Monthly Archives: April 2015

Wendell Stanley: First to Crystallize a Virus

In 1935 Wendell Stanley crystallized tobacco mosaic virus (TMV); an accomplishment for which he was awarded a share of the 1946 Nobel Prize in Chemistry. As a matter of history, Stanley’s Nobel award was the first ever bestowed on a virologist.

Wendel Stanley. 1946 Nobel Prize photo.
Wendel Stanley. 1946 Nobel Prize photo.

I have long considered Stanley’s achievement to be one of the most important developments in virology and, indeed, in biology in general. To appreciate why Stanley’s feat might have been so significant, we need to consider how little was known in the mid 1930s—and for the next two decades as well—about the chemical nature of genes. In fact, it was still widely assumed that genes are comprised of proteins. That was so because before James Watson and Francis Crick solved the structure of DNA in 1953, that molecule was thought to be structurally simple; rather like a starch. In contrast, proteins were structurally complex, and their wide variety seemed to provide for a virtually unlimited number of genes. But, as you might suppose, attempts to explain how proteins might be replicated led to rather unsatisfying models. Consequently, many serious biologists of the day adhered to the vitalist belief that life could not be explained by known laws of physics and chemistry.

The 1928 experiments of Frederick Griffith, involving the bacterium Diplococcus pneumoniae, provided the groundwork for later experiments by others that would cause some to consider that DNA indeed might be the genetic material. Griffith demonstrated that exposing live avirulent pneumococcal cells to an extract prepared from heat-killed virulent cells could transform the avirulent cells into virulent ones. [These experiments were part of Griffith’s efforts to create a vaccine against D. pneumoniae. He died in 1941, never knowing that his work would constitute one of the keystones of molecular biology.]

Griffith’s 1928 experiments were followed by the 1944 experiments of Oswald Avery, Colin MacLeod, and Maclyn McCarty, who identified the transforming activity in the extracts of virulent pneumococcal cells. Avery, MacLeod, and McCarty fractionated the extract into its various macromolecular constituents—protein, lipid, polysaccharide, and DNA. Next, they asked which of these fractions might have transforming activity. To the surprise of almost everyone, only the DNA fraction transformed avirulent, non-encapsulated pneumococcal cells into virulent encapsulated ones.

The remarkable findings from these transformation experiments were met with widespread skepticism. That was so because it was difficult for the classically trained geneticists of the day to accept these strange, seemingly bizarre experiments. Classical geneticists experimented by crossing organisms; not by transforming them with extracts. What’s more, they thought in terms of hereditary units called “genes,” rather than in terms of molecules of nucleic acid, or whatever other substance that genes might be comprised of. Moreover, as noted above, DNA was viewed as a rather uninteresting molecule. Thus, most biologists of the day continued to hold the view that genes are comprised of protein.

As to the state of virology in the mid 1930s, most interest in the field was concerned with medical and agricultural issues. Moreover, essentially all that was known about viruses per se was that they are smaller than bacteria and can propagate only within suitable host cells. Thus, virology had not yet advanced biological knowledge in general (see Aside 1.)

[Aside 1: Stanley relates in his 1946 Nobel lecture (1), “…when the work on viruses, which is recognized by the1946 Nobel Prize for Chemistry, was started in 1932, the true nature of viruses was a complete mystery. It was not known whether they were inorganic, carbohydrate, hydrocarbon, lipid, protein or organismal in nature. It became necessary, therefore, to conduct experiments which would yield information of a definite nature. Tobacco mosaic virus was selected for these initial experiments because it appeared to provide several unusual advantages…”]

Nonetheless, by the mid 1930s biochemists had made great strides in purifying and crystallizing proteins. [Solving the structure of proteins by crystallography was still well beyond the technology of the day.] Inspired by the success of the protein crystallographers, and encouraged by his evidence that TMV is at least partly a protein, Stanley proceeded to crystallize TMV (see Asides 2 and 3).

[Aside 2: Stanley’s evidence that viruses are comprised of protein was recounted in his Nobel lecture (1): “…in studies with pepsin it was found that this enzyme inactivated the virus only under conditions under which pepsin is active as a proteolytic agent… It was concluded in 1934 that “the virus of tobacco mosaic is a protein, or very closely associated with a protein, which may be hydrolyzed by pepsin.”’]

