of the chorio-allantoic membrane, which encloses the embryo and provides an air channel into its body. Alice then closed the window with a piece of glass, held in place with Vaseline.

Alice tried to maintain sterility at all stages of her procedure. Yet despite the elegance of her techniques, she had nothing to show for these efforts except dead embryos that were overgrown with mold or bacteria. She then turned to her husband, Eugene, who was working in a separate laboratory, down the hall from her lab.

Alice and Eugene, working together, developed procedures to sterilely remove fowlpox lesions from the heads of chicks. In brief, the chick heads were shaved and then bathed in alcohol. Then, the lesions were excised with sterile instruments. Next, the excised lesions were tested for bacterial or fungal contamination by incubating fragments in nutrient broth. If a lesion was sterile by that test, it was deemed fit to be inoculated into the eggs.

Eugene further contributed to the effort by applying a technique that he developed earlier; picking out individual inclusion bodies from fowlpox-infected cells. When he discovered that the inclusion bodies could be disrupted into individual virus particles by incubating them in trypsin, he was able to provide Alice with virtually pure virus that she could inoculate the eggs with.

As Greer Williams relates in Virus Hunters (5): “Then, one morning when she peeked into the window of an egg that had been incubating for about a week after she had infected it with the virus, she saw something different. This chick embryo was still alive…She removed the embryo from the shell and examined it. It had a swollen claw. ‘Could this be due to fowlpox infection?’…She went to Goodpasture and put the same question to him…”

In Alice’s own words, “I can’t forget the thrill of that moment when Dr. Goodpasture came into my lab, and we stood by the hood where the incubator was installed and I showed him this swollen claw from the inoculated embryo (5).”

The swollen claw indeed resulted from the fowlpox infection. This was shown by the fact that when bits of the swollen tissue were transferred to other embryos, they in turn induced more swollen tissue. Moreover, these swollen tissues contained fowlpox inclusion bodies. Additionally, when transferred to adult chickens, those bits of swollen tissue produced typical fowlpox lesions.

During the next year, Goodpasture, Alice Woodruff, and Gerritt Budding (a lab assistant, who dropped out of medical school to participate in the chick embryo work) reported that cowpox and herpes simplex viruses could also be grown in the embryonated chicken eggs.

Later studies by Goodpasture and Buddingh showed that each embryonated chicken egg could produce enough vaccinia to produce more than 1,000 doses of smallpox vaccine. They also showed, in a case-study involving 1,074 individuals, that the chick-grown smallpox vaccine works as well in humans as the vaccine produced by inoculating the skin of calves. Regardless, the chick vaccine never caught on to replace the long-established, but cruder calf-grown vaccine (see Aside 2).

Goodpasture placed Alice’s name ahead of his own on their report describing the propagation of fowlpox in chicken eggs. Alice says that Goodpasture was “over-generous” in that regard. Howevever, much of the day-to-day lab work resulted from her initiatives. Eugene’s name also came before Goodpasture’s on the report describing the inclusion body study.

Shortly after completing these studies, Alice left research to raise a family. Eugene’s name also disappeared from the virus literature. But in his case that was because his interests turned to tuberculosis.

In 1932, soon after the above breakthroughs in Godpasture’s laboratory,  Max Theiler and Eugen Haagen developed their yellow fever vaccine (6), which initially was generated in embryo tissue from mice and chickens. But, starting in 1937, production of the yellow fever vaccine was switched to the embryonated egg method, in part, to “cure” the live yellow fever vaccine of its neurotropic tendencies.

Recall our introductory comments regarding the warning that individuals allergic to eggs should get medical advice before receiving the standard flu vaccine. In 1941, Thomas Francis, at the University of Michigan, used embryonated chicken eggs to produce the first influenza vaccine (see Asides 5 and 6). Remarkably, even today, in the era of recombinant DNA and proteomics, this seemingly quaint procedure is still the preferred means for producing the standard trivalent flu vaccine (see Aside 1).

[Aside 5: Thomas Francis produced his 1941influenza vaccine in response to urging by U.S. Armed Forces Epidemiological Board. With the Second World War underway in Europe and Asia, and with the 1918 influenza pandemic in mind, there was fear that if an influenza epidemic were to emerge during the upcoming winter, it might impede the military training that might be necessary. An epidemic did not materialize that winter, but the vaccine was ready, and we were at war.]

[Aside 6: Thomas Francis was one of the great pioneers of medical virology. The same year that he developed his flu vaccine, Jonas Salk (recently graduated from NYU medical school) came to his laboratory for postgraduate studies. Francis taught Salk his methodology for vaccine development, which ultimately enabled Salk to develop his polio vaccine (7).]

Next, Hillary Koprowski developed a safer, less painful and more effective rabies vaccine that is grown in duck eggs, and that is still widely used. Why duck eggs? The reason is that duck eggs require four weeks to hatch, instead of the three weeks required by chicken eggs. So, duck eggs give the slow-growing rabies virus more time to replicate.

By any measure, the procedures for growing viruses in embryonated chicken eggs, developed by Ernest Goodpasture and Alice Woodruff, were a major step forward in vaccine development. Sir Macfarlane Burnet (a Nobel laureate for his work on immunological tolerance) commented 25 years later, “Nearly all the later practical advances in the control of viral diseases of man and animals sprang from this single discovery.”

Addendum 1: Several major advances in cell and tissue culture (the other means for growing viruses outside of an animal) happened after Woodruff and Goopasture reported the development of their embryonated egg method in 1931. For the sake of completeness, several of these are noted.

In 1933, George Gey, at Johns Hopkins, developed the roller tube technique, in which the tissue is placed in a bottle that is laid on its side and continuously rotated around its cylindrical axis. In that way, the media continually circulates around the tissue. Compared to the older process of growing tissues in suspension, the roller culture method allowed the prolonged maintenance of the tissues in an active state and, consequently, the growth of large amounts of virus. The roller tube technique also works very well for cell cultures that attach to the sides of the bottle. [Incidentally, Gey is probably best known for having established the HeLa line of human carcinoma cells from cancer patient, Henrietta Lacks. HeLa cells comprise the first known human immortal cell line and they have served as one of the most important tools for medical research. (See The Immortal Life of Henrietta Lacks, by Rebecca Skloot, 2010.)]

In 1948, John Enders, and colleagues Thomas Weller and Frederick Robbins, used Gey’s methods, to demonstrate for the first time that poliovirus could be grown in non-nervous tissue. This was significant because the potential hazard of injecting humans with nervous tissue was holding up the development of a polio vaccine.

Next, Renato Dulbecco and Marguerite Vogt, working at Caltech, developed procedures to grow large amounts poliovirus in cell culture, adding to the feasibility of an eventual polio vaccine (8). Additionally, Dulbecco and Vogt developed a plaque assay procedure to measure the titer of animal viruses grown in cell culture (7).

