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

2019 ◽  
Vol 94 (4) ◽  
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.

scholarly journals HIV-1 accessory protein Vpr interacts with REAF/RPRD2 to mitigate its antiviral activity

2018 ◽  
Author(s):  
Joseph M Gibbons ◽  
Kelly M Marno ◽  
Rebecca Pike ◽  
Wing-yiu Jason Lee ◽  
Christopher E Jones ◽  
...  
Keyword(s):  
T Cells ◽  
Viral Replication ◽  
Viral Entry ◽  
Reverse Transcription ◽  
Early Phase ◽  
Early Stage ◽  
Target Cells ◽  
Accessory Protein ◽  
Hiv 1

AbstractThe Human Immunodeficiency Virus type 1 (HIV-1) accessory protein Vpr enhances viral replication in both macrophages and in cycling T cells to a lesser extent. Virion packaged Vpr is released in target cells shortly after entry, suggesting its requirement 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 intactvpris more highly restricted by REAF and, using delivery by 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 detect, by co-immunoprecipitation assay, an interaction between Vpr protein and endogenous REAF. Vpr acts very quickly during the early phase of replication and induces the degradation of REAF within 30 minutes of viral entry. Using Vpr F34I and Q65R viral mutants, we show that nuclear localisation and interaction with cullin4A-DBB1 (DCAF1) E3 ubiquitin ligase is 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, which impedes HIV infection in macrophages.ImportanceFor 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-1in 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 which inhibits virus replication early during reverse transcription. REAF is degraded by Vpr within 30 minutes of virus entry, in a manner dependent on the nuclear localization of Vpr and its interaction with the cell’s protein degradation machinery.

scholarly journals HIV-1 Exploits a Dynamic Multi-aminoacyl-tRNA Synthetase Complex To Enhance Viral Replication

2017 ◽  
Vol 91 (21) ◽  
Author(s):  
Alice A. Duchon ◽  
Corine St. Gelais ◽  
Nathan Titkemeier ◽  
Joshua Hatterschide ◽  
Li Wu ◽  
...  
Keyword(s):  
Viral Replication ◽  
Nuclear Localization ◽  
Reverse Transcription ◽  
Transcriptase Inhibitor ◽  
Trna Synthetase ◽  
Mitogen Activated Protein Kinase ◽  
Heat Inactivation ◽  
Target Cells ◽  
Aminoacyl Trna Synthetase ◽  
Hiv 1

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.

scholarly journals Modulation of the CCR5 Receptor/Ligand Axis by Seminal Plasma and the Utility of In Vitro versus In Vivo Models

2019 ◽  
Vol 93 (11) ◽  
Author(s):  
Jennifer A. Juno ◽  
Kathleen M. Wragg ◽  
Anne B. Kristensen ◽  
Wen Shi Lee ◽  
Kevin J. Selva ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Seminal Plasma ◽  
Target Cells ◽  
Ccr5 Expression ◽  
Ccr5 Receptor ◽  
The Impact ◽  
Hiv 1

ABSTRACT Sexual HIV-1 transmission occurs primarily in the presence of semen. Although data from macaque studies suggest that CCR5+ CD4+ T cells are initial targets for HIV-1 infection, the impact of semen on T cell CCR5 expression and ligand production remains inconclusive. To determine if semen modulates the lymphocyte CCR5 receptor/ligand axis, primary human T cell CCR5 expression and natural killer (NK) cell anti-HIV-1 antibody-dependent beta chemokine production was assessed following seminal plasma (SP) exposure. Purified T cells produce sufficient quantities of RANTES to result in a significant decline in CCR5bright T cell frequency following 16 h of SP exposure (P = 0.03). Meanwhile, NK cells retain the capacity to produce limited amounts of MIP-1α/MIP-1β in response to anti-HIV-1 antibody-dependent stimulation (median, 9.5% MIP-1α+ and/or MIP-1β+), despite the immunosuppressive nature of SP. Although these in vitro experiments suggest that SP-induced CCR5 ligand production results in the loss of surface CCR5 expression on CD4+ T cells, the in vivo implications are unclear. We therefore vaginally exposed five pigtail macaques to SP and found that such exposure resulted in an increase in CCR5+ HIV-1 target cells in three of the animals. The in vivo data support a growing body of evidence suggesting that semen exposure recruits target cells to the vagina that are highly susceptible to HIV-1 infection, which has important implications for HIV-1 transmission and vaccine design. IMPORTANCE The majority of HIV-1 vaccine studies do not take into consideration the impact that semen exposure might have on the mucosal immune system. In this study, we demonstrate that seminal plasma (SP) exposure can alter CCR5 expression on T cells. Importantly, in vitro studies of T cells in culture cannot replicate the conditions under which immune cells might be recruited to the genital mucosa in vivo, leading to potentially erroneous conclusions about the impact of semen on mucosal HIV-1 susceptibility.