[Aside 3: The theme of this posting is the significance of Stanley’s feat of crystallizing tobacco mosaic virus. See Stanley’s 1946 Nobel lecture (1) for details on the heroic effort that went into that achievement.]

Importantly, and to the surprise of many, Stanley’s protein-containing TMV crystals retained the infectious activity of the actual virus! Also, it is crucially important that crystals are exquisitely pure. This key fact enabled Frederick Bawden and Norman Pirie in 1936 to demonstrate unequivocally that TMV is not a pure protein. Instead, TMV contains about 6% ribonucleic acid (RNA) (3). Consequently, whatever it is about TMV that enables it to produce copies of itself, that ability resides in its protein, or in its nucleic acid, or in a combination of its two macromolecular constituents (see Aside 4). [Aficionados might note that the ability of TMV to form crystals also implied that the virus has a regular structure.]

[Aside 4: Stanley may have initially believed that TMV is comprised entirely of protein. In his 1935 Science paper (2), he notes: “Although it is difficult, if not impossible, to obtain conclusive positive proof of the purity of a protein, there is strong evidence that the crystalline protein herein described is either pure or is a solid solution of proteins.”]

The finding that TMV is comprised of protein and RNA also gave rise to the notion that a virus is more complex than a mere chemical, even if not quite an organism. But note that Max Schlesinger in 1933 was actually the first one to find nucleic acid in a virus. Making use of new high-speed centrifuges, Schlesinger purified a bacteriophage to high purity and demonstrated that it is comprised of 50% protein and 50% DNA. However, Schlesinger did not study crystalline material, as Bawden and Pirie had done. Moreover, no one at the time knew quite what to make of Schlesinger’s findings. Consequently his work did not get as much attention as that of Bawden and Pirie.

Although Stanley’s work would eventually be recognized by the Nobel committee, when it first appeared many scientists could not accept that a crystal might actually possess a key property that we associate with life—the ability to replicate. And other researchers failed to see how an infectious mottling illness in tobacco plants could be relevant to disease in humans. [This point is reminiscent of the medical community’s disinterest in Peyton Rous’ 1911 discovery of a transmissible cancer in chickens. Medical researchers of the day could not see its relevance to malignancies in humans (4).]

Recall that many serious biologists and chemists in those earlier years still adhered to the belief that some “vital” force outside the known laws of chemistry and physics would be needed to explain the phenomenon of life. Yet if viruses are so simple that they that they could be crystallized like table salt, and still express that most fundamental property of living systems—the ability to replicate—then there might be reason to believe that the nature of biological replication indeed might be understandable in terms of conventional chemistry and physics. Moreover, note that crystallography is a very precise science. Thus, taken together, the facts that TMV could be crystallized, and yet retain biological activity, strongly implied to at least some scientists that conventional physics and chemistry would suffice to explain life.

Spurred on by this line of thought, a somewhat atypical group of investigators sought to understand the nature of genes. These researchers were atypical in that they generally had little or no knowledge of traditional genetics, or of biochemistry, or, in fact, of biology of any sorts. Many were physicists by background. But, they had a single goal in mind: to understand the physical basis of the gene. What’s more, several of these investigators recognized the advantages of focusing their research efforts on viruses.

This odd group’s interest in genes, and its focus on viruses, would lead to discoveries of singular overwhelming importance. Indeed, their research approaches and the results they generated gave rise to molecular biology. Thus, Stanley’s achievement would mark the death knell of vitalism and spur the beginning of the field of molecular biology (see Aside 5). And, when Watson and Crick solved the structure of DNA in 1953, it became clear that the expression and replication of the genetic material would be accounted for by the known laws of physics and chemistry.

[Aside 5: Physicist Max Delbruck was a key player in this atypical group of researchers, and he is recognized as one of the principal founders of the new science of molecular biology. Yet it is ironic that Delbrück was initially drawn to biology by the belief that it might reveal new concepts of physics. For more on Delbruck and the “phage group” he founded at CalTech, see reference 5.]

