scholarly journals Viral protein X reduces the incorporation of mutagenic noncanonical rNTPs during lentivirus reverse transcription in macrophages

2019 ◽  
Vol 295 (2) ◽  
pp. 657-666
Author(s):  
Adrian Oo ◽  
Dong-Hyun Kim ◽  
Raymond F. Schinazi ◽  
Baek Kim
Keyword(s):  
Simian Immunodeficiency Virus ◽  
Reverse Transcription ◽  
Viral Protein ◽  
Cell Types ◽  
Dntp Pool ◽  
Sam Domain ◽  
Primary Monocyte ◽  
Immunodeficiency Virus ◽  
Protein X ◽  
Hiv 1

Unlike activated CD4+ T cells, nondividing macrophages have an extremely small dNTP pool, which restricts HIV-1 reverse transcription. However, rNTPs are equally abundant in both of these cell types and reach much higher concentrations than dNTPs. The greater difference in concentration between dNTPs and rNTPs in macrophages results in frequent misincorporation of noncanonical rNTPs during HIV-1 reverse transcription. Here, we tested whether the highly abundant SAM domain– and HD domain–containing protein 1 (SAMHD1) deoxynucleoside triphosphorylase in macrophages is responsible for frequent rNTP incorporation during HIV-1 reverse transcription. We also assessed whether Vpx (viral protein X), an accessory protein of HIV-2 and some simian immunodeficiency virus strains that targets SAMHD1 for proteolytic degradation, can counteract the rNTP incorporation. Results from biochemical simulation of HIV-1 reverse transcriptase–mediated DNA synthesis confirmed that rNTP incorporation is reduced under Vpx-mediated dNTP elevation. Using HIV-1 vector, we further demonstrated that dNTP pool elevation by Vpx or deoxynucleosides in human primary monocyte-derived macrophages reduces noncanonical rNTP incorporation during HIV-1 reverse transcription, an outcome similarly observed with the infectious HIV-1 89.6 strain. Furthermore, the simian immunodeficiency virus mac239 strain, encoding Vpx, displayed a much lower level of rNTP incorporation than its ΔVpx mutant in macrophages. Finally, the amount of rNMPs incorporated in HIV-1 proviral DNAs remained unchanged for ∼2 weeks in macrophages. These findings suggest that noncanonical rNTP incorporation is regulated by SAMHD1 in macrophages, whereas rNMPs incorporated in HIV-1 proviral DNA remain unrepaired. This suggests a potential long-term DNA damage impact of SAMHD1-mediated rNTP incorporation in macrophages.

scholarly journals The Susceptibility of Primate Lentiviruses to Nucleosides and Vpx during Infection of Dendritic Cells Is Regulated by CA

2015 ◽  
Vol 89 (7) ◽  
pp. 4030-4034 ◽  
Author(s):  
Véronique Barateau ◽  
Xuan-Nhi Nguyen ◽  
Fanny Bourguillault ◽  
Grégory Berger ◽  
Stéphanie Cordeil ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Dendritic Cells ◽  
Simian Immunodeficiency Virus ◽  
Viral Protein ◽  
Immunodeficiency Virus ◽  
Protein X ◽  
Primate Lentiviruses ◽  
Species Specific ◽  
Hiv 1

The block toward human immunodeficiency virus type 1 (HIV-1) infection of dendritic cells (DCs) can be relieved by Vpx (viral protein X), which degrades sterile alpha motif-hydroxylase domain 1 (SAMHD1) or by exogenously added deoxynucleosides (dNs), lending support to the hypothesis that SAMHD1 acts by limiting deoxynucleoside triphosphates (dNTPs). This notion has, however, been questioned. We show that while dNs and Vpx increase the infectivity of HIV-1, only the latter restores the infectivity of a simian immunodeficiency virus of macaques variant, SIVMACΔVpx virus. This distinct behavior seems to map to CA, suggesting that species-specific CA interactors modulate infection of DCs.

scholarly journals Limiting deoxynucleoside triphosphate concentrations emphasize the processivity defect of lamivudine-resistant variants of human immunodeficiency virus type 1 reverse transcriptase.

