Twenty-four visits to Stockholm: a concise history of the Rockefeller Nobel Prizes.

Part XVI: David Baltimore, 1975 Prize in Physiology or Medicine.

By Joseph Luna

On June 19th 1946, a captive rhesus monkey in the Mengo district near the town of Entebbe, Uganda developed unexplained hind-limb paralysis. British and American scientists, part of the local Yellow Fever Research Institute, financed in part by The Rockefeller Foundation, soon isolated what they believed to be a virus as the cause. The named it Mengo Encephalitis Virus, later shortened to just Mengovirus. The virus was quickly isolated in mosquitoes, and found in at least one person, but generally it posed no major risks to human health. Mengovirus was but an additional member of a constellation of RNA viruses known as picornaviruses, of which poliovirus was far and away the star. After a few reports demonstrating that Mengovirus could induce characteristic paralysis in mice as an animal model, interest died down.

A decade later, as mammalian cell culture techniques matured, many viruses were tested for their ability to replicate in a plate of cells instead of a whole animal. And one early and surprising finding was that just the RNA genetic information of Mengovirus was capable of launching an infection if artificially introduced into a cell. Furthermore, whereas normal cellular RNA production occurred almost exclusively in the nucleus, Mengovirus set up shop and made RNA only in the cytoplasm. And the biggest surprise: if cells were treated with the drug Actinomycin D, which prevented normal cellular RNA production from a DNA template, Mengovirus didn’t care, and went on producing copies of its own RNA as if nothing had happened.

For a young MIT graduate student named David Baltimore taking a course at Cold Spring Harbor Laboratory, this became an enthralling problem. So enthralling in fact that Baltimore left MIT to join the lab of the lecturer that day, Richard Franklin, at The Rockefeller University. There, Baltimore’s graduate school project was to develop an in vitro system to characterize the nature of Mengovirus RNA synthesis from an RNA template. He did so by taking Mengovirus-infected cells, grinding them up, and discarding the nuclei (where cellular RNA synthesis occurs from DNA). To the remaining cytoplasmic fraction, where there was no DNA and where Mengovirus could replicate, he added radioactive RNA nucleotides (A, C, G, and U) one-by-one, in combination, or leaving one out. The idea was that if there was an RNA-dependent RNA polymerase (a “replicase”), it should be able to link radioactive nucleotides together to make an RNA copy that would fall out of solution when placed in acid. By taking a Geiger counter and measuring if the radioactivity went into this “acid insoluble” fraction, Baltimore could conclude that a polymerase had acted on existing Mengovirus RNA to make an RNA copy composed of whatever radioactive nucleotides he added.

With this basic assay, Baltimore could test different conditions to characterize how the Mengovirus RNA polymerase behaved. As expected, Actinomycin D had no effect on nucleotide incorporation, but the polymerase was dependent on magnesium, and could be inhibited by manganese, both features in common with cellular RNA polymerases. By the time Baltimore defended his PhD (completed in a mere 18 months), he had shown that an RNA-dependent RNA polymerase (an “RdRp”) of viral origin existed in infected cells. After his pioneering study of Mengovirus, similar results with poliovirus soon followed. David Baltimore left the RU graduate program as a capable enzymologist and virologist.

Because of their lack of the much more famous molecule DNA, RNA viruses always seemed like exceptions to the hypothesis that information flowed from DNA to RNA to protein. And among themselves, RNA viruses were not always so consistent: whereas the RNA of Mengo and polioviruses were infectious all by themselves, other RNA viruses such as vesicular stomatitis virus (VSV), were not. VSV infection started with viral RNA that was antisense to what was needed to make viral proteins. Now at MIT, Baltimore and his lab tackled this strange paradox: how did VSV and related viruses, which entered cells with RNA instructions in reverse, manage to launch an infection? There were two main hypotheses. Either the viral RNA used some unknown cellular RdRp to make a correct copy, or the intact virus entered the cell with its own RdRp to do the job. Focusing on the latter hypothesis, Baltimore with Alice Huang and Martha Stampfer, discovered an RdRp within VSV in 1969.

This result, alongside many others, hinted at the diversity of strategies that RNA viruses employed to replicate. It was from this mindset that Baltimore next chose to go after other RNA viruses to see if they too carried with them a necessary polymerase to get an infection going. And settling on an RNA virus called Rauscher murine leukemia virus (R-MLV), he noticed something quite odd: it never incorporated radioactive RNA nucleotides as he had seen with Mengo, polio or even VSV, but could incorporate radioactive DNA nucleotides. Thus, in early May of 1970, using only a slight variant of the assay he developed while in graduate school, Baltimore tracked down a new polymerase that made DNA out of an RNA template: a reverse transcriptase.

Whether a household name or a footnote, whether a society’s plague or a single virologist’s model toy, isolated in far-off places like the Ebola river, the Zika forest, LaCrosse, Wisconsin, or the New York City subway system, viruses old and new have incredible things to teach us. David Baltimore’s journey from a long-forgotten picornavirus to one of the greatest discoveries in modern biology (certainly one of the coolest enzymes) is a perfect illustration of this. Viruses unknown to science, yet to be named after their symptoms or towns or rivers of origin, will find a bit of David Baltimore in the graduate student or postdoc who decides to study them.