Functional Neutralization of HIV-1 Vif Protein by Intracellular Immunization Inhibits Reverse Transcription and Viral Replication

ABSTRACT A hallmark of retroviruses such as human immunodeficiency virus type 1 (HIV-1) is reverse transcription of genomic RNA to DNA, a process that is primed by cellular tRNAs. HIV-1 recruits human tRNALys3 to serve as the reverse transcription primer via an interaction between lysyl-tRNA synthetase (LysRS) and the HIV-1 Gag polyprotein. LysRS is normally sequestered in a multi-aminoacyl-tRNA synthetase complex (MSC). Previous studies demonstrated that components of the MSC can be mobilized in response to certain cellular stimuli, but how LysRS is redirected from the MSC to viral particles for packaging is unknown. Here, we show that upon HIV-1 infection, a free pool of non-MSC-associated LysRS is observed and partially relocalized to the nucleus. Heat inactivation of HIV-1 blocks nuclear localization of LysRS, but treatment with a reverse transcriptase inhibitor does not, suggesting that the trigger for relocalization occurs prior to reverse transcription. A reduction in HIV-1 infection is observed upon treatment with an inhibitor to mitogen-activated protein kinase that prevents phosphorylation of LysRS on Ser207, release of LysRS from the MSC, and nuclear localization. A phosphomimetic mutant of LysRS (S207D) that lacked the capability to aminoacylate tRNALys3 localized to the nucleus, rescued HIV-1 infectivity, and was packaged into virions. In contrast, a phosphoablative mutant (S207A) remained cytosolic and maintained full aminoacylation activity but failed to rescue infectivity and was not packaged. These findings suggest that HIV-1 takes advantage of the dynamic nature of the MSC to redirect and coopt cellular translation factors to enhance viral replication. IMPORTANCE Human tRNALys3, the primer for reverse transcription, and LysRS are essential host factors packaged into HIV-1 virions. Previous studies found that tRNALys3 packaging depends on interactions between LysRS and HIV-1 Gag; however, many details regarding the mechanism of tRNALys3 and LysRS packaging remain unknown. LysRS is normally sequestered in a high-molecular-weight multi-aminoacyl-tRNA synthetase complex (MSC), restricting the pool of free LysRS-tRNALys. Mounting evidence suggests that LysRS is released under a variety of stimuli to perform alternative functions within the cell. Here, we show that HIV-1 infection results in a free pool of LysRS that is relocalized to the nucleus of target cells. Blocking this pathway in HIV-1-producing cells resulted in less infectious progeny virions. Understanding the mechanism by which LysRS is recruited into the viral assembly pathway can be exploited for the development of specific and effective therapeutics targeting this nontranslational function.

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A Novel Leu92 Mutant of HIV-1 Reverse Transcriptase with a Selective Deficiency in Strand Transfer Causes a Loss of Viral Replication

Journal of Virology ◽

10.1128/jvi.00809-15 ◽

2015 ◽

Vol 89 (16) ◽

pp. 8119-8129 ◽

Cited By ~ 4

Author(s):

Eytan Herzig ◽

Nickolay Voronin ◽

Nataly Kucherenko ◽

Amnon Hizi

Keyword(s):

