OVERVIEW

Oncogenic retrovirusea are great for molecular biologists - they have packaged for our study the genes that cause cancer. The discovery that cellular genes moved into retroviruses are responsible for transformation was a revolution in the thinking about the disease and won the Nobel Prize for Varmus and Bishop.

THE BASICS OF RETROVIRAL STRUCTURE

All oncogenic RNA viruses are retroviruses and all but one are defective. Let's review defective viruses - they have lost the ability to perform some function required for replication. In order for them to replicate they must be co-infected with a helper virus that supplies this function in trans.

The critical information for figuring out the mechanism by which these viruses are oncogenic requires our knowing something about their genome organization. Within the virion there are two copies of the (+) RNA genome, held together by the cell-coded tRNAs that serve as primers for reverse transcription (Fig. 27.1).

The retroviral genome is linear in the virion, and after conversion to dsDNA, can integrate into the host cell's chromosomal DNA. As you all know from the last test, the genome circularizes during replication. Here is a diagram of how this happens (Fig.27 2). (You're not responsible for these details - this figure is inserted to show how clever retroviruses are!)

Fig 27.3 shows the termini of the integrated form, called the provirus, are different from the termini of the provirus. The form in the provirus, direct repeats of sequences call U3, R and U5, is call the long terminal repeat or LTR. There is a promoter in R, that is one of strongest promoters known.

The genes of retroviruses fall into four groups, called gag, pol and env, plus the regulatory genes. Fig 27.3 conains a diagram of their locations on the (+) polarity, polyadenylated RNA. Gag stands for group antigen - the proteins on the inside of the virus. The availability of antibodies against these proteins allow them to be classified into groups, hence the name. Pol, not surprisingly, stands for the polymerase or reverse transcriptase and env stands for the peoplomers or envelope proteins.

Gag-pol is translated as a polyprotein, cleaved by protease activity. Pol is not cleaved from gag until the encapsidation step, to ensure that reverse transcriptase actually becomes a structural protein and is available for the next round of replication. Env in produced from the promoter in the LTR; it's mRNA is spliced and partially from a different reading from gag-pol to avoid the gag-pol stop codon.

THE NON-DEFECTIVE ONCOGENIC RETROVIRUS - ROUS SARCOMA VIRUS

Rous sarcoma virus was first isolated in about 1910 by Peyton Rous who worked at the Rockefeller Institute. He did the classic experiment --he was able to transfer production of a solid tumor, a sarcoma, from one chicken to another, using a filtrate.

By the 1970s, scientists were investigating variants of Rous sarcoma virus that we NOT oncogenic. What do you think caused them to lose their ability to cause cancer?

To quote from a paper by M. Lai et al., 1970. PNAS 70:2266-2270.

We have found that the RNAs of sarcoma viruses contain most, if not all, sequences present in transformation-deficient viruses. In addition, sarcoma virus RNA contains some sequences that appear to be characteristic of transforming ability for fibroblasts.

These results were obtained by a primitive method of sequenceing RNA called "fingerprinting" - using enzymes to break the ³²P-labeled molecules into small pieces and electrophoresing them on paper using extremely high voltage in huge tanks of pyridine - and believe me, that smelled wonderful and was really healthy to be around!

What do you think the nature of these sequences was? The protein coded by these sequences was called src (pronounced sark). How do you think it was initially characterized?

What's odd about src is this it is phosphorylated on tyrosine, and that it can autophosphorylate. This means that if the protein is mixed with gamma-labeled ATP, the phosphate is transferred to a particular tyrosine residue on the protein. The exciting thing about this observation is that this phosphate can also be transferred to other proteins. What do you think this transfer can do to the recipient protein?

WE HAVE MET THE ENEMY AND IT IS US (POGO)

The brilliant hypothesis of Harold Varmus (now Director of the National Institutes of Health) and J. Michael Bishop concerned the origin of the serc gene. They believed and confirmed that src was also coded by the cellular genome. How do you think this was done - in 1976?

More important than the technical details was the revolution this caused in thinking about cancers. If a cellular gene can cause cauncer, then abberations of normal cellular metabolism can cause cancer - hence the quote that introduces this section - which Bishop himself used.

HOW MANY ENEMIES?

Taking advantage of this way of thinking about cancer, virologists began to look at the defective retroviruses. What do you think they found? Yup - they found variants of normal cellular genes.

Transforming genes in retroviruses are generically called viral oncogenes or v-oncs or onc^vs.

Their cellular homologs are called cellular oncogenes or proto-oncogenes or c-oncs or onc^cs.

Where in the retroviral genome do you think these v-oncs are located? They displace some of the normal genetic information of the retroviruses, most often in the gag region. A "new" gene is generated by fusion of the coding sequences for the viral protein and the coding sequences for the proto-oncogene.

THIS IS IMPORTANT! The viral oncogene is created from a normal cellular gene - not the other way around!

Table 27.1 shows many of these oncogenes. Their proteins are designated as p (for protein) or gp (for glycoprotein) or pp (for phosphoprotein) followed by a number that gives their molecular weight in daltons x 10 ^-3 and a superscript that identifies which normal viral gene is linked to which proto-oncogene. Among the cellular genes that end up as v-oncs are genes for regulatory proteins such as G protein (ras), transcription factors (myc, jun), and anti-apoptosis factors (bcl-2).

The interesting thing about the v-oncs is that they have lost the introns that are present in the c-oncs. Fig. 27.4 shows a model for how the v-oncs could be generated and how the introns could be eliminated.

ANOTHER EFFECT OF RETROVIRUS INTEGRATION

One of the reasons that v-oncs are so deadly is that they are transcribed from the LTR, which is one of the strongest promoters known. Even retroviruses without transforming genes (called weakly transforming retroviruses as opposed to acutely transforming retroviruses - those with v-oncs) can potentially cause transformation. How do you think this occurs? Remember that integration of retroviruses is random.

SUMMARY

All oncogenic RNA viruses are retroviruses and all but one are defective. The non-defective retrovirus Rous sarcoma virus was shouwn to contain an extra gene whose protein product, pp60src, is capable of being pohsphorylated on a tyrosine reside and of transferring this phosphate group to another protein. Eventually it was shown that the gene for src was derived from a cellular gene. This led to the hypothesis that cancer is not some bizarre disease, but an aberration or dis-regulation of normal cellular metabolism.

Similar derivatives of cellular genes were sought in other retroviruses. These were indeed found, but their inclusion in the virus rendered it defective, requiring co-infection with a helper for replication. In these viruses, the cellular genes are found as fusion proteins with (usually) gag and their coding sequences lack introns. These cellular genes include those for regulatory proteins and transcription factors.

Remember that retroviruses have one of the strongest promoters know in their LTRs, that are generated during reverse transcription. This means that the ongogenic proteins in retroviruses are produced at an extremely high level. The integration of even a retrovirus without a v-onc can cause transformation if the LTR activates a c-onc.