Novel interactions between human cell proteins and human immunodeficiency virus type 1

Human immunodeficiency virus type 1 (HIV-1) replication requires a complex series of physical and functional interactions between host cell and viral components. Additional molecular insight into these interactions will yield not only an increased understanding of fundamental retroviral biology, but may also identify new opportunities for therapeutic intervention in the treatment of AIDS.; One distinctive feature of HIV-1 is its ability to infect non-dividing cells such as macrophages. Recently it was reported that the viral integrase (IN) protein directed HIV-1 preintegration complex (PIC) nuclear import. To gain insight into PIC nuclear import, the intracellular transport of IN was examined. IN was found to be a karyophilic protein in human cells, but not for the reasons previously assumed. Instead, a model is presented in which IN nuclear accumulation results from its cytoplasmic degradation and specific nuclear retention due to strong chromatin interactions.; To extend these studies, IN-interacting proteins were identified by affinity purification and mass spectrometry. A chromatin-associated protein, p75/LEDGF, was shown to bind wild-type but not a mutant V165A IN protein. To probe the role of p75/LEDGF in HIV-1 replication, RNAi was employed to transiently downregulate p75/LEDGF. A novel retroviral vector was also developed to deliver small hairpin RNA (shRNA) gene cassettes, which enabled generation of stable p75/LEDGF "knock-down" cells. HIV-1 efficiently infected both categories of p75/LEDGF knock-down cells. These results suggest either that p75/LEDGF is not an important determinant of HIV-1 replication in this context, or that the level of p75/LEDGF silencing achieved was insufficient to reveal a replication block.; In a separate project, a potential connection between human serine-threonine kinases and HIV-1 was investigated. HIV-1 particles were shown to incorporate the related NDR1 and NDR2 kinases. The viral-encoded protease was shown to process NDR1 and NDR2, an event that occurred both within virus particles and the HIV-1 producer cells. Protease processing altered NDR2 subcellular localization and altered the enzymatic activity of both NDR1 and NDR2. The potential roles of the NDR1 and NDR2 kinases in HIV-1 biology are discussed, as are experiments identifying Mob2 as a novel cellular regulator of the NDR kinases.