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Four Nobel Prizes for a chicken virus

The Rous sarcoma virus (RSV) was the starting point of the discovery that cancer can be brought on by infections. The enzyme “reverse transcriptase”, which led to a rethinking and technological revolution in the field of molecular biology, was discovered in this retrovirus along with oncogenes that led to a completely new concept of cancer development in molecular genetics and eventually to the development of a new generation of cancer therapeutics.

Nowadays, the transcription of RNA sequences into DNA using reverse transcriptase is standard practice in many basic molecular biology techniques such as cloning, PCR (polymerase chain reaction) and in proteome research. However, only a few scientists who work with the enzyme are probably aware of the controversy associated with the process that led to the use of the enzyme in molecular biology.

Attack on the “central dogma”

Howard Temin © Nobel Prize Committee

In 1958, Francis Crick formulated the central dogma of molecular biology according to which genetic information flows from DNA to RNA and from there to proteins - but cannot be transferred back. The term "central dogma" somehow implies a canonical claim, and is therefore taken by the majority of researchers as the irrevocable truth. In contrast to the common opinion that science is revolutionary, it is actually very conservative, and progress is made slowly and cautiously, with the point of departure being widely recognised beliefs. Revolutionary ideas usually meet with major resistance.

At the beginning of the 1960s, Howard Temin, a young virologist at the University of Wisconsin in Madison, came up with the "provirus hypothesis", a discovery that was more intuitive than based on solid experiments. According to the provirus hypothesis, a DNA provirus is synthesised after an RNA virus has entered a host cell. In more detail, this means that single-stranded RNA is transcribed into double-stranded DNA by RNA tumour viruses when they have infected the host cell. This double-stranded DNA, now termed "provirus", then inserts itself into a host cell chromosome and is propagated along with the host DNA during cell division. The provirus DNA, which is integrated in the chromosome, has the same effect as normal cellular DNA: it is used for the synthesis of new virus particles through the transcription of new viral RNA and for the translation of viral protein with the help of host enzymes. Temin worked with the Rous sarcoma virus (RSV), which, 50 years previously, had been identified as an infective agent in experiments in which Peyton Rous induced cancer in chicken by infecting them with a cell-free extract. It was not until 1961 that it was definitively shown that RSV was a single-stranded RNA virus. In 1966, Rous was awarded the Nobel Prize at the age of 87 for his discovery that sarcomas in chicken are caused by a filterable agent - 55 years after he had published his groundbreaking findings.

Reverse transcriptase and retroviruses

Temin’s provirus hypothesis was heavily attacked, because it was in conflict with the central dogma of molecular genetics, and because no enzyme that was able to copy RNA into DNA was known as well as being impossible to find the provirus DNA Temin had postulated. The breakthrough came in 1970 when Temin and his postdoctoral student Satoshi Mizutani announced that they had found an enzyme which was able to transcribe single-stranded viral RNA into DNA. Temin and Mizutani found the enzyme for RSV and David Baltimore from the Massachusetts Institute of Technology found the enzyme for another virus of the same type, the mouse leukaemia virus (MLV). The enzyme, which became known as reverse transcriptase, was not localised in the cells but in the viruses: the RNA tumour viruses were called retroviruses. The discovery of reverse transcriptase was a decisive event in the development of biotechnology. Only five years after its characterisation, Temin and Baltimore were awarded the Nobel Prize in Physiology or Medicine in 1975. Francis Crick made it clear that the reverse transcriptase was not in conflict with the central dogma, which implied that information cannot be transferred back from protein to nucleic acids. With the new enzyme, it was possible to transcribe messenger RNA directly into DNA, and this newly synthesised DNA could be used for a broad range of applications: the amplification of DNA in bacteria or other cells, the production of radioactive DNA as gene-specific probes, in particular in molecular hybridisations, PCR, the cloning of cDNA of medically important proteins such as insulin and growth hormones by Axel Ulrich and Peter Seeburg, which was the basis for the first and most successful biotechnology company, Genentech.