Wendell Stanley carried out his ground breaking research on TMV at the Rockefeller Institute (now the Rockefeller University). He passed away at a scientific conference in Salamanca, Spain in June, 1971.

References:

(1) Stanley, W.M., The isolation and properties of crystalline tobacco mosaic virus,
Nobel Lecture, December 12, 1946

(2) Stanley, W. 1935. Isolation of a crystalline protein possessing the properties of tobacco-mosaic virus. Science 81:644-645.

(3) Bawden F.C., N.W. Pirie, J.D. Bernal, and I. Fankuchen. 1936. Liquid crystalline substances from virus-infected plants. Nature 138: 1051–1055.

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

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

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Carlton Gajdusek, Kuru, and Cannibalism

Today’s posting features an especially intriguing infectious disease called kuru, and Carlton Gajdusek, the man who won a Nobel Prize in 1976 for his study of its rather shocking epidemiology, only to have a shadow cast over his reputation when he was later convicted of child molestation.

Carlton Gajdusek in 1997
Carlton Gajdusek in 1997

Gajdusek graduated from Harvard Medical School in 1946 and then carried out postdoctoral studies at Caltech under Linus Pauling and Max Delbruck, and at Harvard under John Enders. [All three of Gajduseks postdoctoral mentors became Nobel laureates. See reference 1 for additional examples of leading scientists who had preeminent scientists for mentors.]

In 1954 Gajdusek was in Melbourne, as a visiting investigator under the direction of another future Nobel laureate, the Australian immunologist Sir Macfarlane Burnet. In 1957 Burnet sent Gajdusek to New Guinea, to take part in a multinational study of disease in the native populations. Thus it came to pass that Gajdusek heard about a mysterious illness called kuru, which affected a tribe of the Fore people of the eastern highlands of New Guinea.

The term “kuru” means “shivering” or “trembling” in the Fore language, reflecting an outward symptom of the disease. In the late 1950s, as the Fore people were just emerging from their Stone Age way of life, stories about their strange disease began to leak out to the modern world. Gajdusek was fascinated by these stories and, accordingly, he traveled to the Fore people to see the disease for himself.

Before continuing our account of Gajdusek and the Fore people, we pause to note that kuru is actually not caused by a virus per se. Instead, kuru, like scrapie in sheep, is caused by a prion (a term derived from “proteinaceous infectious particle.”) Prions, like viruses, pass through filters that block bacteria. However, prions are much smaller than even viruses and, in fact, do not contain genomes! Stanley Pruisner, who coined the term “prion,” won a Nobel Prize in 1997 for his breakthrough studies of the nature of prions and their means of replication (topics for a future posting). Other prion diseases include bovine spongiform encephalopathy (known colloquially as “mad cow disease”) and Creutzfeldt-Jacob disease (CJD) in humans. These diseases are also called transmissible spongiform encephalopathies; reflecting their infectious nature and their characteristic neuropathology. Each is invariably fatal. See Aside 1.

[Aside 1: Bovine spongiform encephalopathy (BSE) was a hot news item several years ago after the disclosure that British health officials allowed BSE-affected cattle to enter into England’s food supply, for as long as two years after those officials knew that BSE was spreading in British cattle. The subsequent discovery, that eating meat from BSE-affected cows can give rise to CJD in humans, led to a panic. Health officials were accused of putting the interests of the British meat industry above those of the public.]

When Gajdusek arrived among the Fore, their population consisted of about 35,000 individuals, about 1% of who were afflicted with kuru. Strangely, the disease was most prevalent in women. In fact, the discrepancy between affected women and affected men was so great that in some Fore villages men outnumbered women by 3 to 1!

Children with kuru were rare. Later, it would be understood that this was because of the long incubation period for kuru; a characteristic of the transmissible spongiform encephalopathies in general. The incubation periods for kuru and CJD can be as long as 30 years. But, once symptoms appear, patients generally succumbed within a year.

A Fore child with advanced kuru in 1957. She was sedentary for several months and was reaching the terminal stage of the disease.
A Fore child with advanced kuru in 1957. She was sedentary for
several months and was reaching the terminal stage of the disease.

