Human immunodeficiency virus contains six open reading frames that have not been observed previously in retrovirus genomes. These """"""""accessory genes"""""""" encode proteins that are thought to regulate viral replication and to contribute to the progression of AIDS in an infected individual. Studying these genes is important, since this will lead to an understanding of the mechanisms by which viral replication is regulated and will lead to new targets for therapeutic intervention. Among the least well understood of the accessory genes are vpr and the closely related gene of HIV-2/SIV, vpx. These genes encode proteins whose sequence is conserved throughout HIV/SIV strains, but whose role in viral replication is unknown. Unlike the proteins encoded by the other accessory genes, Vpr and Vpx are virion structural proteins; they are present in virions in molar amounts equivalent to Gag. For this project, we developed an in vitro system in which an epitope tagged Vpr molecule produced by cells transfected with appropriate recombinant expression vectors is efficiently incorporated into HIV-1 virions. This system enables us to study the mechanisms by which Vpr is incorporated into virions; it also allows us to determine the cellular and virion localization of Vpr. Furthermore, it provides a convenient method for preparing purified Vpr in quantities sufficient for structural and biochemical studies. Our preliminary studies have shown that the viral determinant required for incorporation of Vpr into virions is the G- terminal Gag protein, p6. We have begun to define the site on Vpr that interacts with p6 during viral assembly. We have also shown that in cells Vpr (as well as Vpx) is concentrated in the nucleus. The broad goals of this project are: To understand the molecular mechanisms by which Vpr is incorporated into virions. To target a protein to HIV particles, particularly one that will inhibit viral replication. To provide purified Vpr protein for use in structural and biochemical studies. To understand the mechanism by which Vpr is concentrated in the nucleus of cells. To localize Vpr in virions and determine with which elements of the virion Vpr interacts.
