The purpose of this project is to investigate the biological and biochemical functions of HIV accessory proteins, in particular Vif and Vpu, and to understand their precise role in virus replication and in virus-host interaction. One of our goals is to characterize cellular factors involved in Vif and Vpu function. From our studies on Vpu we expect to gain insights into general principles of protein-protein interactions and into mechanisms involving late stages of virus production such as the involvement of lipid rafts in the secretory pathway. Our studies on Vif will provide insights into the function of this viral factor and teach us more about the role of the cytoskeleton in virus replication, about the interactions of cellular and viral factors during viral assembly or maturation, and about the role of host factors in restricting viral replication. Results from our research will enable us to assess viral accessory proteins with respect to their potential as novel antiviral targets. The vpu gene is unique to HIV-1 and encodes a small integral membrane protein. Vpu regulates virus release from the cell surface and degradation of CD4 in the endoplasmic reticulum. These two biological activities of Vpu are based on two independent and distinct molecular mechanisms that can be attributed to separable structural domains of Vpu. Vpu-regulated virus release is sensitive to changes in the transmembrane (TM) domain of Vpu and is correlated with an ion channel activity of Vpu. However, the precise mechanism of Vpu-mediated virus release remains unclear. In the course of our studies we found that the requirement of Vpu for efficient virus release is host cell dependent. In permissive cell types, virus release was equally efficient in the presence or absence of Vpu while in non-permissive cell types virus release was significantly impaired in the absence of Vpu. This suggested the involvement of host factors in Vpu function. Biochemical analyses demonstrated that Vpu is associated with cholesterol-rich lipid rafts in the plasma membrane. We found that addition of cholesterol increased the release of Vpu-defective virus from non-permissive cells but had no effect on virus release from permissive cells. Our results suggest that Vpu modulates structures at the cell surface to facilitate shedding of virus particles. It remains to be shown whether Vpu overcomes the inhibitory effects of a cellular factor in non-permissive cells or whether permissive cells are Vpu-independent because of the presence of a cellular Vpu-like factor. Experiments are ongoing to address this issue. Vif is a 23-kDa basic protein, which has an important function in regulating infectivity of progeny virions. Despite the severe impact of Vif defects on virus infectivity, its mechanism of action has remained largely obscure. It is generally accepted, however, that Vif-deficient viruses can attach to and penetrate host cells but are blocked at a post-penetration step early in the infection cycle. Yet, comparison of virion morphology or protein composition between wild type and Vif-defective virions remained inconclusive. About 40 to 100 molecules of Vif are packaged into virions. The majority of Vif, however, remains cell associated. Packaging of Vif protein into virus particles is mediated through an interaction with viral genomic RNA and results in the association of Vif with the nucleoprotein complex. Despite the specificity of this process, calculations of the amount of Vif packaged have produced vastly different results. We compared the packaging efficiency of Vif into virions derived from acutely and chronically infected H9 cells and found that Vif was efficiently packaged into virions from acutely infected cells (60-100 copies per virion) while packaging into virions from chronically infected H9 cells was near the limit of detection (4-6 copies of Vif per virion). Superinfection by an exogenous Vif-defective virus did not rescue packaging of endogenous Vif expressed in the chronically infected culture. In contrast, exogenous Vif expressed by superinfection of wild type virus was readily packaged (30-40 copies per virion). Biochemical analyses suggest that the differences in the relative packaging efficiencies were due to the accumulation of endogenously expressed Vif in a packaging-incompetent insoluble form. The packaging efficiency was correlated with the level of soluble Vif, which was elevated in cells producing exogenous Vif. The accumulation of endogenously expressed Vif in a detergent-insoluble form is at least in part due to the rapid turnover of soluble Vif and the higher stability of insoluble Vif. Despite its low packaging efficiency, endogenously expressed Vif was sufficient to direct the production of viruses with almost wild type infectivity. The results from this study provide novel insights into the biochemical properties of Vif and offer an explanation for the reported differences regarding Vif packaging . As packaging of Vif requires viral genomic RNA, it is likely that Vif recognizes specific sequences in the viral genome similar to those recognized by Tat and Rev proteins. We have continued our analysis of the sequences on the viral genomic RNA required for efficient packaging of Vif. To this end, we have constructed a series of deletion mutants and analyzed each construct for its ability to support packaging of Vif into a helper virus. In the process, we have narrowed down the sequence motif required for Vif packaging to an approximately 600-nucleotide region located near the 5'-end of the viral genome and encompassing a series of known RNA structures, including the TAR element, the primer binding site, and RNA dimerization signals. Final experiments are being performed to control various experimental parameters. We hope to complete this project within the next year. Replication of HIV-1 in most primary cells and some immortalized T cell lines is critically dependent on the activity of Vif. Vif has the ability to counteract a cellular inhibitor, recently identified as CEM15, that blocks replication of Vif-defective HIV-1 variants. CEM15 is identical to APOBEC3G and belongs to a family of proteins involved in RNA and DNA deamination. Recent evidence suggests that APOBEC3G targets nascent viral minus strand cDNA, introducing cytidine to uridine changes throughout the genome. These changes are believed to either destabilize the viral genome by subjecting it to the activity of uracil DNA glycosylase or to induce hypermutation of the viral genome, resulting in the impairment of viral fitness. We cloned APOBEC3G from a human kidney cDNA library and confirmed that the protein acts as a potent inhibitor of HIV replication and is sensitive to the activity of Vif. We found that wild type Vif blocks packaging of APOBEC3G into virus particles in a dose-dependent manner. In contrast, biologically inactive variants carrying in-frame deletions in various regions of Vif or mutation of two highly conserved cysteine residues did not inhibit packaging of APOBEC3G. Interestingly, expression of APOBEC3G in the presence of wild type Vif not only affected viral packaging but also reduced its intracellular expression level. This effect was not seen in the presence of biologically inactive Vif variants. Pulse/chase analyses did not reveal a significant difference in the stability of APOBEC3G in the presence or absence of Vif. However, in the presence of Vif, the rate of synthesis of APOBEC3G was slightly reduced. The reduction of intracellular APOBEC3G in the presence of Vif does not fully account for the Vif-induced reduction of virus-associated APOBEC3G suggesting that Vif may function at several levels to prevent packaging of APOBEC3G into virus particles.