Life Cycle ◽

Viral Replication ◽

Reverse Transcriptase ◽

Dna Polymerase ◽

Reverse Transcription ◽

Polymerase Activity ◽

Rnase H ◽

Strand Transfer ◽

Hiv 1

ABSTRACTThe process of reverse transcription (RTN) in retroviruses is essential to the viral life cycle. This key process is catalyzed exclusively by the viral reverse transcriptase (RT) that copies the viral RNA into DNA by its DNA polymerase activity, while concomitantly removing the original RNA template by its RNase H activity. During RTN, the combination between DNA synthesis and RNA hydrolysis leads to strand transfers (or template switches) that are critical for the completion of RTN. The balance between these RT-driven activities was considered to be the sole reason for strand transfers. Nevertheless, we show here that a specific mutation in HIV-1 RT (L92P) that does not affect the DNA polymerase and RNase H activities abolishes strand transfer. There is also a good correlation between this complete loss of the RT's strand transfer to the loss of the DNA clamp activity of the RT, discovered recently by us. This finding indicates a mechanistic linkage between these two functions and that they are both direct and unique functions of the RT (apart from DNA synthesis and RNA degradation). Furthermore, when the RT's L92P mutant was introduced into an infectious HIV-1 clone, it lost viral replication, due to inefficient intracellular strand transfers during RTN, thus supporting thein vitrodata. As far as we know, this is the first report on RT mutants that specifically and directly impair RT-associated strand transfers. Therefore, targeting residue Leu92 may be helpful in selectively blocking this RT activity and consequently HIV-1 infectivity and pathogenesis.IMPORTANCEReverse transcription in retroviruses is essential for the viral life cycle. This multistep process is catalyzed by viral reverse transcriptase, which copies the viral RNA into DNA by its DNA polymerase activity (while concomitantly removing the RNA template by its RNase H activity). The combination and balance between synthesis and hydrolysis lead to strand transfers that are critical for reverse transcription completion. We show here for the first time that a single mutation in HIV-1 reverse transcriptase (L92P) selectively abolishes strand transfers without affecting the enzyme's DNA polymerase and RNase H functions. When this mutation was introduced into an infectious HIV-1 clone, viral replication was lost due to an impaired intracellular strand transfer, thus supporting thein vitrodata. Therefore, finding novel drugs that target HIV-1 reverse transcriptase Leu92 may be beneficial for developing new potent and selective inhibitors of retroviral reverse transcription that will obstruct HIV-1 infectivity.

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The Human Immunodeficiency Virus Type 1 (HIV-1) Vif Protein Is Located in the Cytoplasm of Infected Cells and Its Effect on Viral Replication Is Equivalent in HIV-2

AIDS Research and Human Retroviruses ◽

10.1089/aid.1993.9.1025 ◽

1993 ◽

Vol 9 (10) ◽

pp. 1025-1030 ◽

Cited By ~ 22

Author(s):

FRANK H. MICHAELS ◽

NAOHIKO HATTORI ◽

ROBERT C. GALLO ◽

GENOVEFFA FRANCHINI

Keyword(s):

Human Immunodeficiency Virus ◽

Viral Replication ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Immunodeficiency Virus ◽

Infected Cells ◽

Vif Protein ◽

Hiv 1

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Incomplete Suppression of HIV-1 by SAMHD1 Permits Efficient Macrophage Infection

Pathogens and Immunity ◽

10.20411/pai.v3i2.263 ◽

2018 ◽

Vol 3 (2) ◽

pp. 197 ◽

Cited By ~ 1

Author(s):

Timothy Plitnik ◽

Mark E. Sharkey ◽

Bijan Mahboubi ◽

Baek Kim ◽

Mario Stevenson

Keyword(s):

