The long-term goals of this project are: 1) to evaluate the feasibility of generating live attenuated virus vaccines of HIV-1 and HIV-2 that are rendered non-pathogenic by mutation of accessory genes, either individually or in combination; and 2) to explore the possibility that the accessory gene proteins can be targets for anti-viral therapy. Recent studies using the SIV system have shown that viruses defective in the production of Nef are non-pathogenic in monkeys and confer protection to those monkeys from subsequent challenge with wild-type SIV. Mutations in the other accessory genes, vif, vpr and vpu of HIV-1 and vif, vpr and vpx of HIV-2, have yet to be explored in animal model systems. As prerequisites to both the development of candidate live attenuated virus vaccines and the development of anti-HIV drugs directed against the accessory gene products, we have been engaged on studies to determine the role of these proteins in the life cycle of HIV-1 and HIV-2 in vitro, since a knowledge of how they function is critical to both goals. Our earlier work had demonstrated the critical role of HIV-1 Vif to virus replication in primary T cells (peripheral blood mononuclear cells, PBMC), although this protein is not obligatory for HIV-1 replication in most CD4-positive cell lines. We have gone on to show that Vif also plays a critical role for HIV-1 replication in primary monocyte-derived macrophages (MDM). In the case of Nef, there has been a controversy as to whether this protein positively or negatively influences virus growth. We have shown that whether or not Nef has a measurable effect on virus replication depends on the particular virus-host system used. While Nef mutants of several HIV-1 strains all replicate slightly less well than wild type in PBMC and, in the case of a macrophage-tropic HIV-1, in MDM, there can be either no effect or dramatic reductions in virus replication when Nef mutants of HIV-1 and HIV-2 are assayed in CD4-positive cell lines. For Vpr, we have shown that there is little consequence of mutating this gene in HIV-1 and HIV-2 for the replication in CD4-positive cell lines or PBMC, although this protein appears to be a positive factor for growth in MDM. We have commenced studies to investigate the molecular mechanism of Nef, Vif and Vpr. Results so far have indicated that expression of these proteins in several mammalian cells results in the cessation of cell growth (cytostasis), and we are currently investigating the cellular pathways that bring this about. As part of our goal to develop attenuated HIV vaccine candidates, we have modified an HIV-1 genome to allow the insertion of different genes into the nef open reading frame. Using this virus, we plan to investigate the consequences of ectopic expression of the vpr and vif genes on virus replication and whether different HIV-1 and HIV-2 alleles of these genes and nef can complement the appropriate Vif, Vpr or Nef mutants of HIV-1.
