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 lack catalytic activities but instead seem to function as adaptors to link viral or cellular factors to pre-existing cellular pathways. In FY12, we continued studies to improve our understanding of the functional interactions between HIV-1 Vif and the host restriction factor APOBEC3G (A3G). In collaboration with the Maynard lab at USUHS we studied mechanistic details of Vif oligomerization. Analytical ultracentrifugation to examine the oligomeric properties of Vif in solution revealed that, contrary to previous reports, Vif oligomerization does not require the conserved PPLP motif. Instead, our data suggest a more complex mechanism involving interactions among the HCCH motif, the BC box, and downstream residues in Vif. In particular, mutation of residues near the PPLP motif (S165 and V166) affected the oligomeric properties of Vif and weakened the ability of Vif to bind and induce the degradation of APOBEC3G. We propose that Vif oligomerization may represent a mechanism for regulating interactions with APOBEC3G Techtmann (2012). During FY12, we also continued our analysis of Vpu and its functional interaction with the virus-release antagonist BST-2. BST-2 (also known in the literature as CD317, HM1.24, or tetherin) is a B-cell antigen overexpressed on the surface of myeloma cell lines and on neoplastic plasma cells of patients with multiple myeloma. Functionally, BST-2 restricts the secretion of retroviruses, including HIV-1, as well as members of the herpesvirus, filovirus, and arenavirus families, presumably by tethering nascent virions to the cell surface. Antibodies to BST-2 are in clinical trial for the treatment of multiple myeloma and are considered for treatment of solid tumors with high BST-2 antigen levels. We found that in HIV-infected cells, BST-2 antibody treatment facilitates virus release from BST-2(+) cells by interfering with the tethering activity of BST-2. BST-2 antibodies were unable to release already tethered virions and were most effective when added early during virus production. BST-2 antibody treatment did not affect BST-2 dimerization, which we earlier described to be critical for inhibition of virus release, and did not reduce cell surface expression of BST-2. Interestingly, BST-2 antibody treatment also reduced non-specific shedding of BST-2 and limited encapsidation of BST-2 into virions. Finally, floatation analyses indicate that BST-2 antibodies affect the distribution of BST-2 within membrane rafts. Our data suggest that BST-2 antibody treatment may enhance virus release by inducing a redistribution of BST-2 at the cell surface thus preventing it from accumulating at the sites of virus budding (Miyagi 2011). In FY12, we further continued a project investigating the effect of deletions or insertions in the BST-2 ectodomain on interference with virus release. This project was in part stimulated by reports showing that synthetic constructs can have BST-2-like function as long as certain structural features are preserved. A large portion of the BST-2 ectodomain is predicted to assume a coil-coil structure. We found the insertions or deletions in this domain can either be tolerated with little impact on BST-2 function or can completely abrogate BST-2s ability to antagonize virus release. Our data suggest that the effect of a deletion or insertion is less dependent on the size of the deletion/insertion rather than the positioning within the molecule. In fact, we were able to remove an entire seven-heptad repeat motif thought to be critical for the formation of BST-2 coiled-coil dimers, without loss of function. We hypothesize that insertions or deletions in the BST-2 ectodomain impose structural constraints that have variable effects on protein function. We are currently performing molecular modeling studies based on X-ray crystallographic data of wild type BST-2 to study the effects of our mutations on BST-2 structure. We hope to complete this project in FY13. In FY12, we also initiated two new projects. The first new project involves the functional characterization of SAMHD1, a novel host factor involved in the inhibition of virus replication in dendritic and myeloid cells. SAMHD1 is a dNTPase that presumably reduces the cellular dNTP levels to levels too low for retroviral reverse transcription to occur. However, HIV-2 and SIV encoded Vpx counteracts the antiviral effects of SAMHD1 by targeting the protein for proteasomal degradation. SAMHD1 is encoded by a multiply spliced mRNA and consists of 16 exons. We identified two naturally occurring splice variants lacking exons 8-9 and 14, respectively. Like wildtype SAMHD1, both splice variants localize primarily to the nucleus, interact with Vpx, and retain sensitivity to Vpx-dependent degradation. However, the splice variants differ from full-length SAMHD1 in their metabolic stability and catalytic activity. While full-length SAMHD1 is metabolically stable in uninfected cells, both splice variants were inherently metabolically unstable and were rapidly degraded even in the absence of Vpx. Nevertheless, Vpx further enhanced the degradation of these proteins to a rate comparable to that seen for full-length SAMHD1. Experiments are ongoing to study the functional properties of the SAMHD1 splice variants and their role in virus restriction. Our second newly initiated project involves a host factor involved in the control of APOBEC3G by Vif. Vif is a lentiviral accessory protein known to inhibit the virion incorporation of APOBEC3G, a cellular cytidine deaminase that can cause fatal editing of retroviral genomes. Recent studies indicate that the effect of Vif on APOBEC3G is modulated by a newly identified host factor, CBF-beta. We have cloned CBF-beta from human cells. Also, since CBF-beta is ubiquitously expressed in most cell types, we created stable CBF-beta knock-down cell lines to be able to perform experiments on a CBF-beta negative background. Our preliminary results indicate that CBF-beta affects the expression of Vif. Indeed, steady-state levels of Vif were 5-10x lower in cells lacking CBF-beta as compared to the parental CBF-beta positive controls. Experiments are ongoing to study the mechanistic details of the Vif-CBF-beta relationship.
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