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 catalytic activity. Instead, these proteins function primarily if not exclusively as molecular adaptors to link viral or cellular factors to pre-existing cellular pathways. In FY13 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 our study on the characterization of SAMHD1 splice variants and we initiated a new project on the characterization of the functional significance of SAMHD1 phosphorylation for its antiviral activity. Finally, we continued projects related to Vif and its interaction with the host restriction factor APOBEC3G, focusing in particular on the role of a recently identified host factor CBF. BST-2/Vpu: We found that the functional antagonism of rhesus macaque BST-2 by HIV-1 Vpu is mediated by cytoplasmic domain interactions. We knew that Vpu encoded by NL4-3, a widely used HIV-1 laboratory strain, antagonizes human BST-2 but not monkey or murine BST-2 leading to the impression that BST-2 antagonism by Vpu is species-specific. However, in collaboration with Malcolm Martins lab we recently identified several primary Vpu isolates, such as DH12, capable of antagonizing both human and rhesus BST-2. We subsequently found that while Vpu interacts with human BST-2 primarily through their respective transmembrane domains, antagonism of rhesus BST-2 by Vpu involved an interaction of their cytoplasmic domains. Importantly, a Vpu mutant carrying two mutations in its transmembrane domain rendering it incompetent for interaction with human BST-2 was able to interact with human BST-2 carrying the rhesus BST-2 cytoplasmic domain and partially neutralized the ability of this BST-2 variant to inhibit viral release. Interaction of Vpu and rhesus BST-2 involved a five-residue motif in the cytoplasmic domain of BST-2 previously identified as important for antagonism of monkey and great ape BST-2 by SIV Nef. Thus, our study identifies a novel mechanism of antagonism of rhesus BST-2 by Vpu that targets the same motif in BST-2 used by SIV Nef and might explain the expanded host-range observed for Vpu isolates in our previous study. In a parallel study, we investigated the importance of cysteine positioning for functional dimerization of BST-2. BST-2 consists of an N-terminal cytoplasmic domain, a TM domain, an ectodomain, and C-terminal membrane anchor. The N-terminal half of the BST-2 ectodomain contains three cysteine residues, each of which can contribute to the formation of cysteine-linked dimers. In FY13 we started to determine the importance of the positioning of the cysteine bridge for BST-2 function. Starting with a cysteine-free monomeric form of BST-2, individual cysteine residues were reintroduced at various locations throughout the ectodomain. Resulting BST-2 variants were tested for expression, dimerization, surface presentation, and inhibition of HIV-1 virus release. Consistent with the finding that BST-2 dimerization was not a prerequisite for cell surface expression, the vast majority of our BST-2 variants were expressed at the cell surface at wild type levels or above. Our results also demonstrate significant flexibility in the positioning of cysteine residues with regard to 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. We expect to complete this project in FY14. SAMHD1/Vpx: In FY12 we initiated a new project involving the functional characterization of 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. In FY13 we initiated a new SAMHD1-related project that addresses the role of post-translational modifications in SAMHD1 for its antiviral activity. We used 32P labeling to identify SAMHD1 as a phosphoprotein, both endogenously in THP-1 cells and after exogenous expression in HeLa cells. Several amino acids in SAMHD1 were identified to be sites of phosphorylation using direct mass spectrometry Mutation of these residues to alanine, to prevent phosphorylation, or to glutamic acid, to mimic phosphorylation, had no effect on the nuclear localization of SAMHD1. Similarly, like the wildtype protein, these SAMHD1 phospho-site mutants were still able to be efficiently degraded by SIVvpx. Interestingly, while neither alanine nor glutamic acid substitutions severely affected SAMHD1 dNTPase activity in an in vitro assay, we identified one phospho site important for the ability of SAMHD1 to restrict HIV-1. Our results therefore indicate that SAMHD1 phosphorylation could be a regulator of SAMHD1 restriction activity. This project is ongoing and further analysis of SAMHD1 post-translational modifications could give important insights into the mechanisms of SAMHD1 function and/or regulation. APOBEC3G/Vif: In FY12 we initiated a project studying the role of a newly identified host factor, CBF, in the control of APOBEC3G by Vif. Recent studies indicate that the effect of Vif on APOBEC3G is modulated by CBF. We have cloned CBF from human cells. Also, since CBF is ubiquitously expressed in most cell types, we created stable CBF knock-down cell lines to be able to perform experiments on a CBF negative background. Several studies noted reduced Vif expression in CBF knockdown cells while others saw no significant impact of CBF on Vif stability. Indeed, we confirmed that CBF increases Vif steady-state levels. This effect was seen for Vif expressed from a full-length molecular clone of HIV-1 as well as for Vif expressed from a codon-optimized vector. Interestingly, while CBF increased cellular levels of Vif, virus-associated levels of Vif did not increase. CBF itself was excluded from virions suggesting that CBF-Vif complexes are sequestered within cells. CBF neither affected expression of viral Gag nor Vpu protein indicating that CBF regulates Vif expression post-transcriptionally. Kinetic studies revealed effects of CBF both on metabolic stability and on the rate of Vif biosynthesis. These effects were dependent on the ability of CBF to interact with Vif pointing to a chaperone function of CBF. Importantly, at comparable Vif levels, CBF further enhanced A3G degradation suggesting that CBF facilitates A3G degradation by increasing the levels of Vif and by independently augmenting the ability of Vif to target A3G for degradation. We propose that CBF acts like a chaperone to stabilize Vif during and after synthesis and to facilitate interaction of Vif with cellular cofactors required for the degradation of A3G.

Project Start
Project End
Budget Start
Budget End
Support Year
21
Fiscal Year
2013
Total Cost
$1,139,940
Indirect Cost
City
State
Country
Zip Code
Andrew, Amy; Strebel, Klaus (2014) HIV-1 accessory proteins: Vpu and Vif. Methods Mol Biol 1087:135-58
Miyagi, Eri; Kao, Sandra; Yedavalli, Venkat et al. (2014) CBF* enhances de novo protein biosynthesis of its binding partners HIV-1 Vif and RUNX1 and potentiates the Vif-induced degradation of APOBEC3G. J Virol 88:4839-52
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Watashi, Koichi; Khan, Mohammad; Yedavalli, Venkat R K et al. (2008) Human immunodeficiency virus type 1 replication and regulation of APOBEC3G by peptidyl prolyl isomerase Pin1. J Virol 82:9928-36