Addendum 2: The following excerpt tells of the chance encounter that led Howard Temin to become a virologist (4). Temin was the Nobel laureate who first proposed the retroviral strategy of replication, and who co-discovered reverse transcriptase.

“Howard Temin began working on Rous sarcoma virus in the 1950s, while a graduate student in Renato Dulbecco’s laboratory at Caltech (see reference 7 for more on Dulbecco). However, he worked under the direct supervision of Harry Rubin, an early star in the field, who was, at the time, a postdoctoral fellow in the Dulbecco lab. Nothing was known as yet about the replication of the RNA tumor viruses, as the retroviruses were then known. Moreover, little more was known about the molecular basis of cancer in the 1950s than was known in 1911, when Rous first isolated his virus; a state of affairs that would be much alleviated by future studies of the oncogenic retroviruses.

Rubin was a veterinarian by training, perhaps accounting for his somewhat unique appreciation of an oncogenic virus of chickens, well after even Rous himself had lost interest. And, Rubin was responsible for introducing other young investigators to the RNA tumor virus field, both at Caltech and later at UC Berkely.

Rubin’s mentorship of Temin began somewhat fortuitously, as follows. When they first met, Temin was actually doing his graduate research in another laboratory at Caltech, looking into the embryology of the innkeeper worm, Urechis caupo. But he was also serving as a laboratory assistant in the Caltech general biology course. In that capacity, he was dispatched to Dulbecco’s laboratory to obtain some fertilized chicken eggs for use in the general biology lab. Harry Rubin supplied the chicken eggs. But the chance visit from Temin gave Rubin the opportunity to tell Temin about the chicken sarcoma viruses that were being studied in the Dulbecco laboratory.

Rubin had just recently found that he could induce the neoplastic transformation of a normal chicken cell with a single Rous sarcoma virus particle. He then demonstrated that the transformed cell produced hundreds more transformed daughter cells in a week’s time. During their chance conversation, Rubin suggested to Temin that he (Temin) might make use of that observation to develop a quantitative tissue culture assay for Rous sarcoma virus. Sufficiently intrigued by Rubin’s proposition, Temin switched from embryology to virology and proceeded to develop a focus-forming cell culture assay for Rous sarcoma virus; an assay analogous in principle to a plaque assay. But instead of forming plaques of dead cells, the non-cytocidal Rous sarcoma virus induces the growth of visible foci of morphologically transformed neoplastic cells.”

[Addendum 3: Today, viruses are usually cultivated in readily available continuous cell lines. That said, when I first entered the field in 1970, as a postdoctoral studying the murine polyomavirus, my first task of the week was to prepare the baby-mouse-kidney and mouse-embryo primary cell cultures, which at that time served as the cellular host for that virus. This rather unpleasant chore was a reason I eventually turned to SV40, since I could grow that virus in continuous lines of monkey kidney cells.

References:

1. Edward Jenner and the Smallpox Vaccine, posted on the blog September 16, 2014.

2. Leonard C. Norkin, Virology: Molecular Biology and Pathogenesis, ASM Press, 2010. Chapter 1 tells how viruses were discovered and how their distinctive nature was brought to light.

3. Opening Pandora’s Box: Resurrecting the 1918 Influenza Pandemic Virus and Transmissible H5N1 Bird Flu, posted on the blog April 15, 2014.

4. Howard Temin: “In from the Cold,” posted on the blog December 14, 2013.

5. Greer Williams, Virus Hunters, Alfred A. Knopf, 1960.

6. The Struggle Against Yellow Fever: Featuring Walter Reed and Max Theiler, posted on the blog May 12, 2014.

7. Renato Dulbecco and the Beginnings of Quantitative Animal Virology, posted on the blog December 3, 2013.

8. Jonas Salk and Albert Sabin: One of the Great Rivalries of Medical Science, posted on the blog March 27, 2014.

How the Human Immunodeficiency Deficiency Virus (HIV) Got Its Name

This story began with the clash between Luc Montagnier and Robert Gallo over priority of discovery and, with it, the right to name the virus. In the midst of this controversy, Harold Varmus seized the initiative to find a universally accepted name for the virus that causes AIDS.

Our previous posting (Who Discovered HIV?) told how Robert Gallo, at the U.S. National Institutes of Health (NIH), and Luc Montagnier, at the Pasteur Institute in Paris, vied to be recognized as the sole discoverer of the AIDS virus. Montagnier named his isolate of the virus “lymphadenopathy associated virus” or LAV, because it came from a patient presenting with lymphadenopthy. 1 Gallo, in contrast, named the virus “human T-cell lymphotropic virus III” or HTLV-III, based on his belief that it was a variant of the human T-cell leukemia viruses-I and –II, which were isolated earlier in his laboratory.

It soon became clear that LAV was quite distinct from HTLV-I and –II. 2 Moreover, and improbably, HTLV-III was found to be identical to another LAV isolated in Montagnier’s laboratory. What’s more, Montagnier had sent a sample of his virus to Gallo before Gallo reported isolating HTLV-III. These events led to recriminations flying back and forth between Montagnier and Gallo, and, not surprisingly, to a bitter rivalry between them, as each held fast to his claim for priority of the discovery.

For the sake of completeness, Jay Levy, at the University of California, San Francisco (UCSF), was also among the first to isolate the AIDS virus, which he named the “AIDS-associated retrovirus” or ARV. Levy did not take part in the dispute between Gallo and Montagnier and, consequently, did not receive the publicity that they did. And, while Levy did not contend for recognition with the fervor of Gallo and Montagnier, his designation for the AIDS virus, and other proposals as well, also had to be considered in the deliberations described below.

The discoverer of a new virus is generally accorded the privilege of naming it. Consequently, the name that the scientific community might ultimately adopt for the AIDS virus could have implications beyond merely providing an appropriate designation for it. Specifically, if the scientific community were to acknowledge LAV or HTLV-III as the name for the virus, it would have been tantamount to recognizing Montagnier or Gallo, respectively, as its discoverer. Thus, any individuals entrusted with resolving the naming issue had to be wary of inadvertently advancing the claims of one, or the other, of the two main protagonists. There was even more at stake for Gallo, since his integrity was being called into question and, consequently, his reputation as well. Moreover, the national pride of both the United States and France were also at issue, as well patent rights to the blood test for the virus.

Although it was clear to all that HTLV-III (or LAV) is distinct from HTLV-I and –II, and that HTLV-III and LAV are one and the same virus, Gallo still went all-out to preserve HTLV-III as the designation for the virus. So, for a time, the awkward solution of the scientific community was to call the virus LAV/HTLV-III, as was recommended by the World Health Organization, or HTLV-III/LAV, as preferred by the U.S. government.

