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
"Retroviruses from the genus lentivirus, including HIV-1 and at least 40 different lineages of SIVs, infect non-dividing cells such as resting CD4+ T cells and myeloid cells. One hallmark of these non-dividing cell types is a concentration of deoxynucleotides (dNTPs) below the threshold required for reverse transcription [5-8]. The cellular enzyme SAMHD1 depletes the intracellular nucleotide pool in non-cycling cells by converting deoxynucleotides into deoxynucleosides and inorganic triphosphates . "
[Show abstract][Hide abstract] ABSTRACT: SIVMAC/HIV-2 Vpx recruits the CUL4A-DCAF1 E3 ubiquitin ligase complex to degrade the deoxynucleotide hydrolase SAMHD1. This increases the concentration of deoxynucleotides available for reverse transcription in myeloid cells and resting T cells. Accordingly, transduction of these cells by SIVMAC requires Vpx. Virus-like particles containing SIVMAC Vpx (Vpx-VLPs) also increase the efficiency of HIV-1 transduction in these cells, and rescue transduction by HIV-1, but not SIVMAC, in mature monocyte-derived dendritic cells (MDDCs). Differences in Vpx mechanism noted at that time, along with recent data suggesting that SAMHD1 gains additional restriction capabilities in the presence of type I IFN prompted further examination of the role of Vpx and SAMHD1 in HIV-1 transduction of mature MDDCs.
When challenged with Vpx-VLPs, SAMHD1 was degraded in MDDCs even after cells had been matured with LPS, though there was no increase in deoxynucleotide levels. Steady-state levels of HIV-1 late reverse transcription products in mature MDDCs were increased to the same extent by either Vpx-VLPs or exogenous nucleosides. In contrast, only Vpx-VLPs increased the levels of 2-LTR circles and proviral DNA in myeloid cells. These results demonstrate that exogenous nucleosides and Vpx-VLPs both increase the levels of HIV-1 cDNA in myeloid cells, but only Vpx-VLPs rescue 2-LTR circles and proviral DNA in myeloid cells with a previously established antiviral state. Finally, since trans-acting Vpx-VLPs provide long-lasting rescue of HIV-1 vector transduction in the face of the antiviral state, and exogenous nucleosides do not, exogenous nucleosides were used to achieve efficient transduction of MDDCs by vectors that stably encode Vprs and Vpxs from a collection of primate lentiviruses. Vpr from SIVDEB or SIVMUS, Vpx from SIVMAC251 or HIV-2, but not SIVRCM, degraded endogenous SAMHD1, increased steady-state levels of HIV-1 cDNA, and rescued HIV-1 from the antiviral state in MDDCs.
Inhibition of deoxynucleotide hydrolysis by promoting SAMHD1 degradation is not the only mechanism by which Vpx rescues HIV-1 in MDDCs from the antiviral state. Vpx has an additional effect on HIV-1 transduction of these cells that occurs after completion of reverse transcription and acts independently of deoxynucleotide levels.
[Show abstract][Hide abstract] 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.
"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. "
[Show abstract][Hide abstract] 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.
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