MERIT Award Recipient: Stephen P. Goff, Ph.D.
Construction and Analysis of Retrovirus Mutants
Retroviruses are a large family of RNA viruses found in all eukaryotes, and include such important pathogens as the human immunodeficiency viruses (HIVs). These viruses have an extraordinary life cycle: the viral RNA genome is converted into a double-stranded DNA, which is inserted into the host genome and then transcribed to make new viral RNAs. As a direct result of the integration of the viral DNA into the host genome, retroviruses can cause an impressive variety of effects in an infected host. The viruses persist in the genome for the lifetime of an infected cell; they can even be transmitted to offspring in the germ line as integrated DNA. Many cause leukemias and other malignancies by inappropriately activating genes near the site of viral DNA insertion. They can also acquire host genes and carry them into newly infected animals, causing a range of distinctive and aggressive tumors. These properties make retroviruses formidable pathogens.
The Goff laboratory is interested in the replication of a simple retrovirus, the Moloney murine leukemia virus, and the interaction of the virus with the host. The ultimate goal of the research is to determine the roles of each of the viral proteins in the complex viral life cycle, and to identify the impact of these proteins on host cellular machinery. Retroviral genomes are compact, and carry only three major structural genes: gag, encoding virion structural proteins; pol, encoding the reverse transcriptase (RT) and integrase enzymes; and env, encoding the virion envelope protein. The major approach being taken in the lab to determine the functions of these proteins is to generate mutations in cloned proviral DNAs, to recover virus after transformation of mammalian cells with the altered DNA, and to analyze the resulting virus for its ability to replicate in culture. Major efforts are focused on the functions of the Gag polyprotein in retrovirus assembly and disassembly; and of the Pol products, reverse transcriptase and integrase, in formation of the proviral DNA. The Goff laboratory has identified portions of Gag required for early events of infection, including synthesis of viral DNA and trafficking into the nucleus; and also for late events, including virion assembly, release, and RNA packaging. The group has similarly mapped out many critical features of the reverse transcriptase enzyme.
A very active area of research is centered on the use of the yeast two-hybrid system to detect and characterize protein-protein interactions between viral and host gene products. The laboratory has shown that a portion of Gag (termed CA, for capsid) interacts with Ubc9 and PIAS proteins, which modify their targets by addition of a small polypeptide called SUMO; this binding is required during the early steps of infection. Gag also interacts with several host proteins that are involved in virus assembly, including the endophilins, which induce membrane curvature, and IQGAPs, which regulate the cytoskeleton. In another example, the laboratory has found that reverse transcriptase regulates its own synthesis by an interaction with the eukaryotic release factor 1 (eRF1), the protein that recognizes stop codons during translation. These many unanticipated interactions all define new viral functions and, in many cases, reveal unknown host functions. This work is therefore expanding the understanding of the viruses and their gene products, and perhaps more importantly, is uncovering new aspects of cell biology that are exploited by the retroviruses to drive their replication. These viral and cellular functions are likely to provide new targets for antiviral intervention and thus new treatments for retroviral diseases.