scholarly journals HIV-1 Vpr Accelerates Viral Replication during Acute Infection by Exploitation of Proliferating CD4+ T Cells In Vivo

2013 ◽  
Vol 9 (12) ◽  
pp. e1003812 ◽  
Author(s):  
Kei Sato ◽  
Naoko Misawa ◽  
Shingo Iwami ◽  
Yorifumi Satou ◽  
Masao Matsuoka ◽  
...  
Keyword(s):  
T Cells ◽  
Viral Replication ◽  
Cd4 T Cells ◽  
Acute Infection ◽  
Hiv 1

scholarly journals Migration ofAntigen-Specific T Cells Away from CXCR4-Binding Human ImmunodeficiencyVirus Type 1gp120

2004 ◽  
Vol 78 (10) ◽  
pp. 5184-5193 ◽  
Author(s):  
Diana M. Brainard ◽  
William G. Tharp ◽  
Elva Granado ◽  
Nicholas Miller ◽  
Alicja K. Trocha ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Cell ◽  
Active Movement ◽  
Target Cells ◽  
Cell Mediated Immunity ◽  
Cell Trafficking ◽  
Hiv 1

ABSTRACT Cell-mediated immunity depends in part on appropriate migration and localization of cytotoxic T lymphocytes (CTL), a process regulated by chemokines and adhesion molecules. Many viruses, including human immunodeficiency virus type 1 (HIV-1), encode chemotactically active proteins, suggesting that dysregulation of immune cell trafficking may be a strategy for immune evasion. HIV-1 gp120, a retroviral envelope protein, has been shown to act as a T-cell chemoattractant via binding to the chemokine receptor and HIV-1 coreceptor CXCR4. We have previously shown that T cells move away from the chemokine stromal cell-derived factor 1 (SDF-1) in a concentration-dependent and CXCR4 receptor-mediated manner. Here, we demonstrate that CXCR4-binding HIV-1 X4 gp120 causes the movement of T cells, including HIV-specific CTL, away from high concentrations of the viral protein. This migratory response is CD4 independent and inhibited by anti-CXCR4 antibodies and pertussis toxin. Additionally, the expression of X4 gp120 by target cells reduces CTL efficacy in an in vitro system designed to account for the effect of cell migration on the ability of CTL to kill their target cells. Recombinant X4 gp120 also significantly reduced antigen-specific T-cell infiltration at a site of antigen challenge in vivo. The repellant activity of HIV-1 gp120 on immune cells in vitro and in vivo was shown to be dependent on the V2 and V3 loops of HIV-1 gp120. These data suggest that the active movement of T cells away from CXCR4-binding HIV-1 gp120, which we previously termed fugetaxis, may provide a novel mechanism by which HIV-1 evades challenge by immune effector cells in vivo.

scholarly journals Nef enhances HIV-1 replication and infectivity independently of SERINC3 and SERINC5 in CEM T cells

2020 ◽  
Author(s):  
Peter W. Ramirez ◽  
Aaron A. Angerstein ◽  
Marissa Suarez ◽  
Thomas Vollbrecht ◽  
Jared Wallace ◽  
...  
Keyword(s):  
Growth Rate ◽  
T Cells ◽  
T Cell ◽  
Viral Replication ◽  
Cell Line ◽  
Host Protein ◽  
Target Cells ◽  
Viral Fusion ◽  
Viral Growth ◽  
Hiv 1

AbstractThe lentiviral nef gene encodes several discrete activities aimed at co-opting or antagonizing cellular proteins and pathways to defeat host defenses and maintain persistent infection. Primary functions of Nef include downregulation of CD4 and MHC class-I from the cell surface, disruption or mimicry of T-cell receptor signaling, and enhancement of viral infectivity by counteraction of the host antiretroviral proteins SERINC3 and SERINC5. In the absence of Nef, SERINC3 and SERINC5 incorporate into virions and inhibit viral fusion with target cells, decreasing infectivity. However, whether Nef’s counteraction of SERINC3 and SERINC5 is the cause of its positive influence on viral growth-rate in CD4-positive T cells is unclear. Here, we utilized CRISPR/Cas9 to knockout SERINC3 and SERINC5 in a leukemic CD4-positive T cell line (CEM) that displays robust nef-related infectivity and growth-rate phenotypes. As previously reported, viral replication was severely attenuated in CEM cells infected with HIV-1 lacking Nef (HIV-1ΔNef). This attenuated growth-rate phenotype was observed regardless of whether or not the coding regions of the serinc3 and serinc5 genes were intact. Moreover, knockout of serinc3 and serinc5 failed to restore the infectivity of HIV-1ΔNef virions produced from infected CEM cells in single-cycle replication experiments using CD4-positive HeLa cells as targets. Taken together, our results corroborate a recent study using another T-lymphoid cell line (MOLT-3) and suggest that Nef modulates a still unidentified host protein(s) to enhance viral growth rate and infectivity in CD4-positive T cells.ImportanceHIV-1 Nef is a major pathogenicity factor in vivo. A well-described activity of Nef is the enhancement of virion-infectivity and viral propagation in vitro. The infectivity-effect has been attributed to Nef’s ability to prevent the cellular, antiretroviral proteins SERINC3 and SERINC5 from incorporating into viral particles. While the activity of the SERINCs as inhibitors of retroviral infectivity has been well-documented, the role these proteins play in controlling HIV-1 replication is less clear. We report here that genetic disruption of SERINC3 and SERINC5 rescues neither viral replication-rate nor the infectivity of cell-free virions produced from CD4-positive T cells of the CEM lymphoblastoid line infected with viruses lacking Nef. This indicates that failure to modulate SERINC3 and SERINC5 is not the cause of the virologic attenuation of nef-negative HIV-1 observed using this system.

