HIV-1 encodes genes that are crucial for replication in primate cells exerting functions 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 FY16 we continued projects relating to the functional properties of SAMHD1 and its mechanism of virus restriction. Further, we continued projects related to Vif and its interaction with the host proteins APOBEC3G and CBFb. We completed a project involving the characterization of yet another host factor, SLFN11, and its role in virus replication and are in the process of preparing a manuscript for publication. A3G/Vif: In FY16, we continued a project studying the role of CBFb in the Vif-dependent control of the host restriction factor APOBEC3G (A3G). We previously reported 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. To further characterize these functions of CBFb, we assessed the importance of CBFb for dominant negative interference with the function of wt Vif by previously described Vif variants. Our data point to a positive correlation between the ability of Vif variants to bind CBFb and their dominant-negative properties. We are in the process of finalizing experiments and have started with preparations for publication. SAMHD1/Vpx: In FY16 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. We now investigated the importance of cellular dNTP levels for virus restriction by SAMHD1. Unlike other restriction factors such as APOBEC3G that renders normally permissive HeLa cells restrictive for HIV-1, exogenous expression of SAMHD1 in HeLa cells shows little to no restrictive phenotype. It is possible that the continued synthesis of dNTPs in dividing HeLa does not allow SAMHD1 dNTPase activity to sufficiently lower the cellular dNTP pool for lentiviral restriction to occur. Alternatively, it cannot be ruled out that SAMHD1 exerts its antiviral effect in conjunction with additional host factor(s) not expressed in non-myeloid or dividing cell types. To address these questions, we employed SAMHD1 variants together with hydroxyurea treatment to modulate dNTP levels in HeLa cells. Hydroxyurea inhibits ribonucleotide reductase and thus reduces the cellular dNTP pool at the synthesis step. Determination of cellular dATP levels confirmed that hydroxyurea dramatically reduced the baseline dNTP pool in treated HeLa cells with SAMHD1 exhibiting negligible additional effects on the dNTP pool. Using the hydroxyurea strategy of lowering baseline dNTP levels we were able to demonstrate SAMHD1 antiviral activity in HeLa cells whereas no additional antiviral activity was demonstrable in untreated cells. These results suggest that the lack of antiviral activity of SAMHD1 in normal HeLa cells is not due to the lack of additional cellular proteins but are due to the high dNTP levels in these cells. Thus, low cellular dNTP levels appear to be necessary for SAMHD1 restriction activity. However, our results also provide further evidence that SAMHD1 may possess an additional dNTP-independent function that contributes to lentiviral restriction but a contribution of a possible exonuclease activity could not be confirmed. The results from these experiments have been recently published (Welbourn et al 2016). Vpr: in FY16, we continued a project involving the characterization of HIV and SIV Vpr proteins and their contributions to the control of host restriction factors. We performed mass spectrometry to identify Vpr-interacting host factors using conditions that avoided the loss of potential targets of Vpr by protein degradation. We identified several proteins and confirmed specific binding to Vpr by co-IP analysis for several of them. When transiently expressed in mammalian cells, wild type Vpr but not a DCAF1-binding mutant (Q65R) induced a reduction of steady-state expression of some of these proteins. We will continue to characterize these host factors and their potential role in HIV pathogenesis. In collaboration with the Hirsch lab, we investigated the correlation between BST-2 antagonism by SIV and neurovirulence. The study was based on the observation that current antiretroviral therapy (ART) is not sufficient to completely suppress disease progression in the CNS, as indicated by the rising incidence of HIV-1-associated neurocognitive disorders among infected individuals on ART. It is not clear why some HIV-1infected patients develop HAND, despite effective repression of viral replication in the circulation. We identified 4 amino acid substitutions in the cytoplasmic tail of the envelope glycoprotein gp41 of the neurovirulent virus SIVsm804E that enhance replication in macrophages and are correlated with enhanced antagonism of BST-2. The results of our study are consistent with a potential role for BST-2 in the CNS microenvironment and suggest that BST-2 antagonists may serve as a possible target for countermeasures against HIV-1-associated neurocognitive disorders. The results from this collaboration were recently published (Matsuda et al, 2016). In collaboration with Paul ONeill (University of Liverpool, UK) we studied chemical variants of abacavir to assess their antiviral potential in comparison to abacavir. Abacavir is a reverse transcriptase inhibitor (NRTI class) that is often used in patients resistant to AZT or 3TC. It is generally well tolerated but can cause severe T-cell-mediated hypersensitivity reactions in individuals carrying human leukocyte antigen (HLA)-B*57:01. We investigated whether chemical modification of abacavir can produce a molecule with antiviral activity that does not bind to HLA-B*57:01 and activate T cells. We found that the closely related analogues N-isopropyl and N-methyl isopropyl abacavir were devoid of T-cell activity but retained antiviral activity. Our studies demonstrate that the unwanted T-cell activity of abacavir can be eliminated whilst maintaining the favorable antiviral profile. The results from this collaboration were recently published (Naisbitt et al, 2015).
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