Harold Varmus now steps up to become the key player in the resolution of the naming dispute. But first, here is his bio in brief. Varmus, born in 1939, shared a 1989 Nobel Prize with Michael Bishop for demonstrating that retroviral oncogenes (e.g., v-src) have their counterparts (proto-oncogenes; e.g., c-src) in normal cells. 3 In turn, this led to the realization that mutations in particular host genes, or the inappropriate expression of those genes, might be the underlying basis for human cancers.

varmusHarold Varmus (1981)

To appreciate the huge significance of Varmus’ and Bishop’s 1976 findings, bear in mind that most of the scientific community of the day were skeptical of the notion that cancer had a genetic basis, until Varmus and Bishop provided direct evidence in its support.  Moreover, as Varmus later stated: “In recent years, after our prize was awarded, mutant proto-oncogenes and the proteins they encode have become critical tools for the classification of cancers and promising targets for drugs and antibodies-treatments that have, in some cases, proven to be effective for a significant and growing number of cancers, including leukemias and lymphomas, lung, gastrointestinal, and kidney cancers: and cancers of the breast.” 4

Varmus was a professor at UCSF during the happenings recounted here. Later, between 1993 through 1999, he served as Director of the U.S. National Institutes of Health, and from 2000 through 2010, as President of the Memorial Sloan Kettering Cancer Center. He is currently Director of the National Cancer Institute. On a personal note; I got the idea for this posting from Varmus’ brief account in his book, The Art and Politics of Science (2009). This is a marvelous book that I strongly recommend to all readers of this blog.

At the time of our story, Varmus also was serving as chairman of the Retrovirus Study Group of the International Committee on Taxonomy of Viruses (ICTV). [The ICTV, through its various study groups, has the task of developing and maintaining the commonly accepted virus taxonomy.] As chairman of his study group, Varmus assumed responsibility for resolving the AIDS virus naming dispute. To advise him in that effort, he created an international panel of eminent retrovirologists, which included Howard Temin, 5 Peter Vogt, Myron Essex, Ashley Haase, Steven Oroszlan, Natalie Teich, Kumao Toyoshima, Robin Weiss, John Coffin, and Jay Levy, as well as Gallo and Montagnier. Moreover, Varmus solicited written opinions from more than fifty additional prominent scientists and clinicians, not on his panel.

The panel was soon considering more than a dozen names. Some of these were suggested within the panel, while others were suggested by Varmus’ outside correspondents.

After the panel invested more than a year deliberating these proposed names, which included the two that Montagnier and Gallo originally adopted, it finally settled on “human immunodeficiency virus,” or HIV, as the AIDS virus is now universally known. In reaching its conclusion, the panel considered many issues, including the controversy over priority of discovery, the phylogentic relationship between the AIDS virus and HTLV-I and -II, 2 the immunosuppressive properties of the virus, and the desirability of including the term “AIDS” in its designation. Finally, the panel considered how its preferences squared with established naming conventions and precedent. Varmus, of course, mediated all discussions within his panel.

Notwithstanding all the arguments and compromises that the panel considered, Gallo was not satisfied when all was said and done, nor did the outcome end his dispute with Montagnier. 6 Although the panel’s end result essentially nullified the right of Montagnier and his group to name the virus which they believed they had discovered, Montagnier was already prepared to accept an alternative name, although not HTLV-III. In contrast, since the panel rejected Gallo’s claim that the virus was a variant of HTLV, he, unlike Montagnier, would not sign-off on the May 1986 letter the panel sent to Nature, which proposed that the AIDS virus be called human immunodeficiency virus, or HIV. [The panel also recommended subcategories of HIV. HIV-1 designates the more common type of HIV, which Gallo and Montagnier each claimed to have discovered. HIV-2 designates the less common variety seen in West Africa, which Montagnier is acknowledged to have discovered. 6]

As Varmus later related, “However difficult this process was-with leaks to the press by Montagnier, belligerent letters to me from Gallo that were copied to most of our nation’s leaders, surly and aggressive behavior by the two rivals, and refusals to sign the final statement by Gallo and his close colleague Max Essex, a virologist at Harvard’s School of Public Health-it was interesting intellectually and socially.” 4 [It’s been said that the diplomatic skills, which Varmus acquired while leading the effort to solve the AIDS virus naming dispute, served him well later in his role as Director of the NIH. For much more on Varmus in that later role see: Varmus, H. 2009. The Art and Politics of Science. Norton Books, New York, NY.]

Some of the thorny issues that Varmus’ panel had to come to grips with with were enumerated above. Those issues and additional others, were also discussed in Varmus’s written correspondences with members of his panel, as well as with the outside experts whom he consulted. 7 We now draw on those communications to glimpse the multiple points of view that Varmus and his panel had to wrestle with.

We begin by considering why the term “AIDS” was not included in the panel’s designation for the virus. This is particularly interesting, especially in view of the naming precedent for viruses such as poliovirus, hepatitis A virus, hepatitis B virus, and the influenza viruses; all cases where the virus is designated by the clinical syndrome that it is associated with. Moreover, that naming convention is generally accepted, despite the fact that in these and other such instances, only a small minority of infected individuals ever manifest the disease. What is more, taking the cases of Hepatitis A and B viruses as an example; these are two phylogenetically unrelated viruses that have nothing whatsoever in common, other than that each causes liver disease. And, as Varmus, himself, noted: “Traditional retroviral nomenclature has worked well in this regard. The convention has been to name viruses according to the host species and the prominent pathology associated with the prototypic isolate of a single type; two examples of such names are ‘feline leukemia virus’ and ‘mouse mammary tumor virus.’” 8 And, even more to the point, there are the examples of the human T-cell leukemia viruses, which have already featured prominently in this tale, and in our previous one (Who Discovered HIV?). So, why then did the panel not choose to simply call the etiologic agent of AIDS “the AIDS virus”?

Michael Gottlieb was one of Varmus’ correspondents who spoke out strongly on this issue. He, and his colleagues at UCLA, command our attention, since, in 1981, they were the first to realize that individuals suffering from persistent infections with the protozoan Pneumocystis carinini, and those with the rare cancer, Kaposi’s sarcoma, were all afflicted with the same underlying disease that specifically targeted their CD4 T cells for destruction. That is, they were the first to recognize and report the existence of the disease that subsequently was named AIDS. Here, then, is an excerpt from Gottlieb’s April 25, 1985 letter to Varmus.

