HIV-1 encodes a number of genes that are crucial for replication in primate cells. Gag, Pol, and Env products represent the main virion components, while Tat and Rev regulate intracellular transcriptional and post-transcriptional events for the controlled expression of viral genes. Of particular interest are the HIV accessory proteins Vif, Vpr, Vpu, Vpx, and Nef, which are unique to primate lentiviruses. There is increasing evidence that these proteins operate in conjunction with specific host factors. In fact, most if not all, of the accessory proteins appear to lack catalytic activities but instead seem to function as adaptors to link viral or cellular factors to pre-existing cellular pathways. In FY 2009, we continued studies to improve our understanding of the functional interactions between HIV-1 Vif and the host restriction factor APOBEC3G. One aspect we studied was the relative sensitivity of endogenous and exogenously expressed APOBEC3G to degradation by Vif. We found that newly synthesized APOBEC3G was more sensitive to degradation than pre-existing endogenous APOBEC3G. Nevertheless, endogenous and newly synthesized APOBEC3G were packaged with similar efficiency into vif-deficient HIV-1 virions and Vif inhibited encapsidation of both forms of APOBEC3G into HIV particles equally well. These results suggest that HIV-1 Vif preferentially induces degradation of newly synthesized APOBEC3G but indiscriminately inhibits encapsidation of old and new APOBEC3G. These results have important implications for the in vivo relevance of APOBEC3G degradation by Vif. APOBEC3G is a stable cellular protein, whose turnover rate and rate of de novo synthesis is slow. The vast majority of APOBEC3G present in virus-producing cells will therefore be old and will be insensitive to degradation. Thus, we predict that in vivo the degradation-independent inhibition of APOBEC3G by Vif may be more critical than protein degradation. During FY2009 we also rekindled our interest in Vpu and its regulation of virus release. We performed two studies one of which involved the characterization of endogenous BST-2/tetherin and its role in Vpu-dependent virus release and the second one was a biochemical characterization of BST-2. BST-2/CD317/tetherin is a host factor whose inhibitory activity on viral release is counteracted by Vpu. A current working model proposes that BST-2 inhibits virus release by tethering viral particles to the cell surface. We analyzed endogenous BST-2 with respect to its effect on virus release from HeLa cells, T cells, and macrophages. We noted significant cell type-dependent variation in BST-2 expression. Vpu caused a reduction in BST-2 expression in transfected HeLa cells and long-term infected macrophages. However, Vpu expression did not result in cell-surface down-modulation of BST-2 or a reduction in intracellular BST-2 expression during acute infection of CEMx174 or H9 cells. Yet, virus replication in these cells was Vpu-responsive. Surprisingly, BST-2 was undetectable in cell-free virions that were recovered from the surface of HeLa cells by physical shearing suggesting that a tethering model may not explain all the functional properties of BST-2. Thus, enhancement of virus release by Vpu does - at least in CEMx174 and H9 cells - not require cell-surface down-modulation or intracellular depletion of BST-2 nor does it entail exclusion of BST-2 from viral particles. In our second study we defined structural properties of BST-2 required for inhibition of virus release and for sensitivity to Vpu. We found that BST-2 is modified by N-linked glycosylation at two sites in the extracellular domain. However, N-linked glycosylation was not important for inhibition of HIV-1 virus release nor did it affect surface expression or sensitivity to Vpu. Rodent BST-2 was previously found to form cysteine-linked dimers. Analysis of single, double, or triple cysteine mutants revealed that any one of three cysteine residues present in the BST-2 extracellular domain was sufficient for BST-2 dimerization, for inhibition of virus release, and sensitivity to Vpu. In contrast, BST-2 lacking all three cysteines in its ectodomain was unable to inhibit release of wild type or Vpu-deficient HIV-1 virions. This defect was not caused by a gross defect in BST-2 trafficking as the mutant protein was expressed at the cell surface of transfected 293T cells and was down-modulated by Vpu similar to wild type BST-2. Thus, formation of cysteine-linked BST-2 dimers is important for inhibition of virus release. However lack of dimerization does not prevent surface expression or Vpu sensitivity of BST-2 suggesting Vpu sensitivity and inhibition of virus release are separable properties of BST-2.
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