scholarly journals Cyclophilin A Prevents HIV-1 Restriction in Lymphocytes by Blocking Human TRIM5α Binding to the viral Core

2019 ◽  
Author(s):  
Anastasia Selyutina ◽  
Mirjana Persaud ◽  
Angel Bulnes-Ramos ◽  
Cindy Buffone ◽  
Alicia Martinez-Lopez ◽  
...  
Keyword(s):  
T Cells ◽  
Reverse Transcription ◽  
Human Lymphocytes ◽  
Cyclophilin A ◽  
Inhibitory Effect ◽  
Jurkat Cells ◽  
Human Pbmcs ◽  
The Core ◽  
Hiv 1 ◽  
Cypa Binding

ABSTRACTDisruption of cyclophilin A (CypA)-capsid interactions affects HIV-1 replication in human lymphocytes. To understand the mechanism, we used Jurkat cells, human PBMCs, and human CD4+T cells. Our results showed that the inhibition of HIV-1 infection caused by disrupting CypA-capsid interactions is dependent on human TRIM5α (TRIM5αhu), suggesting that TRIM5αhurestricts HIV-1. Accordingly, we found that TRIM5αhubinds to the HIV-1 core. Disruption of CypA-capsid interactions failed to affect HIV-1-A92E infection, correlating with the loss of TRIM5αhubinding to HIV-1-A92E cores. Disruption of CypA-capsid interactions in PBMCs and CD4+T cells had a greater inhibitory effect on HIV-1 when compared to Jurkat cells. HIV-1-A92E infection of PBMCs and CD4+T cells was unaffected by disruption of CypA-capsid interactions. Consistent with TRIM5α restriction, disruption of CypA-capsid interactions in CD4+T cells inhibited reverse transcription. Overall, our results showed that CypA binding to the core protects HIV-1 from TRIM5αhurestriction.

scholarly journals Human Immunodeficiency Virus Type 1 Replication Is Modulated by Host Cyclophilin A Expression Levels

1998 ◽  
Vol 72 (8) ◽  
pp. 6430-6436 ◽  
Author(s):  
Lei Yin ◽  
Douglas Braaten ◽  
Jeremy Luban
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cells ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Cyclophilin A ◽  
Jurkat Cells ◽  
Expression Levels ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) Gag and the cellular protein cyclophilin A form an essential complex in the virion core: virions produced by proviruses encoding Gag mutants with decreased cyclophilin A affinity exhibit attenuated infectivity, as do virions produced in the presence of the competitive inhibitor cyclosporine. The A224E Gag mutant has no effect on cyclophilin A affinity but renders HIV-1 replication cyclosporine resistant in Jurkat T cells. In contrast, A224E mutant virus is dead in H9 T cells, although replication is rescued by cyclosporine or by expression in cis of a Gag mutant that decreases cyclophilin A-affinity. The observation that disruption of the Gag-cyclophilin A interaction rescues A224E mutant replication in H9 cells prompted experiments which revealed that, relative to Jurkat cells, H9 cells express greater quantities of cyclophilin A. The resulting larger quantity of cyclophilin A shown to be packaged into virions produced by H9 cells is presumably disruptive to the A224E mutant virion core. Further evidence that increased cyclophilin A expression in H9 cells is of functional relevance was provided by the finding that Gag mutants with decreased cyclophilin A affinity are dead in Jurkat cells but capable of replication in H9 cells. Similarly, cyclosporine concentrations which inhibit wild-type HIV-1 replication in Jurkat cells stimulate HIV-1 replication in H9 cells. These results suggest that HIV-1 virion infectivity imposes narrow constraints upon cyclophilin A stoichiometry in virions and that infectivity is finely tuned by host cyclophilin A expression levels.

scholarly journals PF74 Inhibits HIV-1 Integration by Altering the Composition of the Preintegration Complex