“I am writing to convey my concerns as a clinician about sentiment for nomenclature which would identify the agent as the ‘AIDS virus.’ I believe that this nomenclature would be unfortunate. It is estimated that over one million persons in the U.S. alone have serum antibodies. The fully expressed AIDS syndrome is well publicized to be a lethal intractable illness associated with considerable suffering. In my view the term ‘AIDS virus’ would create considerable distress among all individuals found to have previous exposure…I am hopeful that your Study Group will also wish to avoid creating widespread social distress…”  [My note: Gottlieb’s comments, as well as others quoted below, reflect that it was not yet appreciated that virtually all HIV-infected individuals would eventually succumb to AIDS. That disheartening state of affairs would begin to change dramatically with the development of antiretroviral therapy. 6]

Mark Kaplan (North Shore University Hospital), Jerome Groopman (New England Deaconess Hospital), and several other clinicians spoke on the same issue in their April 29, 1985 letter to Varmus:

“The last major aspect to consider in determining the nomenclature of this virus must be the emotions of the patient who is infected with this agent. Patients told that they have infection with the AIDS virus develop devastating psychological symptoms that have been witnessed by all clinicians dealing with these patients and their families. It is a cruel name for the virus for it leaves no hope for the patient, implying that the patient will inevitably develop and die from AIDS. If we were to have called the EB virus by the disease it was first felt to produce, it would have been called the Burkitts Lymphoma virus. By analogy, one can imagine the distress caused to a patient with EBV if told that he had the Burkitts lymphoma virus…”  [My note: EBV, for the Epstein-Barr virus, is a ubiquitous herpesvirus that occasionally causes the non-fatal illness, infectious mononucleosis. It also is associated with Burkitt’s lymphoma, a malignant B-cell lymphoma seen in children living in equatorial Africa and New Guinea.]

Addressing the same issue in her April 22, 1985 letter to Varmus, panel member Natalie Teich, at the Imperial Cancer Research Fund Laboratories, wrote the following :

“Poliovirus was acceptable even though the vast majority of infected persons remained asymptomatic. However, with AIDS, the social and economic implications and stigma may be too overriding.”

Yet in the case of this issue, and others as well, there was no immediate consensus among those contributing to the discussion. Here is what Jay Levy, also a panel member, wrote in his May 10, 1985 letter to Varmus:

“The concern about frightening individuals with the term ‘AIDS’ virus should not be a consideration…no matter what term is given to the AIDS retrovirus, individuals will easily recognize its connotation.”

Levy adds the following: “I favor classifying the AIDS virus in a category by itself. It is most likely the prototype of a human lentivirus and should not be confused with other human retroviruses. My group prefers to maintain our initial nomenclature, that of AIDS-associated retrovirus (ARV) as it best defines the agent linked to this distinct clinical disease.”

Irrespective of whatever scientific merits Levy’s proposal may have brought to the table, it was not seriously considered by the discussants. For example, Natalie Teich dismissed it as follows: “With due regard for Jay, this was clearly a ‘johnny-come-lately’ claim.”

And, William Haseltine, at the Dana-Farber Cancer Institute, wrote the following in his August 7, 1985 letter to Varmus. “…Dr. Jay Levy’s proposed name has no merit as his report merely repeated the original isolations using previously published methods.”

Notice that both Teich and Haseltine rebuff Levy’s preference solely on the basis of right-of-discovery. With that in mind, here is Haseltine’s take on the appropriateness of calling the virus HTLV-III:

“I strongly favor the name HTLV-III for the virus. I would not oppose the name HTLV-III/LAV or LAV/HTLV-III. My reasons are as follows:…Unless there is good reason to the contrary, the original discoverers of the virus should have the right to call the virus the name they chose. Both the laboratories of Drs. Gallo and Montagnier have valid claims to be original discoverers of the virus. Although the Paris laboratory published first, I am convinced that Gallo had, in fact, isolated the virus at or before late 1982 to very early 1983 as did the Paris Laboratory…Given what must be considered to be a lack of consensus of the committee on the appropriate nomenclature, there is no compelling reason not to abide by the choice of the discoverers themselves…HTLV-III is a far better name than LAV. LAV refers to a specific disease state. HTLV-III does not.” [My note: This passage underscores that the controversy between Montagnier and Gallo, over priority of discovery, was still very much alive at this time.]

Anthony Fauci, as Director of the National Institute of Allergy and Infectious Diseases, also commanded attention. In his May 3, 1985 letter to Varmus, Fauci noted that he typically refers to the virus as the “AIDS retrovirus.” However, he argues against adopting that name, not quite for the reasons expressed above, but seemingly because of the mistaken belief at the time that many infected individuals will not develop AIDS. Nevertheless, even if that belief were correct, the very vast majority of individuals infected with poliovirus, and the hepatitis A and B viruses, and other viruses likewise named for the pathology with which they are associated, do not develop those diseases, as was noted above.

Fauci’s most interesting comments may be those concerned with naming the virus either “LAV” or “HTLV-III.” Regarding “LAV,” he says: “…I do believe it would be inappropriate to call this the lymphadenopathy-associated virus (LAV). The reasons for this should be obvious. First, the virus causes more than lymphadenopathy…”

Regarding “HTLV-III,” he says: “Although there are accumulating data, of which you are aware or more aware than I am, that there are significant dissimilarities between this virus and HTLV-I and –II, I still believe that there is enough reason to maintain this virus within the HTLV nomenclature that this should be continued. The reasons for this are that it surely is a human virus (H), it is a T-lymphotropic virus (TL), and it is a virus (V). Therefore, I would think that HTLV itself is a reasonable abbreviation for the virus. For that reason I would suggest naming it either HTLV-III alone or HTLV-III/LAV. However, for reasons given above concerning the disadvantage of using the terminology LAV, I would elect to call it HTLV-III.”

Fauci does not neglect to point out: “I am well aware of all the difficulties and the emotional issues that are interjected into this vis-à-vis who will get more credit related to the name that is chosen. I will try to disassociate myself from any of that and give you as objective a viewpoint as I possibly can concerning the nomenclature….”

The above comments are from but a small subset of Varmus’ correspondences. And, the comments cited above are merely a subset of the positions and arguments stated in them. Yet they enable us to better appreciate Varmus’ accomplishment in arriving at an acceptable and appropriate name for the AIDS virus, and one which did not stir up further discord. As he succinctly stated in his January 17, 1986 memorandum to his panel: “I and several committee members have come to favor HIV: it is simple; it is novel (and hence does not inflame controversies); and it is based upon the name of the disease with which the virus is readily identified, without including the term AIDS.”

I end this posting with the text of a December 19, 1984 letter from Varmus to David Kingsbury at Oxford, in which Varmus informs Kingsbury of the progress of his ICTV Retrovirus Study Group towards revising the retrovirus phylogeny. Varmus’ letter is followed by a portion of Kingsbury’s January 4, 1985 response. [Kingsbury is best known for his research on influenza viruses. I presume that Varmus was corresponding with Kingsbury here, in part because of the latter’s stature within the ICTV, which put Kingsbury in a position to help Varmus gain approval from the ICTV’s higher leadership for his study group’s recommendations.]

