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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