1997 ◽  
Vol 41 (11) ◽  
pp. 2484-2491 ◽  
Author(s):  
N K Back ◽  
B Berkhout
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcription ◽  
Cell Types ◽  
Primary Cells ◽  
Dntp Pool ◽  
Immunodeficiency Virus ◽  
Deoxynucleoside Triphosphate ◽  
Dntp Level ◽  
Hiv 1

The nucleoside drug lamivudine (3TC) triggers the selection of resistant forms of the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) with a substitution of amino acid 184Met. The 3TC-resistant RT enzymes 184Val and 184Ile exhibit a processivity defect in in vitro assays that correlates with reduced replication of the corresponding virus variants in primary cells. However, no replication defect is apparent for these two mutants in the transformed T-cell line SupT1. One obvious difference between the two cell types is the intracellular deoxynucleoside triphosphate (dNTP) level. Primary cells have a much smaller dNTP pool, and this cellular condition may emphasize the processivity defect of the codon 184 RT variants. Alternatively, cell-specific cofactors that influence the process of reverse transcription may exist. Such accessory factors may be packaged into the virion to exert an effect on the RT enzyme. To discriminate between these possibilities we performed additional assays with the wild-type and mutant RT enzymes. The RT proteins were either isolated from virions produced by primary and transformed cell types or expressed as recombinant protein. We also performed infection assays with cells treated with a drug that reduces the intracellular dNTP pool. Furthermore, reverse transcription was studied within virus particles in the endogenous assay, which allows for the manipulation of the dNTP level. The combined results indicate that the enzymatic defect of the 3TC-resistant HIV-1 variants is stressed at low dNTP concentrations.

scholarly journals Heterologous Human Immunodeficiency Virus Type 1 Lentiviral Vectors Packaging a Simian Immunodeficiency Virus-Derived Genome Display a Specific Postentry Transduction Defect in Dendritic Cells

2003 ◽  
Vol 77 (17) ◽  
pp. 9295-9304 ◽  
Author(s):  
Caroline Goujon ◽  
Loraine Jarrosson-Wuilleme ◽  
Jeanine Bernaud ◽  
Dominique Rigal ◽  
Jean-Luc Darlix ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Human Immunodeficiency Virus ◽  
Dendritic Cells ◽  
Simian Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Cell Types ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Heterologous lentiviral vectors (LVs) represent a way to address safety concerns in the field of gene therapy by decreasing the possibility of genetic recombination between vector and packaging constructs and the generation of replication-competent viruses. Using described LVs based on human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus MAC251 (SIVMAC251), we asked whether heterologous virion particles in which trans-acting factors belonged to HIV-1 and cis elements belonged to SIVMAC251 (HIV-siv) would behave as parental homologous vectors in all cell types. To our surprise, we found that although the heterologous HIV-siv vector was as infectious as its homologous counterpart in most human cells, it was defective in the transduction of dendritic cells (DCs) and, to a lesser extent, macrophages. In DCs, the main postentry defect was observed in the formation of two-long-terminal-repeat circles, despite the fact that full-length proviral DNA was being synthesized and was associated with the nucleus. Taken together, our data suggest that heterologous HIV-siv vectors display a cell-dependent infectivity defect, most probably at a post-nuclear entry migration step. As homologous HIV and SIV vectors do transduce DCs, we believe that these results underscore the importance of a conserved interaction between cis elements and trans-acting viral factors that is lost or suboptimal in heterologous vectors and essential only in the transduction of certain cell types. For gene therapy purposes, these findings indicate that the cellular tropism of LVs can be modulated not only through the use of distinct envelope proteins or tissue-specific promoters but also through the specific combinatorial use of packaging and transfer vector constructs.

scholarly journals Effect of induced dNTP pool imbalance on HIV-1 reverse transcription in macrophages

Retrovirology ◽  
2019 ◽  
Vol 16 (1) ◽  
Author(s):  
Caitlin Shepard ◽  
Joella Xu ◽  
Jessica Holler ◽  
Dong-Hyun Kim ◽  
Louis M. Mansky ◽  
...  
Keyword(s):  
Target Cell ◽  
Copy Number ◽  
Transduction Efficiency ◽  
Cell Type ◽  
Single Cycle ◽  
Dntp Pool ◽  
Sam Domain ◽  
Primary Monocyte ◽  
Viral Mutant ◽  
Hiv 1

