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 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 HIV-1 Reverse Transcriptase A62V Mutation Influences Replication Fidelity and Viral Fitness in the Context of Multi-Drug-Resistant Mutations

Viruses ◽  
2018 ◽  
Vol 10 (7) ◽  
pp. 376 ◽  
Author(s):  
José Maldonado ◽  
Louis Mansky
Keyword(s):  
Drug Resistance ◽  
Amino Acid ◽  
Reverse Transcriptase ◽  
Viral Fitness ◽  
Multi Drug Resistance ◽  
Single Cycle ◽  
Replication Fidelity ◽  
Viral Mutant ◽  
Hiv 1 ◽  
Mutant Frequency

Emergence of human immunodeficiency virus type 1 (HIV-1) drug resistance arises from mutation fixation in the viral genome during antiretroviral therapy. Primary mutations directly confer antiviral drug resistance, while secondary mutations arise that do not confer drug resistance. The A62V amino acid substitution in HIV-1 reverse transcriptase (RT) was observed to be associated with multi-drug resistance, but is not known to be a resistance-conferring mutation. In particular, A62V was observed in various multi-dideoxynucleoside resistant (MDR) mutation complexes, including the Q151M complex (i.e., A62V, V75I, F77L, F116Y, and Q151M), and the T69SSS insertion complex, which has a serine–serine insertion between amino acid positions 69 and 70 (i.e., M41L, A62V, T69SSS, K70R, and T215Y). However, what selective advantage is conferred to the virus remains unresolved. In this study, we hypothesized that A62V could influence replication fidelity and viral fitness with viruses harboring the Q151M and T69SSS MDR mutation complexes. A single-cycle replication assay and a dual-competition fitness assay were used to assess viral mutant frequency and viral fitness, respectively. A62V was found to increase the observed lower mutant frequency identified with each of the viruses harboring the MDR mutation complexes in the single-cycle assay. Furthermore, A62V was observed to improve viral fitness of replication-competent MDR viruses. Taken together, these observations indicate an adaptive role of A62V in virus replication fidelity and viral fitness, which would likely enhance virus persistence during drug-selective pressure.

scholarly journals Modulation of HIV-1 infectivity and cyclophilin A-dependence by Gag sequence and target cell type

Retrovirology ◽  
2009 ◽  
Vol 6 (1) ◽  
pp. 21 ◽  
Author(s):  
Saori Matsuoka ◽  
Elisabeth Dam ◽  
Denise Lecossier ◽  
François Clavel ◽  
Allan J Hance
Keyword(s):  
Target Cell ◽  
Cyclophilin A ◽  
Cell Type ◽  
Hiv 1

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.

scholarly journals Evolutionary Genetics of Mycobacterium tuberculosis and HIV-1: “The Tortoise and the Hare”

2021 ◽  
Vol 9 (1) ◽  
pp. 147
Author(s):  
Ana Santos-Pereira ◽  
Carlos Magalhães ◽  
Pedro M. M. Araújo ◽  
Nuno S. Osório
Keyword(s):  
Genetic Diversity ◽  
Drug Resistance ◽  
Mycobacterium Tuberculosis ◽  
Evolutionary Dynamics ◽  
Evolutionary Genetics ◽  
Host Cells ◽  
Whole Genome Sequencing Data ◽  
Host Immune System ◽  
Viral Mutant ◽  
Hiv 1

The already enormous burden caused by Mycobacterium tuberculosis and Human Immunodeficiency Virus type 1 (HIV-1) alone is aggravated by co-infection. Despite obvious differences in the rate of evolution comparing these two human pathogens, genetic diversity plays an important role in the success of both. The extreme evolutionary dynamics of HIV-1 is in the basis of a robust capacity to evade immune responses, to generate drug-resistance and to diversify the population-level reservoir of M group viral subtypes. Compared to HIV-1 and other retroviruses, M. tuberculosis generates minute levels of genetic diversity within the host. However, emerging whole-genome sequencing data show that the M. tuberculosis complex contains at least nine human-adapted phylogenetic lineages. This level of genetic diversity results in differences in M. tuberculosis interactions with the host immune system, virulence and drug resistance propensity. In co-infected individuals, HIV-1 and M. tuberculosis are likely to co-colonize host cells. However, the evolutionary impact of the interaction between the host, the slowly evolving M. tuberculosis bacteria and the HIV-1 viral “mutant cloud” is poorly understood. These evolutionary dynamics, at the cellular niche of monocytes/macrophages, are also discussed and proposed as a relevant future research topic in the context of single-cell sequencing.