Viral Replication ◽

Reverse Transcriptase ◽

Aspartic Acid ◽

Reverse Transcription ◽

Differential Sensitivity ◽

Macrophage Infection ◽

Dividing Cells ◽

The Face ◽

Acid Domain ◽

Hiv 1

Background: Sterile alpha motif and histidine/aspartic acid domain-containing protein (SAMHD1) is a dNTP triphosphorylase that reduces cellular dNTP levels in non-dividing cells, such as macrophages. Since dNTPs are required for reverse transcription, HIV-2 and most SIVs encode a Vpx protein that promotes proteasomal degradation of SAMHD1. It is unclear how HIV-1, which does not appear to harbor a SAMHD1 escape mechanism, is able to infect macrophages in the face of SAMHD1 restriction.Methods: To assess whether HIV-1 had a mechanism to negate SAMHD1 activity, we compared SAMHD1 and dNTP levels in macrophages infected by HIV-1 and SIV. We examined whether macrophages infected by HIV-1 still harbored antiviral levels of SAMHD1 by assessing their susceptibility to superinfection by vpx-deleted SIV. Finally, to assess whether HIV-1 reverse transcriptase (RT) has adapted to a low dNTP environment, we evaluated SAMHD1 sensitivity of chimeric HIV-1 and SIV variants in which the RT regions were functionally exchanged.Results: Here, we demonstrate that HIV-1 efficiently infects macrophages without modulating SAMHD1 activity or cellular dNTP levels, and that macrophages permissive to HIV-1 infection remained refractory to superinfection by vpx-deleted SIV. Furthermore, through the use of chimeric HIV/SIV, we demonstrate that the differential sensitivity of HIV-1 and SIV to SAMHD1 restriction is not dictated by RT.Conclusions: Our study reveals fundamental differences between HIV-1 and SIV in the strategy used to evade restriction by SAMHD1 and suggests a degree of resistance of HIV-1 to the antiviral environment created by SAMHD1. Understanding how these cellular restrictions antagonize viral replication will be important for the design of novel antiviral strategies.Keywords: HIV-1/ macrophages/ SAMHD1

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The Role of APOBECs in Viral Replication

Microorganisms ◽

10.3390/microorganisms8121899 ◽

2020 ◽

Vol 8 (12) ◽

pp. 1899

Author(s):

Wendy Kaichun Xu ◽

Hyewon Byun ◽

Jaquelin P. Dudley

Keyword(s):

Viral Replication ◽

Mammalian Species ◽

Specific Gene ◽

Viral Inhibition ◽

Cytidine Deaminases ◽

Catalytically Active ◽

Antiviral Function ◽

Selection For ◽

Vif Protein ◽

Hiv 1

Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) proteins are a diverse and evolutionarily conserved family of cytidine deaminases that provide a variety of functions from tissue-specific gene expression and immunoglobulin diversity to control of viruses and retrotransposons. APOBEC family expansion has been documented among mammalian species, suggesting a powerful selection for their activity. Enzymes with a duplicated zinc-binding domain often have catalytically active and inactive domains, yet both have antiviral function. Although APOBEC antiviral function was discovered through hypermutation of HIV-1 genomes lacking an active Vif protein, much evidence indicates that APOBECs also inhibit virus replication through mechanisms other than mutagenesis. Multiple steps of the viral replication cycle may be affected, although nucleic acid replication is a primary target. Packaging of APOBECs into virions was first noted with HIV-1, yet is not a prerequisite for viral inhibition. APOBEC antagonism may occur in viral producer and recipient cells. Signatures of APOBEC activity include G-to-A and C-to-T mutations in a particular sequence context. The importance of APOBEC activity for viral inhibition is reflected in the identification of numerous viral factors, including HIV-1 Vif, which are dedicated to antagonism of these deaminases. Such viral antagonists often are only partially successful, leading to APOBEC selection for viral variants that enhance replication or avoid immune elimination.

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Subtype-associated differences in HIV-1 reverse transcription affect the viral replication

Retrovirology ◽

10.1186/1742-4690-7-85 ◽

2010 ◽

Vol 7 (1) ◽

Cited By ~ 17

Author(s):

Sergey Iordanskiy ◽

Mackenzie Waltke ◽

Yanjun Feng ◽

Charles Wood

Keyword(s):

Viral Replication ◽

Reverse Transcription ◽

Hiv 1

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Detailed Characterization of Early HIV-1 Replication Dynamics in Primary Human Macrophages

Viruses ◽

10.3390/v10110620 ◽

2018 ◽

Vol 10 (11) ◽

pp. 620 ◽

Cited By ~ 13

Author(s):

David Bejarano ◽

Maria Puertas ◽

Kathleen Börner ◽

Javier Martinez-Picado ◽

Barbara Müller ◽

...