Gajdusek was fascinated and puzzled by several aspects of kuru, including the strange tendency of the disease to affect women, and its slow progression. Moreover, he observed that there was no inflammation or fever associated with kuru or, indeed, any sign of infection or post-infection phenomena. For these reasons, Gajdusek at first thought that kuru might be caused by a sex-linked genetic factor, or perhaps by malnutrition. See Aside 2.

[Aside 2: Conventional virus infections of the human brain (e.g., subacute sclerosing panencephalitis (measles), progressive multifocal leukoencephalopathy (JC virus), cytomegalovirus brain infection, etc,) are associated with an inflammatory response, a rise in serum protein, and the presence of virus particles in electron micrographs; none of which are seen in the transmissible spongiform encephalopathies.]

Then, in 1959, while Gajdusek was searching for the cause of kuru in New Guinea, William Hadlow fortuitously came on the scene in London. Hadlow was a veterinary pathologist from the United States Department of Agriculture, who, at the time, was studying scrapie in England. By chance, Hadlow was visited in London by his friend, parasitologist William Jellison, from the Rocky Mountain Laboratory in Montana, where Hadlow too had worked before joining the USDA.

During his visit with Hadlow, Jellison casually mentioned an exhibit on the neuropathology of kuru at the Wellcome Medical Museum in London (see Aside 3). Jellison thought that Hadlow might find the exhibit interesting since Hadlow was studying scrapie which, like kuru, is a neurodegenerative disease. Thus, it came to pass that five days later Hadlow visited the exhibit and was struck by the remarkable similarity between the neuropathology of kuru in humans and scrapie in sheep. Importantly, it was already known that scrapie in sheep is a transmissible disease.

[Aside 3: The London exhibit was prepared by Igor Klatzo, Head of the Neuropathology Section of the National Institute of Neurological Diseases at the NIH. Gajdusek sent brains from kuru victims back to the NIH for analysis. That is how Klatzo came to describe the neuropathology of kuru and, in addition, to note its similarity to the more widespread Creutzfeldt-Jakob disease. The importance of Klatzo’s observation concerning CJD and kuru became clearer after Gajdusek discovered that kuru is a transmissible disease (see below). Subsequently, CJD too was found to be transmissible. Later in his career, Klatzo carried out experiments which laid the groundwork for studies of the pathogenesis of Alzheimer’s disease.]

In 1959 Hadlow published a letter in the Lancet noting his observation of the similarities between the neuropathologies of kuru and scrapie (2). Hadlow sent a copy of the letter to Gajdusek, while also suggesting that somebody should inoculate primates with kuru brain tissue to test whether kuru, like scrapie, might be transmissible.

Gajdusek quickly wrote back to Hadlow, telling him that the experiment was underway. However, it was not until 1966 when Gajdusek, now working at the NIH, reported that he indeed succeeded in transmitting kuru to chimpanzees. Perhaps the lag between the start of Gajdusek’s experiment in 1959, and his report of success in 1966, was due in part to the length of the kuru incubation period, which, in chimpanzees, ranges from 14 to 82 months. At any rate, Gajdusek had established that kuru is a transmissible disease. Moreover, the exceptionally long incubation period between inoculation and the first appearance of symptoms, and the absence of fever and inflammation, suggested to Gajdusek that the kuru might be caused by a previously unrecognized class of infectious agents. But, what might account for the predilection of the disease to affect women? The answer would be startling.

In 1961 American anthropologists Ann and J. L. Fischer reported that the Fore people had a custom of eating the corpses of dead relatives (3). In view of the Fischers’ finding, and not yet knowing that kuru is transmissible, in 1963 Gajdusek hypothesized that kuru is a hypersensitivity disease, triggered by consuming human tissue. Gajdusek also proposed that the strange tendency of kuru to strike women might be explained if the tribe’s women consumed more human flesh than the men did. See Aside 4.

[Aside 4: The Fischers were quite prescient in their 1961 report, speculating that kuru is due to a transmissible agent, and that cannibalism might be the way by which it is transmitted between the Fore people. The Fischers also suggested, before Gajdusek did, that more human flesh might have been eaten by women than by men of the tribe, thus accounting for the demographics of kuru.]