scholarly journals Novel Cell type-specific aptamer-siRNA delivery system for HIV-1 therapy

2007 ◽  
Author(s):  
Jiehua Zhou ◽  
Haitang Li ◽  
Shirley Li ◽  
John Zaia ◽  
John Rossi
Keyword(s):  
Delivery System ◽  
Sirna Delivery ◽  
Cell Type ◽  
Cell Type Specific ◽  
Hiv 1 ◽  
Sirna Delivery System

scholarly journals Retinal cell type expression specificity of HIV-1-derived gene transfer vectors upon subretinal injection in the adult rat: influence of pseudotyping and promoter

2005 ◽  
Vol 7 (10) ◽  
pp. 1367-1374 ◽  
Author(s):  
Alexis-Pierre Bemelmans ◽  
Sébastien Bonnel ◽  
Leïla Houhou ◽  
Noëlle Dufour ◽  
Emeline Nandrot ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Retinal Cell ◽  
Cell Type ◽  
Adult Rat ◽  
Transfer Vectors ◽  
Hiv 1 ◽  
Subretinal Injection

scholarly journals Tissue-Specific Transcriptional Targeting of a Replication-Competent Retroviral Vector

2002 ◽  
Vol 76 (24) ◽  
pp. 12783-12791 ◽  
Author(s):  
Christopher R. Logg ◽  
Aki Logg ◽  
Robert J. Matusik ◽  
Bernard H. Bochner ◽  
Noriyuki Kasahara
Keyword(s):  
Tumor Cells ◽  
Viral Vectors ◽  
High Efficiency ◽  
Leukemia Virus ◽  
Transduction Efficiency ◽  
Promoter Strength ◽  
Cell Type ◽  
Cell Type Specificity ◽  
Tissue Specific

ABSTRACT The inability of replication-defective viral vectors to efficiently transduce tumor cells in vivo has prevented the successful application of such vectors in gene therapy of cancer. To address the need for more efficient gene delivery systems, we have developed replication-competent retroviral (RCR) vectors based on murine leukemia virus (MLV). We have previously shown that such vectors are capable of transducing solid tumors in vivo with very high efficiency. While the natural requirement of MLV infection for cell division imparts a certain degree of specificity for tumor cells, additional means for confining RCR vector replication to tumor cells are desirable. Here, we investigated the parameters critical for successful tissue-specific transcriptional control of RCR vector replication by replacing various lengths of the MLV enhancer/promoter with sequences derived either from the highly prostate-specific probasin (PB) promoter or from a more potent synthetic variant of the PB promoter. We assessed the transcriptional specificity of the resulting hybrid long terminal repeats (LTRs) and the cell type specificity and efficiency of replication of vectors containing these LTRs. Incorporation of PB promoter sequences effectively restricted transcription from the LTR to prostate-derived cells and imparted prostate-specific RCR vector replication but required the stronger synthetic promoter and retention of native MLV sequences in the vicinity of the TATA box for optimal replicative efficiency and specificity. Our results have thus identified promoter strength and positioning within the LTR as important determinants for achieving both high transduction efficiency and strict cell type specificity in transcriptionally targeted RCR vectors.

scholarly journals Nature, Position, and Frequency of Mutations Made in a Single Cycle of HIV-1 Replication

2010 ◽  
Vol 84 (19) ◽  
pp. 9864-9878 ◽  
Author(s):  
Michael E. Abram ◽  
Andrea L. Ferris ◽  
Wei Shao ◽  
W. Gregory Alvord ◽  
Stephen H. Hughes
Keyword(s):  
Viral Replication ◽  
Mutation Rate ◽  
Hot Spots ◽  
High Rate ◽  
Published Data ◽  
Single Cycle ◽  
Efficient System ◽  
Hiv 1