Harold Varmus © Nobel Prize Committee

The identification of RSV provirus DNA in the cellular chromosome, a major focus of Harold Varmus and Michael Bishop at the University of California in San Francisco, turned out to be quite difficult, and had unforeseeable and far-reaching consequences. At first, the researchers encountered a quantitative problem. A single viral RNA molecule, the RNA genome, has about 8,000 nucleotides and the corresponding DNA is about the same length. The genome of RSV-infected chicken cells has approximately 2 billion nucleotides, i.e. is about 250,000 times larger. In order to identify the comparatively low quantity of viral DNA, the researchers had to produce (using reverse transcriptase) highly radioactive (single-stranded) DNA probes for the hybridisation of the two strands. These DNA probes had to be highly specific, which means that they had to enable the faultless pairing of the bases during the synthesis of the DNA double-strand molecules measured.

 

Oncogenes

Varmus and Bishop were surprised to find that normal chicken cells also contained DNA that was similar to the RNA of the infecting virus. Using improved techniques to determine RNA tumour viruses and their proteins, for example with specific antibodies, different groups of researchers found that cells of normal chicken embryos were able to produce viral proteins of this kind and were even able to produce infectious particles.
These findings were reproducible and more or less the only possible conclusion was that animals have “endogenous” RNA tumour virus genes that are embedded in the normal chromosomes and are directly passed on to the progeny. From these observations, the next step on the path led to the final evidence of proviruses in the host chromosome such as had been postulated by Temin, and also to the discovery of viral and cellular oncogenes, i.e. genes that lead to the transformation of normal cells into cancer cells. Although it was a long and difficult path – using the somewhat approximative methods molecular biologists had available in the 1960s and 1970s – it eventually ended in the award of the Nobel Prize to Varmus and Bishop in 1989 for “their discovery of the cellular origin of retroviral oncogenes”. The relatively “innocent” title does not make it at all clear that the Nobel Prize was awarded for the proof of the genetic basis of cancer.

The decisive findings were once again made with the Rous sarcoma virus: using a temperature-sensitive RSV mutant, researchers succeeded in separating the replication properties and the release of virus particles from chicken fibroblasts from the transformation ability (the conversion of fibroblasts into sarcoma cells). The DNA copy of the gene, generated by the enzyme reverse transcriptase, was incorporated into the host cell where, in its transformation into provirus as predicted by Temin, it gave instructions for the production of new viruses. But the host chromosome also had a gene that originated from the viral RNA, which, in its active state, encoded a protein that led to the transformation of fibroblast cells to sarcoma cells. No new virus particles were released in this case. This was the first oncogene (src - from sarcoma) to be identified. It soon turned out that the src gene sequence was also found on the chromosomes of cells that were not infected by RSV.

In order not to make the issue more confusing than it needed to be, clear terms were created: the virus oncogene was termed v-src and the corresponding host gene c-src. The latter is a proto-oncogene. The conversion of proto-oncogenes in cancer genes is the result of mutations (as is also the case when they are incorporated into the retroviral genome), which leads to the strong activation of protein products and disturbs the normal pattern of cell growth, cell differentiation and cell death, eventually causing cancer.

Numerous endogenous oncogenes of retroviral origin have since been discovered. Some occur in hundreds and even thousands of copies in vertebrates such as humans and mice. In 1981, Robert Weinberg from the MIT described the first human oncogene, which is a member of the larger ras-gene superfamily, and he was able to show that it is also present in healthy cells in the resting state. Activating ras mutations are found in almost 25% of all human tumours.

These discoveries led to a new molecular genetic orientation in cancer research. Hormones, growth factors or their receptors and other components at the switchboards of cellular signal transduction have turned out to be the products of viral oncogenes and proto-oncogenes. These include a number of protein kinases, in particular tyrosine kinases, which are among the most important targets for the development of a new generation of anti-cancer drugs such as Glivec, Tarceva, Iressa and Sutent. It is now known that the virus-induced chicken sarcoma described by Peyton Rous in 1911 develops as a result of the effect of a v-src or c-src tyrosine kinase.

Further information:

Harold Varmus: The Art and Politics of Science. W.W. Norton & Company, New York and London

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