Abstract Background Terminally differentiated/nondividing macrophages, a key target cell type of HIV-1, harbor extremely low dNTP concentrations established by a host dNTP triphosphohydrolase, SAM domain and HD domain containing protein 1 (SAMHD1). We tested whether the induction of dNTP pool imbalance can affect HIV-1 replication in macrophages. For this test, we induced a large dNTP pool imbalance by treating human primary monocyte derived macrophages with either one or three of the four deoxynucleosides (dNs), which are phosphorylated to dNTPs in cells, to establish two different dNTP imbalance conditions in macrophages. Results The transduction efficiency and 2-LTR circle copy number of HIV-1 GFP vector were greatly diminished in human primary macrophages treated with the biased dN treatments, compared to the untreated macrophages. We also observed the induced dNTP bias blocked the production of infectious dual tropic HIV-1 89.6 in macrophages. Moreover, biochemical DNA synthesis by HIV-1 reverse transcriptase was significantly inhibited by the induced dNTP pool imbalance. Third, the induced dNTP bias increased the viral mutant rate by approximately 20–30% per a single cycle infection. Finally, unlike HIV-1, the single dN treatment did not significantly affect the transduction of SIVmac239-based GFP vector encoding Vpx in macrophages. This is likely due to Vpx, which can elevate all four dNTP levels even with the single dN treatment. Conclusion Collectively, these data suggest that the elevated dNTP pool imbalance can induce kinetic block and mutation synthesis of HIV-1 in macrophages.

scholarly journals Enhanced enzyme kinetics of reverse transcriptase variants cloned from animals infected with SIVmac239 lacking viral protein X

2020 ◽  
Vol 295 (50) ◽  
pp. 16975-16986
Author(s):  
Si'Ana A. Coggins ◽  
Dong-Hyun Kim ◽  
Raymond F. Schinazi ◽  
Ronald C. Desrosier ◽  
Baek Kim
Keyword(s):  
Steady State ◽  
Viral Protein ◽  
Catalytic Efficiency ◽  
Myeloid Cell ◽  
Immunodeficiency Virus ◽  
Protein X ◽  
Kinetics Of ◽  
Hiv 1

HIV Type 1 (HIV-1) and simian immunodeficiency virus (SIV) display differential replication kinetics in macrophages. This is because high expression levels of the active host deoxynucleotide triphosphohydrolase sterile α motif domain and histidine-aspartate domain–containing protein 1 (SAMHD1) deplete intracellular dNTPs, which restrict HIV-1 reverse transcription, and result in a restrictive infection in this myeloid cell type. Some SIVs overcome SAMHD1 restriction using viral protein X (Vpx), a viral accessory protein that induces proteasomal degradation of SAMHD1, increasing cellular dNTP concentrations and enabling efficient proviral DNA synthesis. We previously reported that SAMHD1-noncounteracting lentiviruses may have evolved to harbor RT proteins that efficiently polymerize DNA, even at low dNTP concentrations, to circumvent SAMHD1 restriction. Here we investigated whether RTs from SIVmac239 virus lacking a Vpx protein evolve during in vivo infection to more efficiently synthesize DNA at the low dNTP concentrations found in macrophages. Sequence analysis of RTs cloned from Vpx (+) and Vpx (−) SIVmac239–infected animals revealed that Vpx (−) RTs contained more extensive mutations than Vpx (+) RTs. Although the amino acid substitutions were dispersed indiscriminately across the protein, steady-state and pre-steady-state analysis demonstrated that selected SIVmac239 Vpx (−) RTs are characterized by higher catalytic efficiency and incorporation efficiency values than RTs cloned from SIVmac239 Vpx (+) infections. Overall, this study supports the possibility that the loss of Vpx may generate in vivo SIVmac239 RT variants that can counteract the limited availability of dNTP substrate in macrophages.

scholarly journals Localization of Viral protein X in Simian Immunodeficiency Virus Macaque Strain and Analysis of its Packaging Requirements