Keyword(s):

Viral Replication ◽

Reverse Transcription ◽

Human Monocyte ◽

Digital Pcr ◽

Productive Infection ◽

Target Cells ◽

Post Entry ◽

Early Replication ◽

Kinetics Of ◽

Hiv 1

Macrophages are natural target cells of human immunodeficiency virus type 1 (HIV-1). Viral replication appears to be delayed in these cells compared to lymphocytes; however, little is known about the kinetics of early post-entry events. Time-of-addition experiments using several HIV-1 inhibitors and the detection of reverse transcriptase (RT) products with droplet digital PCR (ddPCR) revealed that early replication was delayed in primary human monocyte-derived macrophages of several donors and peaked late after infection. Direct imaging of reverse-transcription and pre-integration complexes (RTC/PIC) by click-labeling of newly synthesized DNA further confirmed our findings and showed a concomitant shift to the nuclear stage over time. Altering the entry pathway enhanced infectivity but did not affect kinetics of viral replication. The addition of viral protein X (Vpx) enhanced productive infection and accelerated completion of reverse transcription and nuclear entry. We propose that sterile alpha motif (SAM) and histidine/aspartate (HD) domain-containing protein 1 (SAMHD1) activity lowering deoxyribonucleotide triphosphate (dNTP) pools is the principal factor delaying early HIV-1 replication in macrophages.

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Stage-Dependent Inhibition of HIV-1 Replication by Antiretroviral Drugs in Cell Culture

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01537-09 ◽

2009 ◽

Vol 54 (3) ◽

pp. 1047-1054 ◽

Cited By ~ 35

Author(s):

Daniel A. Donahue ◽

Richard D. Sloan ◽

Björn D. Kuhl ◽

Tamara Bar-Magen ◽

Susan M. Schader ◽

...

Keyword(s):

Viral Load ◽

Viral Replication ◽

Reverse Transcription ◽

Antiretroviral Drugs ◽

Proteolytic Processing ◽

Viral Inhibition ◽

Dependent Inhibition ◽

Infected Cells ◽

Hiv 1

ABSTRACT Recent clinical trials have shown that the use of the HIV-1 integrase (IN) inhibitor raltegravir (RAL) results in drops in the viral load that are more rapid than those achieved by use of the reverse transcriptase (RT) inhibitor efavirenz. Previously, mathematical modeling of viral load decay that takes into account the stage of viral replication targeted by a drug has yielded data that closely approximate the clinical trial results. This model predicts greater inhibition of viral replication by drugs that act later in the viral replication cycle. In the present study, we have added drugs that target entry, reverse transcription, integration, or proteolytic processing to acutely infected cells and have shown modest viral inhibition by entry inhibitors, intermediate levels of inhibition by RT and IN inhibitors, and high levels of inhibition by protease inhibitors relative to the levels of growth for the no-drug controls. When dual or triple combinations of these drugs were added to acutely infected cells, we found that the levels of inhibition achieved by any given combination were comparable to those achieved by the latest-acting drug in the combination. In single-round infections in which the kinetics of reverse transcription and integration had been determined by quantitative PCR, addition of IN inhibitors at various times postinfection resulted in levels of inhibition equal to or greater than those achieved by addition of RT inhibitors. Collectively, our data provide in vitro evidence of the stage-dependent inhibition of HIV-1 by clinically relevant drugs. We discuss how stage-dependent inhibition helps to explain the unique viral load decay dynamics observed clinically with RAL.

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Elimination of Retroviral Infectivity by N-Ethylmaleimide with Preservation of Functional Envelope Glycoproteins

Journal of Virology ◽

10.1128/jvi.79.3.1533-1542.2005 ◽

2005 ◽

Vol 79 (3) ◽

pp. 1533-1542 ◽

Cited By ~ 36

Author(s):

David R. Morcock ◽

James A. Thomas ◽

Tracy D. Gagliardi ◽

Robert J. Gorelick ◽

J. David Roser ◽

...