ABSTRACT There is considerable HIV-1 variation in patients. The extent of the variation is due to the high rate of viral replication, the high viral load, and the errors made during viral replication. Mutations can arise from errors made either by host DNA-dependent RNA polymerase II or by HIV-1 reverse transcriptase (RT), but the relative contributions of these two enzymes to the mutation rate are unknown. In addition, mutations in RT can affect its fidelity, but the effect of mutations in RT on the nature of the mutations that arise in vivo is poorly understood. We have developed an efficient system, based on existing technology, to analyze the mutations that arise in an HIV-1 vector in a single cycle of replication. A lacZα reporter gene is used to identify viral DNAs that contain mutations which are analyzed by DNA sequencing. The forward mutation rate in this system is 1.4 × 10−5 mutations/bp/cycle, equivalent to the retroviral average. This rate is about 3-fold lower than previously reported for HIV-1 in vivo and is much lower than what has been reported for purified HIV-1 RT in vitro. Although the mutation rate was not affected by the orientation of lacZα, the sites favored for mutations (hot spots) in lacZα depended on which strand of lacZα was present in the viral RNA. The pattern of hot spots seen in lacZα in vivo did not match any of the published data obtained when purified RT was used to copy lacZα in vitro.

scholarly journals Functional Mapping of Regions Involved in the Negative Imprinting of Virion Particle Infectivity and in Target Cell Protection by Interferon-Induced Transmembrane Protein 3 against HIV-1

2018 ◽  
Vol 93 (2) ◽  
Author(s):  
Romain Appourchaux ◽  
Mathilde Delpeuch ◽  
Li Zhong ◽  
Julien Burlaud-Gaillard ◽  
Kevin Tartour ◽  
...  
Keyword(s):  
Target Cell ◽  
Transmembrane Protein ◽  
Regulatory Role ◽  
Target Cells ◽  
Restriction Factors ◽  
Cell Protection ◽  
Differential Behavior ◽  
Antiviral Activities ◽  
Divergent Behavior ◽  
Hiv 1

ABSTRACT The interferon-induced transmembrane proteins (IFITMs) are a family of highly related antiviral factors that affect numerous viruses at two steps: in target cells by sequestering incoming viruses in endosomes and in producing cells by leading to the production of virions that package IFITMs and exhibit decreased infectivity. While most studies have focused on the former, little is known about the regulation of the negative imprinting of virion particle infectivity by IFITMs and about its relationship with target cell protection. Using a panel of IFITM3 mutants against HIV-1, we have explored these issues as well as others related to the biology of IFITM3, in particular virion packaging, stability, the relation to CD63/multivesicular bodies (MVBs), the modulation of cholesterol levels, and the relationship between negative imprinting of virions and target cell protection. The results that we have obtained exclude a role for cholesterol and indicate that CD63 accumulation does not directly relate to an antiviral behavior. We have defined regions that modulate the two antiviral properties of IFITM3 as well as novel domains that modulate protein stability and that, in so doing, influence the extent of its packaging into virions. The results that we have obtained, however, indicate that, even in the context of an IFITM-susceptible virus, IFITM3 packaging is not sufficient for negative imprinting. Finally, while most mutations concomitantly affect target cell protection and negative imprinting, a region in the C-terminal domain (CTD) exhibits a differential behavior, potentially highlighting the regulatory role that this domain may play in the two antiviral activities of IFITM3. IMPORTANCE IFITM proteins have been associated with the sequestration of incoming virions in endosomes (target cell protection) and with the production of virion particles that incorporate IFITMs and exhibit decreased infectivity (negative imprinting of virion infectivity). How the latter is regulated and whether these two antiviral properties are related remain unknown. By examining the behavior of a large panel of IFITM3 mutants against HIV-1, we determined that IFITM3 mutants are essentially packaged into virions proportionally to their intracellular levels of expression. However, even in the context of an IFITM-susceptible virus, IFITM3 packaging is not sufficient for the antiviral effects. Most mutations were found to concomitantly affect both antiviral properties of IFITM3, but one CTD mutant exhibited a divergent behavior, possibly highlighting a novel regulatory role for this domain. These findings thus advance our comprehension of how this class of broad antiviral restriction factors acts.

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