Cholecalciferol modulates the phenotype of differentiated monocyte-derived dendritic cells without altering HIV-1 transfer to CD4+ T cells

2019 ◽  
Vol 40 (1) ◽  
Author(s):  
Sandra M. Gonzalez ◽  
Wbeimar Aguilar-Jimenez ◽  
Natalia Alvarez ◽  
Maria T. Rugeles
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
Systemic Infection ◽  
Target Cells ◽  
Viral Particles ◽  
Hiv 1 ◽  
Lps Treatment ◽  
In Vitro Treatment

Abstract Background Dendritic cells (DCs) play a crucial role during HIV-1 transmission due to their ability to transfer virions to susceptible CD4+ T cells, particularly in the lymph nodes during antigen presentation which favors the establishment of systemic infection. As mature dendritic cells (mDCs) exhibit a greater ability to transfer virions, compared to immature DCs (iDCs), maintenance of an iDC phenotype could decrease viral transmission. The immunomodulatory vitamin D (VitD) has been shown to reduce activation and maturation of DCs; hence, we hypothesized that it would reduce viral transference by DCs. Materials and methods We evaluated the effect of in vitro treatment with a precursor of VitD, cholecalciferol, on the activation/maturation phenotype of differentiated monocyte-derived DCs and their ability to transfer HIV-1 to autologous CD4+ T cells. Results Our findings show that although cholecalciferol decreases the activation of iDCs, it did not impact the maturation phenotype after LPS treatment nor iDCs’ ability to transfer viral particles to target cells. Conclusion These findings suggest that despite cholecalciferol potentially modulates the phenotype of mucosal iDCs in vivo, such modulation might not impact the ability of these cells to transfer HIV-1 to target CD4+ T cells.

scholarly journals A Novel Motif in HIV-1 Nef That Regulates MIP-1β Chemokine Release in Macrophages

2010 ◽  
Vol 84 (16) ◽  
pp. 8327-8331 ◽  
Author(s):  
Lue Dai ◽  
Mario Stevenson
Keyword(s):  
Major Histocompatibility Complex ◽  
Viral Replication ◽  
Accessory Protein ◽  
Major Histocompatibility ◽  
Histocompatibility Complex ◽  
Virus Dissemination ◽  
Primate Lentiviruses ◽  
Hiv 1

ABSTRACT All primate lentiviruses encode Nef, an accessory protein that is important for viral pathogenicity in vivo. Lentiviral Nef proteins regulate the release of chemokines (MIP-1 α/β) from infected macrophages, thereby enhancing virus dissemination (S. Swingler, A. Mann, J. Jacque, B. Brichacek, V. G. Sasseville, K. Williams, A. A. Lackner, E. N. Janoff, R. Wang, D. Fisher, and M. Stevenson, Nat. Med. 5:997-1003, 1999). In the current study, we have identified a novel domain within Nef (K92EK) that is required for Nef-dependent MIP-1β production by infected macrophages. Mutations in this domain abrogated MIP-1β induction but did not affect other Nef-ascribed activities, such as CD4 or major histocompatibility complex (MHC) class Ι downregulation. This further underscores Nef as a modular protein with genetically separable activities that may contribute to its role in viral replication and pathogenicity.

scholarly journals Lentivector Knockdown of CCR5 in Hematopoietic Stem and Progenitor Cells Confers Functional and Persistent HIV-1 Resistance in Humanized Mice

2015 ◽  
Vol 89 (13) ◽  
pp. 6761-6772 ◽  
Author(s):  
Renier Myburgh ◽  
Sandra Ivic ◽  
Michael S. Pepper ◽  
Gustavo Gers-Huber ◽  
Duo Li ◽  
...  
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
Immune System ◽  
Humanized Mice ◽  
Target Cells ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Hiv 1 ◽  
Selection Of