1994 ◽  
Vol 75 (11) ◽  
pp. 2955-2962 ◽  
Author(s):  
V. Liska ◽  
D. Spehner ◽  
M. Mehtali ◽  
D. Schmitt ◽  
A. Kirn ◽  
...  
Keyword(s):  
Simian Immunodeficiency Virus ◽  
Viral Protein ◽  
Immunodeficiency Virus ◽  
Protein X

scholarly journals Chimeric Human Immunodeficiency Virus Type 1 Virions That Contain the Simian Immunodeficiency Virus nef Gene Are Cyclosporin A Resistant

2005 ◽  
Vol 79 (5) ◽  
pp. 3211-3216 ◽  
Author(s):  
Mahfuz Khan ◽  
Lingling Jin ◽  
Lesa Miles ◽  
Vincent C. Bond ◽  
Michael D. Powell
Keyword(s):  
Human Immunodeficiency Virus ◽  
Cyclosporin A ◽  
Simian Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Cell Types ◽  
Infectivity Assay ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT We have previously shown that human immunodeficiency virus type 1 (HIV-1) virions which have their own nef gene deleted and are trans complemented to contain HIV-2 or simian immunodeficiency virus (SIV) Nef become resistant to treatment with cyclosporin A. To expand and confirm these studies, we have tested an HIV-1 isolate in which the HIV-1 nef gene has been replaced by the nef gene from SIV in a multiround infectivity assay using more physiologically relevant cell types. Our results confirm that HIV-1 virions that contain SIV nef can replicate in a cyclophilin-independent fashion.

scholarly journals Inhibitory Effects of HIV-2 Vpx on Replication of HIV-1

2018 ◽  
Vol 92 (14) ◽  
Author(s):  
Mohamed Mahdi ◽  
Zsófia Szojka ◽  
János András Mótyán ◽  
József Tőzsér
Keyword(s):  
Cell Culture ◽  
Human Immunodeficiency Virus ◽  
Viral Protein ◽  
Dual Infection ◽  
Cellular Level ◽  
Potential Candidate ◽  
Accessory Gene ◽  
Immunodeficiency Virus ◽  
Protein X ◽  
Hiv 1

ABSTRACTHuman immunodeficiency virus type 1 (HIV-1) and HIV-2 share a striking genomic resemblance; however, variability in the genetic sequence accounts for the presence of unique accessory genes, such as the viral protein X (vpx) gene in HIV-2. Dual infection with both viruses has long been described in the literature, yet the molecular mechanism of how dually infected patients tend to do better than those who are monoinfected with HIV-1 has not yet been explored. We hypothesized that in addition to extracellular mechanisms, an HIV-2 accessory gene is the culprit, and interference at the viral accessory/regulatory protein level is perhaps responsible for the attenuated pathogenicity of HIV-1 observed in dually infected patients. Following simulation of dual infection in cell culture experiments, we found that pretransduction of cells with HIV-2 significantly protects against HIV-1 transduction. Importantly, we have found that this dampening of the infectivity of HIV-1 was a result of interviral interference carried out by viral protein X of HIV-2, resulting in a severe hindrance to the replication dynamics of HIV-1, influencing both its early and late phases of the viral life cycle. Our findings shed light on potential intracellular interactions between the two viruses and broaden our understanding of the observed clinical spectrum in dually infected patients, highlighting HIV-2 Vpx as a potential candidate worth exploring in the fight against HIV-1.IMPORTANCEDual infection with human immunodeficiency virus types 1 and 2 is relatively common in areas of endemicity. For as-yet-unclarified reasons, patients who are dually infected were shown to have lower viral loads and generally a lower rate of progression to AIDS than those who are monoinfected. We aimed to explore dual infection in cell culture, to elucidate possible mechanisms by which HIV-2 may be able to exert such an effect. Our results indicate that on the cellular level, pretransduction of cells with HIV-2 significantly protects against HIV-1 transduction, which was found to be a result of interviral interference carried out by viral protein X of HIV-2. These findings broaden our knowledge of interviral interactions on the cellular level and may provide an explanation for the decreased pathogenicity of HIV-1 in dually infected patients, highlighting HIV-2 Vpx as a potential candidate worth exploring in the fight against HIV.