Keyword(s):

Viral Replication ◽

Zinc Finger ◽

Reverse Transcription ◽

Covalent Modification ◽

Viral Envelope ◽

Envelope Glycoproteins ◽

Cell Capture ◽

Retroviral Infection ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT The zinc finger motifs in retroviral nucleocapsid (NC) proteins are essential for viral replication. Disruption of these Cys-X2-Cys-X4-His-X4-Cys zinc-binding structures eliminates infectivity. To determine if N-ethylmaleimide (NEM) can inactivate human immunodeficiency virus type 1 (HIV-1) or simian immunodeficiency virus (SIV) preparations by alkylating cysteines of NC zinc fingers, we treated infectious virus with NEM and evaluated inactivation of infectivity in cell-based assays. Inactivation was rapid and proportional to the NEM concentration. NEM treatment of HIV-1 or SIV resulted in extensive covalent modification of NC and other internal virion proteins. In contrast, viral envelope glycoproteins, in which the cysteines are disulfide bonded, remained intact and functional, as assayed by high-performance liquid chromatography, fusion-from-without analyses, and dendritic cell capture. Quantitative PCR assays for reverse transcription intermediates showed that NEM and 2,2′-dipyridyl disulfide (aldrithiol-2), a reagent which inactivates retroviruses through oxidation of cysteines in internal virion proteins such as NC, blocked HIV-1 reverse transcription prior to the formation of minus-strand strong-stop products. However, the reverse transcriptase from NEM-treated virions remained active in exogenous template assays, consistent with a role for NC in reverse transcription. Since disruption of NC zinc finger structures by NEM blocks early postentry steps in the retroviral infection cycle, virus preparations with modified NC proteins may be useful as vaccine immunogens and probes of the role of NC in viral replication.

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HIV-1 Accessory Protein Vpr Interacts with REAF/RPRD2 To Mitigate Its Antiviral Activity

Journal of Virology ◽

10.1128/jvi.01591-19 ◽

2019 ◽

Vol 94 (4) ◽

Cited By ~ 1

Author(s):

Joseph M. Gibbons ◽

Kelly M. Marno ◽

Rebecca Pike ◽

Wing-yiu Jason Lee ◽

Christopher E. Jones ◽

...

Keyword(s):

T Cells ◽

Viral Replication ◽

Nuclear Localization ◽

Reverse Transcription ◽

Early Phase ◽

Early Stage ◽

Target Cells ◽

Accessory Protein ◽

Hiv 1

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) accessory protein Vpr enhances viral replication in both macrophages and, to a lesser extent, cycling T cells. Virion-packaged Vpr is released in target cells shortly after entry, suggesting it is required in the early phase of infection. Previously, we described REAF (RNA-associated early-stage antiviral factor; RPRD2), a constitutively expressed protein that potently restricts HIV replication at or during reverse transcription. Here, we show that a virus without an intact vpr gene is more highly restricted by REAF and, using delivery by virus-like particles (VLPs), that Vpr alone is sufficient for REAF degradation in primary macrophages. REAF is more highly expressed in macrophages than in cycling T cells, and we detected, by coimmunoprecipitation assay, an interaction between Vpr protein and endogenous REAF. Vpr acts quickly during the early phase of replication and induces the degradation of REAF within 30 min of viral entry. Using Vpr F34I and Q65R viral mutants, we show that nuclear localization and interaction with cullin 4A-DBB1 (DCAF1) E3 ubiquitin ligase are required for REAF degradation by Vpr. In response to infection, cells upregulate REAF levels. This response is curtailed in the presence of Vpr. These findings support the hypothesis that Vpr induces the degradation of a factor, REAF, that impedes HIV infection in macrophages. IMPORTANCE For at least 30 years, it has been known that HIV-1 Vpr, a protein carried in the virion, is important for efficient infection of primary macrophages. Vpr is also a determinant of the pathogenic effects of HIV-1 in vivo. A number of cellular proteins that interact with Vpr have been identified. So far, it has not been possible to associate these proteins with altered viral replication in macrophages or to explain why Vpr is carried in the virus particle. Here, we show that Vpr mitigates the antiviral effects of REAF, a protein highly expressed in primary macrophages and one that inhibits virus replication during reverse transcription. REAF is degraded by Vpr within 30 min of virus entry in a manner dependent on the nuclear localization of Vpr and its interaction with the cell’s protein degradation machinery.

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