The human immunodeficiency viruses type-1 (HIV-1) and type-2 (HIV-2) are able to infect both dividing and non-dividing (post-mitotic) cells productively. Interestingly, the capacity to replicate in non-dividing cells is a feature of the HIVs that is not shared by oncogenic retroviruses such as murine leukemia virus (MLV). This fundamental difference between the life cycles of these viruses is attributable to the ability of HIV post-entry nucleoprotein complexes (referred to as pre-integration complexes, PICs) to be specifically imported into the nucleus through nuclear pore complexes (NPCs) during interphase. By analogy with cellular nuclear import pathways, not only must specific signals reside within HIV-1 and HIV-2 PICs that target them to the nuclear interior, but cellular factors (proteins) must also be responsible for mediating the import process. in terms of nuclear localization sequences (NLSs) that are present in PICs, we hypothesize that the major signal for HIV-1 resides within integrase (IN) and that the accessory protein Vpr may serve to enhance the efficiency of import further by directly tethering PICs to the NPC. in contrast, the HIV-2 accessory protein Vpx appears to play a major role in PIC import, whereas the possible contribution of IN remains unexplored. The principal goals of this proposed research are, therefore, to expand on these ideas and to investigate the viral and cellular determinants of HIV- 1 and HIV-2 PIC nuclear import - these planned experiments are organized into three complementary areas. One, the NLS of HIV- 1 IN that we have mapped to residues 161 to 173 will be characterized, and the consequences of its disruption for virus infection and replication defined. Two, the NLSs of HIV- 1 Vpr and HIV-2 Vpx, which both appear to mediate import via a novel mechanism(s), will be characterized using site-directed mutagenesis and the construction of chimeric proteins; also, the effects of HIV-2 Vpx NLS disruption on infection will be analyzed. Three, a combination of assorted functional, biochemical and cDNA library screening strategies will be used to identify and study host cell proteins important for the nuclear import of HIV-1 and HIV-2 PICs. importantly, HIV infections of non-dividing cells such as macrophages are critical for the establishment of pathogenic infections in infected persons. Thus, an improved understanding of the viral and cellular factors that govern such infections should provide new insights into HIV pathogenesis and, potentially, suggest new approaches or targets for the treatment of HIV infections and AIDS.