“Dear David:

Thanks for your newsletter. As you probably know, we have updated the summary of Retroviridae for Intervirology (a minor task), and we are anticipating some difficulty with finding a suitable name for the AIDS virus. I am waiting for the dust to settle from the nucleotide sequencing (done or almost done in four labs at least) before convening a subcommittee. But it is clear that the AIDS virus is no more related to HTLV-I than to any other retrovirus on the basis of sequence comparison. Would you like to tell Bob Gallo it shouldn’t be called HTLV-III?

Best regards,

Harold E. Varmus, M.D.”

And Kingsbury’s  reply:

“Dear Harold:

….The news about the AIDS virus is startling! Another family of human retroviruses? When you have adequate data to take a firm position on this I will be happy to tell Bob Gallo the facts. I have no vested interest in the matter.

With best wishes,

David”

Footnotes:

1.  As noted in Who Discovered HIV?, before Montagnier began his search for the AIDS agent, a group of French physicians and scientists suggested to him that the best chance to find and isolate it might be at the start of the disease, before the patient’s T cells had severely declined.The reasoning was that if a virus were found at this early stage of the disease, then it would more likely be its cause, rather than merely a consequence of the immune depression. So Montagnier and co-workers looked for a retrovirus in a lymph-node biopsy from a patient with persistent lymphadenopathy (swollen lymph glands); an early sign in patients progressing towards AIDS, but with little sign yet of the impending severe immunodeficiency.

2.  The following statement appears in Harold Varmus’ draft report (Naming the AIDS Virus), which reviews the deliberations of his panel to find a suitable name for the retrovirus that causes AIDS.

 “If an evolutionary tree is established for retroviruses by comparing the order of amino acids in the protein most characteristic of retroviruses, the enzyme that converts RNA to DNA, it is apparent that the AIDS virus is most closely related to the sheep lentivirus, called visna, whereas the human T cell leukemia viruses are in another limb of the tree, more closely related to other oncogenic viruses, leukemia and sarcoma viruses of various animals, particularly the bovine leukemia virus.”

3.  Stehelin, D., H.E. Varmus, M. Bishop, and P.K. Vogt. 1976. DNA related to transforming gene(s) of avian sarcoma viruses is present in normal avian DNA. Nature 260:170-173.

4.  Varmus, H. 2009. The Art and Politics of Science. Norton Books, New York, NY.

5.   For more on Temin, see: Howard Temin: In From the Cold, on the blog.

 6.  Who Discovered HIV? On the blog.

7. The Harold Varmus Papers, AIDS and HIV: Science, Politics, and Controversy, 1981-1993: Documents

8.  Harold Varmus’ April 10,1986 draft of his report, Naming the AIDS Virus, which reviews the deliberations of his panel to find a suitable name for the retrovirus that causes AIDS.

 

Howard Temin: “In from the Cold”

Howard Temin persevered during years of neglect and ridicule until his iconoclastic provirus hypothesis was at last accepted.  This story also features David Baltimore, and personal recollections of Temin and Baltimore.

Concurrently, but independent of each other, Howard Temin and David Baltimore discovered reverse transcriptase in 1970; one of the most dramatic and important findings in the history of molecular biology. The impact of this discovery was so huge that Temin and Baltimore had to wait a mere five years for their contribution to be recognized by the Nobel Committee. Yet the path to the Nobel Prize was far from easy for Temin. Rather, it is a tale of unwavering conviction, persistence, and courage. We begin though with a bit of earlier history.

In 1911, Peyton Rous reported the existence of a filterable, infectious agent that causes sarcomas in chickens. This agent, eventually named the Rous sarcoma virus, was the first virus known to cause solid tumors (1). Moreover; it was the prototype of a virus family that initially was called the RNA tumor viruses (and also the leukoviruses,1).  But, after Temin’s unexpected and radical provirus hypothesis was finally accepted, the family was named the retroviruses.

Here is how Rous described his findings in his original 1911 report: “A transmissible sarcoma of the chicken has been under observation in this laboratory for the past fourteen months, and it has assumed of late a special interest because of its extreme malignancy and a tendency to widespread metastasis. In a careful study of the growth, tests have been made to determine whether it can be transmitted by a filtrate free of the tumor cells . . . . Small quantities of a cell-free filtrate have sufficed to transmit the growth to susceptible fowl.”

Current students might be surprised that Rous’ singularly novel finding of a tumorigenic virus generated only scant interest back in the day. But, bear in mind that nothing whatsoever was known yet regarding the genetic and molecular basis of cancer or, in fact, of the nature of viruses. What’s more, medical researchers in the early 1900s did not recognize the relevance of a transmissible cancer in chickens to malignancies in humans (1).

Indeed, Rous’s unique observations were not appreciated until the 1950s, when it was shown that other viruses (DNA tumor viruses, as well as other RNA tumor viruses) could cause sarcomas, mammary tumors, and leukemias in mice. Only then did the study of tumor viruses become a respectable and even a mainstream pursuit.

Eventually, in 1966, Rous was recognized by the Nobel Committee for his 1911 discovery (2)! This 55-year hiatus is the longest in the history of the Nobel Prize. Bearing in mind that Nobel Prizes are not awarded posthumously, Rous was fortunate to have longevity on his side. He was 87-years-old when awarded the Prize.

This brief anecdote concerning Peyton Rous is relevant to our main account of Howard Temin for multiple reasons. First, the independent discovery of reverse transcriptase by Temin and Baltimore, and the resultant acceptance of Temin’s provirus hypothesis, were the crucial keys to elucidating the unique replication strategy of the retroviruses, of which Rous’ virus was the prototype. Second, reminiscent of the indifferent response of the scientific community to Rous’ discovery, Temin too had to wait years for his contribution to be acknowledged by his peers. However, unique to Temin’s circumstance, during his nearly ten-year wait, he endured being ridiculed by some, while being ignored by most, yet all the time holding fast to his convictions.

Interestingly, during Temin’s 1975 Nobel lecture, he commented as follows on the indifferent response of the scientific community to Rous’ findings at the time they were first reported, and indeed for the next 40 or more years afterwards as well: “Although Rous and his associates carried out many experiments with Rous sarcoma virus, as the virus is now called, and had many prophetic insights into its behavior, they and other biologists of that time did not have the scientific concepts or the technical tools to exploit his discovery.”