The role of cannibalism in kuru was corroborated by a 1967 report by Robert Glasse, an anthropologist at Queens College of the City University of New York, who at the time was working as an anthropologist for the New Guinea Public Health Department. Glasse discovered a striking correlation between the Fore’s practice of “ritual cannibalism” and the incidence of kuru. The Fore people ate their dead relatives as an act of homage during funeral rites. The bodies of the deceased would be cut up into parts. The men took the meatiest parts for themselves, leaving the pancreas, liver, kidneys, and, importantly, the brain for the women and the children. Many of the women would eventually develop kuru after eating the infected brains. When they died, they too were ritually eaten, thereby escalating the epidemic. [See reference 4 for more on the extraordinary observations of Glasse and his colleagues.]

Based on Glasse’s findings, as well as on his own discovery that kuru could be transmitted to chimpanzees; Gajdusek concluded that kuru was caused by an infectious agent, spread by the Fore practice of consuming the brains of deceased relatives. See Aside 5.

[Aside 5: Who, if indeed any one individual, ought to have priority for the discovery of cannibalism as the means by which kuru was transmitted between the Fore people? Hopefully, I’ve credited all the key players. Historian Richard Rhodes’ 1997 letter to Nature contains a short, but likely accurate accounting of this part of our story (5). For more details, see Chapter 6 of Rhodes book, Deadly Feasts: Tracking the Secrets of a Terrifying New Plague (6).]

The incidence of kuru among the Fore declined dramatically in the late 1950s, after the Australian colonial administration put an end to their practice of ritual cannibalism. The very few adults who came down with kuru, as late as 20 years after the government suppressed cannibalism, reflect the fact that the incubation period for kuru in humans could range up to 20 years or longer. [Despite the scientific advances since the 1950s, most Fore people still believed that their kuru was caused by sorcery.]

Next, we consider the allegations of sexual misconduct against Gajdusek. In 1963 Gajdusek began to bring Fore boys back to the US to live with him. To Gajdusek’s credit, he saw to it that all of the boys went through high school, and he paid for some to attend college and even medical school.

In the 1990s a member of Gajdusek’s lab informed the FBI that Gajdusek might be engaging in inappropriate sexual behavior with the boys. The FBI questioned the boys and found one (a 23-year-old, who, at the time, was attending college under Gajdusek’s sponsorship) who claimed that he and Gajdusek had earlier been masturbating each other. None of the other boys would incriminate Gajdusek. On the contrary, several were willing to give evidence in his favor. However, an incriminating, secretly recorded phone conversation sealed the case against Gajdusek.

In 1996 Gajdusek plead guilty to a child molestation charge. Under a plea bargain, he was sentenced to 12 months in prison. In 1998 he was released and moved to Europe, never to return to the US.

Gajdusek’s supporters included leading scientists, as well as some of his adopted children. Pleading for leniency on his behalf, some supporters argued that Gajdusek’s sexual behaviors with the boys were acceptable and, in fact, widespread among the Fore people. Moreover, these supporters took our society to task for not appreciating that different cultures have different attitudes regarding sex. [For more on the sexual outlook and practices Gajdusek encountered among the Fore people, as well as for an assessment of his character from Sir Macfarlane Burnet, see references 7 and 8.] On the other hand, Gajdusek’s detractors maintained that he used his stature as a renowned scientist in order to exploit the boys.

Gajdusek never expressed any remorse over his transgressions. He passed away on December 12, 2008.

References:

(1) 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.

(2) Hadlow, W.J. (1959) Scrapie and kuru. Lancet 2:289–290.

(3) Fischer, A., and J. L. Fischer. (1961) Culture and epidemiology: A theoretical investigation of Kuru, J Health Hum Behav 2: 16-25.

(4) Lindenbaum, S. (2008) Understanding kuru: the contribution of anthropology and medicine. Philos Trans R Soc Lond B Biol Sci 363:3715–3720.

(5) Rhodes, R. (1997) Gourmet cannibalism in a New Guinea tribe. Nature 389: 11.

(6) Rhodes, R., Deadly Feasts: Tracking the Secrets of a Terrifying New Plague, Simon & Schuster, 1997.

(7) http://www.independent.co.uk/news/the-fall-of-a-family-man-1308277.html

(8) http://www.timeshighereducation.co.uk/…/91481.articl