Anti-HIV Host Factor SAMHD1 Regulates Viral Sensitivity to Nucleoside Reverse Transcriptase Inhibitors via Modulation of Cellular dNTP Levels.
ABSTRACT Newly identified anti-HIV host factor, SAMHD1, restricts replication of lentiviruses such as HIV-1, HIV-2, and SIV in macrophages by enzymatically hydrolyzing and depleting cellular dNTPs, which are the substrates of viral DNA polymerases. HIV-2 and some SIVs express Vpx, which counteracts SAMHD1 and elevates cellular dNTPs, enhancing viral replication in macrophages. Since nucleoside reverse transcriptase inhibitors (NRTIs), the most commonly used anti-HIV drugs, compete against cellular dNTPs for incorporation into proviral DNA, we tested whether SAMHD1 directly affects the efficacy of NRTIs in inhibiting HIV-1. We found that reduction of SAMHD1 levels with the use of virus-like particles (VLPs) expressing Vpx and SAMHD1 specific shRNA, subsequently elevates cellular dNTPs and significantly decreases HIV-1 sensitivity to various NRTIs in macrophages. However, VLP +Vpx treatment of activated CD4+ T cells only minimally reduced NRTI efficacy. Furthermore, our HPLC-based assay could not detect SAMHD1-mediated hydrolysis of various NRTI-triphosphates tested in this study, suggesting that the reduced sensitivity of HIV-1 to NRTIs upon SAMHD1 degradation is most likely caused by the elevation of the cellular dNTP levels.
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ABSTRACT: Background Human Immunodeficiency Virus-type 2 (HIV-2) encodes Vpx that degrades SAMHD1, a cellular restriction factor active in non-dividing cells. HIV-2 replicates in lymphocytes but the susceptibility of monocyte-derived dendritic cells (MDDCs) to in vitro infection remains partly characterized.ResultsHere, we investigated HIV-2 replication in primary CD4+ T lymphocytes, both activated and non-activated, as well as in MDDCs. We focused on the requirement of Vpx for productive HIV-2 infection, using the reference HIV-2 ROD strain, the proviral clone GL-AN, as well as two primary HIV-2 isolates. All HIV-2 strains tested replicated in activated CD4+ T cells. Unstimulated CD4+ T cells were not productively infected by HIV-2, but viral replication was triggered upon lymphocyte activation in a Vpx-dependent manner. In contrast, MDDCs were poorly infected when exposed to HIV-2. HIV-2 particles did not potently fuse with MDDCs and did not lead to efficient viral DNA synthesis, even in the presence of Vpx. Moreover, the HIV-2 strains tested were not efficiently sensed by MDDCs, as evidenced by a lack of MxA induction upon viral exposure. Virion pseudotyping with VSV-G rescued fusion, productive infection and HIV-2 sensing by MDDCs.Conclusion Vpx allows the non-productive infection of resting CD4+ T cells, but does not confer HIV-2 with the ability to efficiently infect MDDCs. In these cells, an entry defect prevents viral fusion and reverse transcription independently of SAMHD1. We propose that HIV-2, like HIV-1, does not productively infect MDDCs, possibly to avoid triggering an immune response mediated by these cells.Retrovirology 01/2015; 12(1):2. DOI:10.1186/PREACCEPT-2035398010150355 · 4.77 Impact Factor
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ABSTRACT: The eradication of HIV necessitates elimination of the HIV latent reservoir. Identifying host determinants governing latency and reservoir size in the setting of antiretroviral therapy (ART) is an important step in developing strategies to cure HIV infection. We sought to determine the impact of cell-intrinsic immunity on the HIV latent reservoir. We investigated the relevance of a comprehensive panel of established anti-HIV-1 host restriction factors to multiple established virologic and immunologic measures of viral persistence in HIV-1-infected, ART-suppressed individuals. We measured the mRNA expression of 42 anti-HIV-1 host restriction factors, levels of cell-associated HIV-1 RNA, levels of total pol and 2-long terminal repeat (2-LTR) circle HIV-1 DNA and immunophenotypes of CD4 T cells in 72 HIV-1-infected individuals on suppressive ART (23 individuals initiated ART less than 1 year postinfection, and 49 individuals initiated ART greater than 1 year post-infection). Correlations were analysed using nonparametric tests. The enhanced expression of a few select host restriction factors, p21, schlafen 11 and PAF1, was strongly associated with reduced CD4 T-cell associated HIV RNA during ART (P < 0.001). In addition, our data suggested that ART perturbs the regulatory relationship between CD4 T-cell activation and restriction factor expression. Lastly, cell-intrinsic immune responses were significantly enhanced in individuals who initiated ART during early versus chronic infection and may contribute to the reduced reservoir size observed in these individuals. Intrinsic immune responses modulate HIV persistence during suppressive ART and may be manipulated to enhance the efficacy of ART and promote viral eradication through reversal of latency in vivo.AIDS (London, England) 01/2015; DOI:10.1097/QAD.0000000000000572 · 6.56 Impact Factor
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ABSTRACT: Development of dNTP-based drugs requires a quantitative understanding of any inhibition, activation or hydrolysis by off-target cellular enzymes. SAMHD1 is a regulatory dNTP-triphosphohydrolase that inhibits HIV-1 replication in human myeloid cells. We describe a coupled-enzyme assay to quantify activation, inhibition and hydrolysis of dNTPs, nucleotide analogues and nucleotide analogue inhibitors by triphosphohydrolase enzymes. The assay facilitates mechanistic studies of triphosphohydrolase enzymes and quantification of off-target effects of nucleotide-based antiviral and chemotherapeutic agents.Antimicrobial Agents and Chemotherapy 10/2014; 59(1). DOI:10.1128/AAC.03903-14 · 4.45 Impact Factor