Howard M. Temin – via Nobelprize.org. Nobel Media AB 2013

Howard Temin began working on Rous sarcoma virus in the 1950s, while a graduate student in Renato Dulbecco’s laboratory at Caltech [See Renato Dulbecco and the Beginnings of Quantitative Animal Virology]. However, he worked under the direct supervision of Harry Rubin, an early star in the field, who was, at the time, a postdoctoral fellow in the Dulbecco lab. Nothing was known as yet about the replication of the RNA tumor viruses, as the retroviruses were then known. Moreover, little more was known about the molecular basis of cancer in the 1950s than was known in 1911, when Rous first isolated his virus; a state of affairs that would be much alleviated by future studies of the oncogenic retroviruses.

Rubin was a veterinarian by training, perhaps accounting for his somewhat unique appreciation of an oncogenic virus of chickens, well after even Rous himself had lost interest. And, Rubin was responsible for introducing other young investigators to the RNA tumor virus field, both at Caltech and later at UC Berkely.

Rubin’s mentorship of Temin began somewhat fortuitously, as follows. When they first met, Temin was actually doing his graduate research in another laboratory at Caltech, looking into the embryology of the innkeeper worm, Urechis caupo. But he was also serving as a laboratory assistant in the Caltech general biology course. In that capacity, he was dispatched to Dulbecco’s laboratory to obtain some fertilized chicken eggs for use in the general biology lab. Harry Rubin supplied the chicken eggs. But the chance visit from Temin gave Rubin the opportunity to tell Temin about the chicken sarcoma viruses that were being studied in the Dulbecco laboratory.

Rubin had just recently found that he could induce the neoplastic transformation of a normal chicken cell with a single Rous sarcoma virus particle. He then demonstrated that the transformed cell produced hundreds more transformed daughter cells in a week’s time. During their chance conversation, Rubin suggested to Temin that he (Temin) might make use of that observation to develop a quantitative tissue culture assay for Rous sarcoma virus. Sufficiently intrigued by Rubin’s proposition, Temin switched from embryology to virology and proceeded to develop a focus-forming cell culture assay for Rous sarcoma virus; an assay analogous in principle to a plaque assay. But instead of forming plaques of dead cells, the non-cytocidal Rous sarcoma virus induces the growth of visible foci of morphologically transformed neoplastic cells.

Temin’s assay is truly quantitative, since the number of foci of transformed cells that appear in a Rous sarcoma virus-infected cell culture is proportional to the concentration of virus particles in the inoculum. Indeed, Temin’s assay was the first quantitative assay for viral transformation in general, and the first quantitative assay for a retrovirus. Its development was all the more impressive at the time because cell culture was still in its infancy. Importantly, Temin’s assay opened up the study of retroviruses in cell culture, enabling others to eventually make the connection between viral carcinogenesis and the genetic basis of cancer. [Most importantly, the oncogenes of the RNA tumor viruses have their counterparts in the normal cell genome.] But Temin’s more far reaching contribution was still to come. Its genesis was as follows.

As noted above, Rous sarcoma virus is not cytocidal. Consequently, it is able to stably transform chicken cells, while at the same time replicating in them. From Temin’s point of view in the late 1950s, these facts were consistent with the attractive but still untested premise that virus-mediated transformation might result from the continuous presence and expression of virus genes in the transformed cells (3). But how then might one account for the fact that Rous sarcoma virus could likewise stably transform rat cells, which do not support replication of the avian Rous sarcoma virus? To do so, one must either abandon the paradigm that continued viral gene expression maintains the transformed cell phenotype, or one must somehow explain how a viral RNA genome might persist in a line of cells that do not support replication of the virus.

At this early time in the history of retrovirology, it was discovered that bacteriophage λ (lambda) could stably integrate its DNA genome into the chromosome of its host bacterium; a state in which the temperate bacteriophage genome might stably persist for many cell generations. So, did the example of the temperate bacteriophages influence the radical hypothesis that Temin was about to put forward? That question is considered below. In any case, Temin proposed that the Rous sarcoma virus genome likewise persists in eukaryotic host cells in a similar integrated state.

Temin of course realized that such an option would not be possible unless the Rous sarcoma virus single-stranded RNA genome might somehow be transcribed into a double-stranded DNA form. Accordingly, that is precisely what Temin hypothesized. In 1964, Temin’s premise became known as the provirus hypothesis, which explicitly proposes that Rous sarcoma virus generates a DNA copy of its RNA genome, which is then integrated into the cellular DNA as a provirus and stably maintained in that state in clones of transformed cells.

Temin’s provirus hypothesis was iconoclastic in the extreme, largely because it was proposed at the time that the “central dogma of molecular biology” was taking hold. The central dogma, as expounded by Francis Crick in 1958, maintained that information in biological systems always “flows” from DNA to RNA and then to protein. Indeed, the central dogma had taken such a strong hold on thinking at the time that Temin’s provirus hypothesis was regarded as the scientific equivalent of heresy. Consequently, Temin had to fight a long, lonely battle against the criticism and ridicule that his hypothesis generated. Even Harry Rubin, who recruited Temin to the RNA tumor virus field and who served as his first mentor, was candid in his disdain for the provirus hypothesis.

It may now seem odd that the central dogma should have exercised such a strong influence, since RNA viruses were already known and appeared to be an exception to the dogma as then stated. In this regard, Temin noted the following in his 1975 Nobel lecture. “Studies with the newly discovered RNA bacteriophage [see The Phage in the Letter] and with animal RNA viruses, especially using the antibiotic actinomycin D, indicated that RNA viruses transferred their information from RNA to RNA and from RNA to protein and that DNA was not directly involved in the replication of these RNA viruses.”

Temin was a serious researcher, and he indeed carried out experiments to test his radical hypothesis during the ten years that he was being ignored or else scoffed at. Moreover, his experimental results were entirely consistent with his proposal. However, the experimental results generated by the technology of the day could not provide the compelling proof that was demanded to verify a hypothesis that seemingly challenged such strongly entrenched beliefs as enunciated by the central dogma.

Temin’s early experimental findings included the following. First, he reported that actinomycin D, which inhibits transcription from a DNA template, impaired replication of Rous sarcoma virus in cultured chicken cells. Yet while this experimental finding is consistent with the provirus hypothesis, the result also might be explained if viral replication were dependent on the expression of particular cellular genes. Second, Temin showed that inhibitors of DNA synthesis impeded Rous sarcoma virus replication. Yet one could argue that cellular DNA synthesis rather than viral DNA synthesis is necessary for virus replication, perhaps as part and parcel of establishing an intracellular milieu conducive to viral replication. Third, Temin carried out nucleic acid hybridization experiments to demonstrate the presence of Rous sarcoma virus DNA in virus-transformed rat cells. Results of those hybridization experiments also supported the provirus hypothesis. Nevertheless, the hybridization technology of the 1960s could not generate data that was compelling enough to convince Temin’s critics.

