Frequent Incorporation of Ribonucleotides during HIV-1 Reverse Transcription and Their Attenuated Repair in Macrophages
ABSTRACT Macrophages are well known long-lived reservoirs of HIV-1. Unlike activated CD4(+) T cells, this nondividing HIV-1 target cell type contains a very low level of the deoxynucleoside triphosphates (dNTPs) required for proviral DNA synthesis whereas the ribonucleoside triphosphate (rNTP) levels remain in the millimolar range, resulting in an extremely low dNTP/rNTP ratio. Biochemical simulations demonstrate that HIV-1 reverse transcriptase (RT) efficiently incorporates ribonucleoside monophosphates (rNMPs) during DNA synthesis at this ratio, predicting frequent rNMP incorporation by the virus specifically in macrophages. Indeed, HIV-1 RT incorporates rNMPs at a remarkable rate of 1/146 nucleotides during macrophage infection. This greatly exceeds known rates for cellular replicative polymerases. In contrast, little or no rNMP incorporation is detected in CD4(+) T cells. Repair of these rNMP lesions is also substantially delayed in macrophages compared with CD4(+) T cells. Single rNMPs embedded in a DNA template are known to induce cellular DNA polymerase pausing, which mechanistically contributes to mutation synthesis. Indeed, we also observed that embedded rNMPs in a dsDNA template also induce HIV-1 RT DNA synthesis pausing. Moreover, unrepaired rNMPs incorporated into the provirus during HIV-1 reverse transcription would be generally mutagenic as was shown in Saccharomyces cerevisiae. Most importantly, the frequent incorporation of rNMPs makes them an ideal candidate for development of a new class of HIV RT inhibitors.
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- "SAMHD1 and innate immune sensing mutation  . Overall, it is less clear how mutations in RNase H2 contribute to AGS. "
ABSTRACT: SAMHD1 is the most recent addition to a unique group of host restriction factors that limit retroviral replication at distinct stages of the viral life cycle. SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase that degrades the intracellular pool of deoxynucleoside triphosphates available during early reverse transcription. SAMHD1 activity is blocked by the Vpx accessory function present in HIV-2 and SIVsm. Mutations in SAMHD1 are associated with the autoimmune disorder Aicardi-Goutières syndrome, thus emphasizing its role in regulation of the immune response. SAMHD1 anti-retroviral activity is modulated by post-translational modifications, cell-cycle dependent functions and cytokine-mediated changes. Innate receptors that sense retroviral DNA intermediates are the focus of intense study, and recent studies have established a link between SAMHD1 restriction, innate sensing of DNA and protective immune responses. Cell-cycle dependent regulation of SAMHD1 by phosphorylation and the increasingly broad range of viruses inhibited by SAMHD1 further emphasize the importance of these mechanisms of host restriction. This review highlights current knowledge regarding SAMHD1 regulation and its impact on innate immune signalling and retroviral restriction.Journal of Molecular Biology 10/2013; 425. DOI:10.1016/j.jmb.2013.10.022 · 4.33 Impact Factor
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- "Indeed, as illustrated in Fig. 1, the scarcity of cellular dNTPs in macrophages, which is engineered by the enzymatic activity of SAMHD1, is a clear example of a metabolic bottleneck that HIV has to counteract during viral infection (Goldstone et al., 2011; Hrecka et al., 2011; Laguette et al., 2011; Lahouassa et al., 2012; Powell et al., 2011). In addition, the poor dNTP availability in macrophages generates a unique metabolic environment that promotes viral mutagenesis induced by frequent rNMP and non-canonical dUMP incorporation (Kennedy et al., 2010, 2011, 2012). These observations caused us to predict that macrophages may serve as a viral reservoir that contributes to the unique viral genomic hypermutability of HIV-1. "
ABSTRACT: Retroviruses consume cellular deoxynucleoside triphosphates (dNTPs) to convert their RNA genomes into proviral DNA through reverse transcription. While all retroviruses replicate in dividing cells, lentiviruses uniquely replicate in nondividing cells such as macrophages. Importantly, dNTP levels in nondividing cells are extremely low, compared to dividing cells. Indeed, a recently discovered anti-HIV/SIV restriction factor, SAMHD1, which is a dNTP triphosphohydrolase, is responsible for the limited dNTP pool of nondividing cells. Lentiviral reverse transcriptases (RT) uniquely stay functional even at the low dNTP concentrations in nondividing cells. Interestingly, Vpx of HIV-2/SIVsm proteosomally degrades SAMHD1, which elevates cellular dNTP pools and accelerates lentiviral replication in nondividing cells. These Vpx-encoding lentiviruses rapidly replicate in nondividing cells by encoding both highly functional RTs and Vpx. Here, we discuss a series of mechanistic and virological studies that have contributed to conceptually linking cellular dNTP levels and the adaptation of lentiviral replication in nondividing cells.Virology 12/2012; 436(2). DOI:10.1016/j.virol.2012.11.010 · 3.28 Impact Factor
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ABSTRACT: HIV-1-specific antibodies and CD8(+) cytotoxic T cells are detected in most HIV-1-infected people, yet HIV-1 infection is not eradicated. Contributing to the failure to mount a sterilizing immune response may be the inability of antigen-presenting dendritic cells (DCs) to sense HIV-1 during acute infection, and thus the inability to effectively prime naive, HIV-1-specific T cells. Recent findings related to DC-expressed innate immune factors including SAMHD1, TREX1, and TRIM5 provide a molecular basis for understanding why DCs fail to adequately sense invasion by this deadly pathogen and suggest experimental approaches to improve T cell priming to HIV-1 in prophylactic vaccination protocols.Cell host & microbe 10/2012; 12(4):408-18. DOI:10.1016/j.chom.2012.10.002 · 12.19 Impact Factor