HIV-1 encodes genes that are crucial for replication in primate cells and whose function is not provided by the host. 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 now strong evidence that these proteins operate in conjunction with specific host factors. In fact, none of the HIV accessory proteins has a known enzymatic activity. Instead, these proteins function primarily if not exclusively as molecular adaptors to link viral or cellular factors to pre-existing cellular pathways. In FY14 we continued projects relating to Vpu and its interaction with the host restriction factor BST-2 as well as the structural characterization of BST-2. We also completed the characterization of the functional significance of SAMHD1 phosphorylation for its antiviral activity and continued to address questions raised in the process. Further, we continued projects related to Vif and its interaction with the host CBFb. Finally, we initiated a project involving the characterization of yet another host factor, SLFN11, and its role in virus replication BST-2/Vpu: We continued our project on the analysis of cysteine residues in the BST-2 ectodomain. Starting with an inactive cysteine-free monomeric form of BST-2, individual cysteine residues were reintroduced throughout the ectodomain at positions predicted to form or to not form disulfides based on the available X-ray crystal structures. Resulting BST-2 variants were tested for expression, dimerization, surface presentation, and inhibition of HIV-1 virus release. Our results demonstrate significant flexibility in the positioning of cysteine residues with regard to functional BST-2 dimerization even though the propensity to catalyze dimerization generally decreased with increasing proximity of the cysteines to the C-terminus of the BST-2 ectodomain. Importantly, our data indicate that BST-2 dimerization is not sufficient for inhibition of virus release since not all dimerization-competent BST-2 variants were functional in our virus release assay. Our results expose new structural constraints governing the functional dimerization of BST-2, a property essential to its role as a restriction factor tethering viruses to the host cell. In FY14, we initiated a new project studying the importance of Vpu oligomerization for its various biological activities. Vpu is a small integral membrane protein, which is known to cause the degradation of the viral receptor CD4 and to enhance virus release from the cell surface. In the case of CD4, Vpu acts as a molecular adaptor to connect CD4 to an E3 ubiquitin ligase complex in the endoplasmic reticulum (ER) resulting in CD4 degradation by cellular proteasomes. This function requires signals located in Vpus cytoplasmic domain. On the other hand, Vpus TM domain is required to antagonize a cellular host factor, BST-2, which otherwise prevents virus release by tethering virus particles to the cell surface. Vpu has the propensity to form homo-oligomeric complexes capable of forming ion conducting membrane pores. However, the importance of Vpu oligomerization for its biological functions has not been experimentally addressed. One of the reasons is that to date no oligomerization-defective mutants of Vpu have been identified. However, it is known that the Vpu TM domain is a driving force in the oligomerization process. To generate oligomerization-defective Vpu mutants, we decided to use a model membrane protein whose oligomerization properties have been well-characterized and for which oligomerization-defective variants have been described. Indeed, transfer of the TM domains of this model protein conferred their oligomerization characteristics to Vpu. Vpu encoding the wt TM domain of our model protein formed dimers as well as higher-order complexes as assessed by immunoblotting whereas Vpu carrying the mutated TM domain remained monomeric. We assessed the ability of monomeric and oligomeric Vpu to induce CD4 degradation in transiently transfected 293T cells and found no significant difference. Thus, degradation of CD4 is clearly not dependent on Vpu oligomerization. This project is ongoing and we will next assess the role of Vpu oligomerization in the regulation of virus release, the deregulation of the NFkB pathway, and other reported functions of Vpu. Vif/CBFb: In FY14, we continued a project studying the role of a newly identified host factor, CBFb. Several reports suggested that knockdown of CBFb results in reduced steady-state expression of Vif whereas others found that CBFb could facilitate Vif-induced A3G degradation without apparent effect on Vif stability. Neither the cause underlying the reduced steady state levels of Vif in the absence of CBFb nor the reason for the discrepant observations regarding the effects of CBFb on Vif expression are currently understood. We therefore performed a series of experiments to assess the role of CBFb in the expression of Vif and its ability to target APOBEC3G. We found that CBFb was critical for the stable expression of Vif. In fact, shRNA mediated knockdown of CBFb in HeLa cells resulted in the reduction of Vif levels. Vif expression was restored by ectopic expression of CBFb. Kinetic studies revealed that CBFb significantly enhanced the rate of de novo Vif biosynthesis and to a more limited extent increased the metabolic stability of Vif. Deletions in the N-terminal region of Vif resulted in loss of CBFb responsiveness. Independent from its effects on Vif expression, CBFb also enhanced the Vif-mediated degradation of A3G in agreement with reports that observed CBFb-enhanced degradation of A3G without effect on Vif expression. Taken together our data suggest that CBFb functions like a molecular chaperone to enhance Vif biosynthesis, to stabilize mature Vif protein, and to facilitate the assembly of an A3G-Vif-Cul5 E3 ligase complex that overall results in more efficient degradation of A3G. SAMHD1/Vpx: In FY14 we continued the functional characterization of SAMHD1. SAMHD1 is a dNTPase that reduces cellular dNTP concentrations to levels too low for retroviral reverse transcription. We previously found that SAMHD1 is a phosphoprotein and that its antiviral activity but not its dNTPase activity are regulated by phosphorylation at a threonine residue located near the C-terminal end of the protein. These results suggested (a) that reducing cellular dNTP levels may be necessary but is not sufficient to inhibit retroviral infection and (b) that SAMHD1 has another intrinsic activity critical for the restriction of retroviruses. The goal of our ongoing experiments is to identify and characterize this additional function of SAMHD1. In particular, a proposed exonuclease activity of SAMHD1 is under analysis. SLFN11: In FY14 we initiated a new project involving the functional characterization of a novel host factor believed to inhibit HIV-1 replication. SLFN11 was recently reported to inhibit HIV-1 replication by depleting cells of rare tRNAs used by viruses for protein synthesis. To this end we cloned SLFN11 from two separate human cellular sources and identified a polymorphism in human SLFN11. Preliminary results indicate that the two haplotypes of SLFN11 identified thus far differ in their functional properties with respect to their ability to interfere with HIV replication. We have created SLFN11 knockdown cell lines in the backbone of 293T for transient studies as well as in H9 cells for multi-round replication studies. These cell lines will be used to further dissect the mechanism by which SLFN11 interferes with HIV replication.
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