In Temin’s Nobel lecture, he said the following about how his experimental findings were received by his colleagues: “Based on the results of these experiments, I proposed the DNA provirus hypothesis at a meeting in the Spring of 1964—the RNA of infecting Rous sarcoma virus acts as a template for the synthesis of viral DNA, the provirus, which acts as a template for the synthesis of progeny Rous sarcoma virus RNA. . . . At this meeting and for the next 6 years this hypothesis was essentially ignored.”

A crucial sticking point preventing acceptance of the provirus hypothesis was that no enzyme was known that could copy RNA into DNA. Thus, the breakthrough that led to widespread acceptance of Temin’s hypothesis came in 1970, when Temin (at the University of Wisconsin) and David Baltimore (at MIT) concurrently, but independently, showed that the previously unknown enzyme, reverse transcriptase, is present in RNA tumor virus particles.Temin found the enzyme in Rous sarcoma virus, while Baltimore found it in the related murine leukemia virus.

In recognition of their independent discoveries, Temin and Baltimore were awarded Nobel Prizes in Physiology or Medicine in 1975. Renato Dulbecco shared in that award for his work on the DNA tumor viruses [see Renato Dulbecco and the Beginnings of Quantitative Animal Virology]. One of Dulbecco’s findings, relevant in the current context, is that neoplastic transformation by the polyomaviruses is associated with the stable integration of their DNA genomes into the host cell genome.

Temin and Baltimore had somewhat different motivations for carrying out their breakthrough experiments. Temin’s entire research career was dedicated to studying the RNA tumor viruses and his primary goal became to validate his provirus hypothesis.  His impetus to seek a reverse transcriptase activity in the retrovirus particle came about as follows. In 1969, in a study that never was reported in its entirety, Temin and his postdoc, Satoshi Mizutani, did experiments demonstrating that the alleged Rous sarcoma virus DNA synthesis could occur in the absence of de novo protein synthesis. This experimental finding implied (to believers at least) that the DNA polymerase activity that catalyzed the synthesis of the viral DNA was present before infection. With no precedent for such a cellular activity, Temin and Mizutani sought the putative polymerase in the virus particle.

In contrast, Baltimore had been studying polymerases generated by other RNA viruses, specifically, ones that transcribe RNA from RNA. Earlier, with his wife, Alice Huang, and other colleagues, he sought to explain why the purified single-stranded RNA genome of vesicular stomatitis virus (VSV) is not infectious, while the purified single-stranded RNA genome of poliovirus is. The contrasting infectivities of the VSV and poliovirus RNA genomes led Baltimore, Huang, and their coworkers to hypothesize that VSV has an obligate requirement for a particle-associated polymerase activity. Earlier, they found that during infection, the poliovirus genome functions as an mRNA after it is released from the virus particle into the cytoplasm. Consequently, the purified poliovirus genome likewise may function as an mRNA in transfection experiments. In contrast to the poliovirus case, they found that the VSV RNA genome is complementary to the mRNAs that encode that virus’s proteins. Accordingly, to account for how VSV might initiate transcription upon infection, they looked for, and found, an RNA-dependent transcriptase activity within VSV particles. From this background and point of view, Baltimore sought a reverse transcriptase activity in retrovirus particles.

David_Baltimore_NIH
David Baltimore in the 1970’s. Image via the National Library of Medicine (image in public domain).

During his Nobel lecture, Baltimore reflected on how his earlier experiences might have led him to his discovery of the retrovirus reverse transcriptase, as follows. “If we look back to virology books of 15 years ago, we find no appreciation yet for the variety of viral genetic systems used by RNA viruses. Since then, the various systems have come into focus, the last to be recognized being that of the retroviruses (“RNA tumor viruses”). As each new genetic system was discovered, it was often the identification of an RNA or a DNA polymerase that could be responsible for the synthesis of virus-specific nucleic acids that gave the most convincing evidence for the existence of the new system. . . .” [This line of thought underlies the Baltimore classification system. See my personal recollection of David Baltimore, below.]

Baltimore spoke after Temin at the Nobel ceremony. The following item from Baltimore’s Nobel lecture is cited here for his comments about his co-award recipient: “In his Nobel lecture, Howard Temin has outlined how he was led to postulate a DNA intermediate in the growth of RNA tumor viruses. Although his logic was persuasive and seems in retrospect to have been flawless, in 1970 there were few advocates and many skeptics. Luckily, I had no experience in the field and so no axe to grind—I also had enormous respect for Howard dating back to my high school days when he had been the guru of the Summer School I attended at the Jackson Laboratory in Maine. So I decided to hedge my bets—I would look for either an RNA or a DNA polymerase in virions of RNA tumor viruses. . . .”

Temin died in 1994 from lung cancer. He was only 59 years of age and never was a smoker. His lung cancer was an adenocarcinoma, a type not linked to smoking. In fact, Temin was a zealous crusader against smoking. Even at the Nobel Prize ceremony in his honor, he reprimanded smokers (including Swedish princesses) in the audience.

After Temin’s death, Baltimore wrote the following: “Ten years in the scientific wilderness is a long time; few have had to bear the silence of their colleagues for so long. I can remember meetings in the 1960s when Howard would present his latest data supporting the provirus notion only to be greeted by either skeptical questions or quiet, polite disbelief. Howard’s conviction that there had to be a provirus never seemed to waver over the whole decade. He knew he was right—and he was—but what fortitude it took to keep looking for the experiment that would show it! My first reaction when I realized that I had seen the reverse transcriptase was to call Howard, because I so much wanted him to know that he was vindicated in his commitment to the idea of a provirus. But he had already found out for himself.”

Howard Temin and David Baltimore published their independently discovered, singularly important finding in back-to-back papers in the British journal Nature (4, 5).

In 1972, Hill and Hillova provided further proof that RNA tumor virus genomes persist in transformed cells in the form of a DNA copy. They did so by demonstrating that purified DNA from a Rous sarcoma virus-transformed cell could produce infectious virus when transfected into a normal cell.

Indisputable evidence of reverse transcription led to the RNA tumor viruses becoming known as the retroviruses. What’s more, it led to the realization that the central dogma, as initially stated by Crick, is not always valid. Exceptions include other viruses such as the hepatitis B viruses, which actually have double-stranded DNA genomes, yet make use of reverse transcription to replicate those genomes. And, there are the cellular retroelements and telomeres, which also bring reverse transcription into play. Importantly, knowledge of the reverse transcription step in the retrovirus life cycle also led to a more complete understanding of HIV infection and the pathogenesis of AIDS. What’s more, the reverse transcriptase enzyme is a critical tool of modern molecular biology.