ABSTRACTGene-engineered CD34+hematopoietic stem and progenitor cells (HSPCs) can be used to generate an HIV-1-resistant immune system. However, a certain threshold of transduced HSPCs might be required for transplantation into mice for creating an HIV-resistant immune system. In this study, we combined CCR5 knockdown by a highly efficient microRNA (miRNA) lentivector with pretransplantation selection of transduced HSPCs to obtain a rather pure population of gene engineered CD34+cells. Low-level transduction of HSPCs and subsequent sorting by flow cytometry yielded >70% transduced cells. Mice transplanted with these cells showed functional and persistent resistance to a CCR5-tropic HIV strain: viral load was significantly decreased over months, and human CD4+T cells were preserved. In one mouse, viral mutations, resulting presumably in a CXCR4-tropic strain, overcame HIV resistance. Our results suggest that HSPC-based CCR5 knockdown may lead to efficient control of HIVin vivo. We overcame a major limitation of previous HIV gene therapy in humanized mice in which only a proportion of the cells in chimeric micein vivoare anti-HIV engineered. Our strategy underlines the promising future of gene engineering HIV-resistant CD34+cells that produce a constant supply of HIV-resistant progeny.IMPORTANCEMajor issues in experimental long-termin vivoHIV gene therapy have been (i) low efficacy of cell transduction at the time of transplantation and (ii) transduction resulting in multiple copies of heterologous DNA in target cells. In this study, we demonstrated the efficacy of a transplantation approach with a selection step for transduced cells that allows transplantation of an enriched population of HSPCs expressing a single (low) copy of a CCR5 miRNA. Efficient maintenance of CD4+T cells and a low viral titer resulted only when at least 70% of the HIV target cells were genetically modified. These findings imply that clinical protocols of HIV gene therapy require a selective enrichment of genetically targeted cells because positive selection of modified cells is likely to be insufficient below this threshold. This selection approach may be beneficial not only for HIV patients but also for other patients requiring transplantation of genetically modified cells.

scholarly journals Rhesus Macaque Resistance to Mucosal Simian Immunodeficiency Virus Infection Is Associated with a Postentry Block in Viral Replication

2002 ◽  
Vol 76 (12) ◽  
pp. 6016-6026 ◽  
Author(s):  
Bo Peng ◽  
Rebecca Voltan ◽  
Lulu Lim ◽  
Yvette Edghill-Smith ◽  
Sanjay Phogat ◽  
...  
Keyword(s):  
T Cells ◽  
Viral Replication ◽  
Rhesus Macaque ◽  
Simian Immunodeficiency Virus ◽  
Reverse Transcription ◽  
Mutation Screening ◽  
Immunodeficiency Virus ◽  
Siv Infection

ABSTRACT Elucidation of the host factors which influence susceptibility to human immunodeficiency virus or simian immunodeficiency virus (SIV) infection and disease progression has important theoretical and practical implications. Rhesus macaque 359, a vaccine control animal, resisted two successive intravaginal challenges with SIVmac251 and failed to seroconvert. Here, after an additional intrarectal SIVmac32H challenge, macaque 359 remained highly resistant to infection. Viral RNA (106 copies/ml) was observed in plasma only at week 2 postchallenge. Virus isolation and proviral DNA were positive only once at week eight postchallenge. The animal remained seronegative and cleared SIV in vivo. Its blood and lymph node cells obtained at 49 weeks after intrarectal challenge did not transmit SIV to a naive macaque. We found that the resistance of macaque 359 to SIV infection was not due to a high level of CD8+ suppressor activity but to an inherent resistance of its CD4+ T cells. To elucidate the basis for the unusually strong resistance of macaque 359 to SIV infection in vivo and in vitro, we investigated early events of viral infection and replication in CD4+ cells of macaque 359, including expression and mutation screening of SIV coreceptors and analysis of viral entry and reverse transcription. Mutation screening revealed no genetic alteration in SIV coreceptors. PCR analysis revealed a significant delay in production of early in vitro reverse transcription intermediates in macaque 359 cells compared to susceptible controls, but cell fusion assays showed that SIV entered the CD4+ CCR5+ cells of macaque 359 as readily as cells of macaques susceptible to SIV infection. Our results suggest that the resistance of macaque 359 to SIV infection is due to a postentry block in viral replication and implicate a cellular inhibitory mechanism in its CD4+ T cells. Identification of this host mechanism will help further elucidate the biochemistry of reverse transcription and may suggest therapeutic strategies. Determining the prevalence of this host resistance mechanism among macaques may lead to better design of SIV pathogenesis and vaccine studies.

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