scholarly journals Human Immunodeficiency Virus Type 1 (HIV-1) Viral Protein R (Vpr) Interacts with Lys-tRNA Synthetase: Implications for Priming of HIV-1 Reverse Transcription

1998 ◽  
Vol 72 (4) ◽  
pp. 3037-3044 ◽  
Author(s):  
Lesley A. Stark ◽  
Ronald T. Hay
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Reverse Transcription ◽  
Viral Protein ◽  
Trna Synthetase ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The vpr gene of human immunodeficiency virus type 1 (HIV-1) encodes a 96-amino-acid 14-kDa protein (viral protein R [Vpr]), which is produced late in the viral life cycle and is incorporated into the virion. Although Vpr is not required for viral replication in transformed cell lines and primary T lymphocytes, it is essential for productive infection of macrophages and monocytes and appears to be important for pathogenesis in vivo. To establish the role of Vpr in HIV-1 replication and pathogenesis, we have isolated cellular proteins with which Vpr interacts. By using the yeast two-hybrid system, Lys-tRNA synthetase (LysRS) was identified as a Vpr-interacting protein. The interaction between Vpr and LysRS was characterized both in vitro and in vivo, and the domains of Vpr required for the interaction were defined. In the presence of Vpr, LysRS-mediated aminoacylation of tRNALys is inhibited. Since tRNALys is the primer for reverse transcription of the HIV-1 genome, this suggests that the interaction between Vpr and LysRS may influence the initiation of HIV-1 reverse transcription.

scholarly journals Vpx overcomes a SAMHD1-independent block to HIV reverse transcription that is specific to resting CD4 T cells

2017 ◽  
Vol 114 (10) ◽  
pp. 2729-2734 ◽  
Author(s):  
Hanna-Mari Baldauf ◽  
Lena Stegmann ◽  
Sarah-Marie Schwarz ◽  
Ina Ambiel ◽  
Maud Trotard ◽  
...  
Keyword(s):  
T Cells ◽  
Reverse Transcription ◽  
Cd4 T Cells ◽  
Single Amino Acid ◽  
Sooty Mangabey ◽  
Dntp Pool ◽  
Sam Domain ◽  
Virion Incorporation ◽  
Intracellular Dntp ◽  
Hiv 1

Early after entry into monocytes, macrophages, dendritic cells, and resting CD4 T cells, HIV encounters a block, limiting reverse transcription (RT) of the incoming viral RNA genome. In this context, dNTP triphosphohydrolase SAM domain and HD domain-containing protein 1 (SAMHD1) has been identified as a restriction factor, lowering the concentration of dNTP substrates to limit RT. The accessory lentiviral protein X (Vpx) proteins from the major simian immunodeficiency virus of rhesus macaque, sooty mangabey, and HIV-2 (SIVsmm/SIVmac/HIV-2) lineage packaged into virions target SAMHD1 for proteasomal degradation, increase intracellular dNTP pools, and facilitate HIV cDNA synthesis. We find that virion-packaged Vpx proteins from a second SIV lineage, SIV of red-capped mangabeys or mandrills (SIVrcm/mnd-2), increased HIV infection in resting CD4 T cells, but not in macrophages, and, unexpectedly, acted in the absence of SAMHD1 degradation, dNTP pool elevation, or changes in SAMHD1 phosphorylation. Vpx rcm/mnd-2 virion incorporation resulted in a dramatic increase of HIV-1 RT intermediates and viral cDNA in infected resting CD4 T cells. These analyses also revealed a barrier limiting HIV-1 infection of resting CD4 T cells at the level of nuclear import. Single amino acid changes in the SAMHD1-degrading Vpx mac239 allowed it to enhance early postentry steps in a Vpx rcm/mnd-2–like fashion. Moreover, Vpx enhanced HIV-1 infection of SAMHD1-deficient resting CD4 T cells of a patient with Aicardi-Goutières syndrome. These results indicate that Vpx, in addition to SAMHD1, overcomes a previously unappreciated restriction for lentiviruses at the level of RT that acts independently of dNTP concentrations and is specific to resting CD4 T cells.

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