Interestingly, the discovery of reverse transcriptase led Crick to state that he never excluded the possibility that RNA might serve as a template to make DNA, but instead only the possibility that a protein might serve as the template to make a nucleic acid. Moreover, Crick’s use of the word “dogma” itself caused him additional embarrassment. In his autobiography, What Mad Pursuit, he wrote the following: “I called this idea the central dogma, for two reasons, I suspect. I had already used the obvious word hypothesis in the sequence hypothesis, and in addition I wanted to suggest that this new assumption was more central and more powerful. … As it turned out, the use of the word dogma caused almost more trouble than it was worth. Many years later Jacques Monod pointed out to me that I did not appear to understand the correct use of the word dogma, which is a belief that cannot be doubted. I did apprehend this in a vague sort of way but since I thought that all religious beliefs were without foundation, I used the word the way I myself thought about it, not as most of the world does, and simply applied it to a grand hypothesis that, however plausible, had little direct experimental support.”

We conclude this part of the posting by returning to an issue raised above: did the discovery of bacteriophage lysogeny prompt Temin to put forward the provirus hypothesis? Here are Baltimore’s comments on this question: “Although the pregnant analogy to known lysogenic bacteriophage might have guided Howard, people who were at Caltech at that time assure me that Howard was unlikely to have arrived at the notion of a DNA intermediate through this route. Apparently, the influence of Max Delbrück (6)—who was totally committed to the study of lytic phages and did not really believe in the importance of phage lysogeny—was so great that there was little discussion of lysogeny at Caltech then. Furthermore, Howard has minimized the importance of lysogeny as a precursor to his concepts. Therefore, he must have arrived at the concept of a DNA intermediate simply from the persuasive power of such a concept to explain the properties of the transformed state. He was particularly influenced by the morphological difference between cells transformed by particular Rous sarcoma virus variants, which he felt had to mean that the viral genome continued forever to affect the transformed cell.” Temin, in his Nobel lecture, indeed cites the mutant studies to which Baltimore refers as a key factor in the genesis of the provirus hypothesis.

Personal Recollections of Howard Temin and David Baltimore

I briefly met Howard Temin in 1971, when he visited my postdoctoral advisor, Tom Benjamin. Several years later, when I was a still young assistant professor at UMass, I had the chance to spend more time with Howard when he came to present a seminar in my department. After the seminar, I had the privilege of taking Howard to lunch where, much to my delight, he asked me to tell him about my own research. What struck me during this incident was how attentive and responsive Howard was to all I was telling him. He instantly understood the implications of each experiment I described and his mind always seemed to be one experiment ahead of me. When we were finished, I mentioned this impression to him. His response, delivered in a most unassuming way, was “Lenny, I’ve seen a lot of science. There are only so many experiments one can do, and I’ve seen them all.”

Shortly afterwards, while investigating simian virus (SV40) entry into cells, I became interested in the intracellular signals that SV40 appeared to transmit from the cell surface. At the time, cell signaling was already a very big field with a vast literature. However, very little was known or published about virus-induced signaling. I soon found myself snowed under by the enormity of the new-to-me cell signaling literature. Then, I suddenly realized that I was seeing one or more of the same four basic experimental approaches in each of the papers I was reading.  Recalling my lunch with Howard; there are only so many experiments one can do, and I’d become familiar with all that I would need.

David Baltimore (born in 1938) is currently Professor of Biology at Caltech, where he earlier served as President. Before that, he served as a professor at MIT and as President of Rockefeller University. I never had the pleasure of meeting David Baltimore. Nevertheless, he influenced my career in a major way as follows. I earned my Ph.D. in 1969 in the area of bacterial genetics. Then, I spent an all too brief two years as a post-doc studying transformation by the mouse polyomavirus. Next, it seemed I was all too suddenly an assistant professor at UMass, where I was expected to teach an animal virology course to advanced undergraduates and graduate students.

Here then was my dilemma. I was still an expert bacterial geneticist. But, while I was knowledgeable regarding transformation by the tumor viruses, I was far from being an expert animal virologist. Moreover, I never actually had a virology course. My predicament was compounded further by the fact that the virology textbooks of the day were for the most part descriptive. Thus, I was at a loss as to how I might deliver three 50-minute virology lectures per week to advanced undergraduates and graduate students.

With my back seemingly against the wall, I rather fortuitously came across a 1971 review article by David Baltimore, in which he put forward his Baltimore classification system (7).  In brief, the Baltimore classification system is based on the notion that the nature of a viral genome (e.g., double-stranded DNA, plus-strand RNA, minus-strand RNA, etc.) largely determines its expression strategy. Moreover, there were only six classes, or basic strategies in the original Baltimore scheme, and each of the formal virus families fit into one of the Baltimore classes. Although the Baltimore system is not a formal means for classifying viruses, I immediately recognized its didactic potential. Indeed, I had my answer regarding how to teach virology.

At first, I followed Baltimore’s review article, initially basing my lectures on the references therein, which I updated with more recent articles over the next several years. But, the field was progressing too rapidly to go on in this way, so I had to turn to textbooks. However, none of these books was organized around the Baltimore classification system, which remained the key concept around which I structured my lectures. Indeed, to this day I remain convinced that the best way to teach virology is to discus viruses in the context of virus families, with the Baltimore system as the organizing principle.

Over the years I fantasized about writing a book that might present the field in this way. Virology: Molecular Biology and Pathogenesis was the fulfillment of that vision.

References and notes:

1. An oncogenic virus, later known to be closely related to the Rous sarcoma virus, actually was discovered 3 years earlier by Vilhelm Ellereman and Olaf Bang. They found that leukemia in birds could be transmitted by a filterable agent from leukemic cells or by serum from leukemic birds. However, leukemia was not then recognized as cancer, so the significance of this discovery, like Rous’, went unrecognized.

2. The 1966 Nobel Prize in Physiology or Medicine was divided equally between Peyton Rous “for his discovery of tumour-inducing viruses” and Charles Brenton Huggins “for his discoveries concerning hormonal treatment of prostatic cancer”.

3. Shortly after the discovery of reverse transcriptase, Steve Martin, at the time a graduate student in Harry Rubin’s laboratory (at UC Berkeley where Rubin was then a professor) reported the isolation of a mutant of Rous sarcoma virus that was temperature-sensitive for transformation. Importantly, cells transformed by the mutant virus at its permissive temperature reacquired the normal cell phenotype when incubated at the non-permissive temperature. Thus, expression of a viral oncogene (src in this instance) is required to both initiate and maintain the transformed cell phenotype.

4. Baltimore, D. 1970. RNA-dependent DNA polymerase in virions of RNA tumor viruses. Nature 226:1209-1211.

5. Temin, H. M., S.Mizutani. 1970. RNA-dependent DNA polymerase in virions of Rous sarcoma virus. Nature 226:1211-1213.

6. See the earlier posting, Max Delbruck, Lise Meitner, Niels Bohr,  and the Nazis.

7. Baltimore, D. 1971. Expression of animal virus genomes. Bacteriol Rev. 35:235-41.