2018 ◽  
Vol 93 (6) ◽  
Author(s):  
Muthukumar Balasubramaniam ◽  
Jing Zhou ◽  
Amma Addai ◽  
Phillip Martinez ◽  
Jui Pandhare ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Integrase Inhibitor ◽  
Antiviral Effect ◽  
Inhibitory Effect ◽  
Clear Understanding ◽  
Nuclear Entry ◽  
Preintegration Complex ◽  
Gradient Based ◽  
Hiv 1

ABSTRACTThe HIV-1 capsid protein (CA) facilitates reverse transcription and nuclear entry of the virus. However, CA’s role in post-nuclear entry steps remains speculative. We describe a direct link between CA and integration by employing the capsid inhibitor PF74 as a probe coupled with the biochemical analysis of HIV-1 preintegration complexes (PICs) isolated from acutely infected cells. At a low micromolar concentration, PF74 potently inhibited HIV-1 infection without affecting reverse transcription. Surprisingly, PF74 markedly reduced proviral integration owing to inhibition of nuclear entry and/or integration. However, a 2-fold reduction in nuclear entry by PF74 did not quantitatively correlate with the level of antiviral activity. Titration of PF74 against the integrase inhibitor raltegravir showed an additive antiviral effect that is dependent on a block at the post-nuclear entry step. PF74’s inhibitory effect was not due to the formation of defective viral DNA ends or a delay in integration, suggesting that the compound inhibits PIC-associated integration activity. Unexpectedly, PICs recovered from cells infected in the presence of PF74 exhibited elevated integration activity. PF74’s effect on PIC activity is CA specific since the compound did not increase the integration activity of PICs of a PF74-resistant HIV-1 CA mutant. Sucrose gradient-based fractionation studies revealed that PICs assembled in the presence of PF74 contained lower levels of CA, suggesting a negative association between CA and PIC-associated integration activity. Finally, the addition of a CA-specific antibody or PF74 inhibited PIC-associated integration activity. Collectively, our results demonstrate that PF74’s targeting of PIC-associated CA results in impaired HIV-1 integration.IMPORTANCEAntiretroviral therapy (ART) that uses various combinations of small molecule inhibitors has been highly effective in controlling HIV. However, the drugs used in the ART regimen are expensive, cause side effects, and face viral resistance. The HIV-1 CA plays critical roles in the virus life cycle and is an attractive therapeutic target. While currently there is no CA-based therapy, highly potent CA-specific inhibitors are being developed as a new class of antivirals. Efforts to develop a CA-targeted therapy can be aided through a clear understanding of the role of CA in HIV-1 infection. CA is well established to coordinate reverse transcription and nuclear entry of the virus. However, the role of CA in post-nuclear entry steps of HIV-1 infection is poorly understood. We show that a CA-specific drug PF74 inhibits HIV-1 integration revealing a novel role of this multifunctional viral protein in a post-nuclear entry step of HIV-1 infection.

scholarly journals Interferon-inducible miR-128 modulates HIV-1 replication by targeting TNPO3 mRNA

2017 ◽  
Author(s):  
Aurore Bochnakian ◽  
Dimitrios G Zisoulis ◽  
Adam Idica ◽  
Anjie Zhen ◽  
Vineet N KewalRamani ◽  
...  
Keyword(s):  
T Cells ◽  
Nuclear Import ◽  
Cd4 T Cells ◽  
Jurkat Cells ◽  
Type I ◽  
Coding Region ◽  
Aids Pandemic ◽  
Mrna And Protein Expression ◽  
Dual Mechanism ◽  
Hiv 1

ABSTRACTThe HIV/AIDS pandemic remains an important threat to human health. We have recently demonstrated that a novel microRNA (miR-128) represses retrotransposon (LINE-1 or L1) by a dual mechanism, by directly targeting the coding region of the L1 RNA and by repressing a required nuclear import factor (TNPO1). We have further determined that miR-128 represses the expression of all three isoforms of TNPO proteins (transportins, TNPO1,-2 and TNPO3). Here, we establish that miR-128 also controls HIV-1 replication by repressing TNPO3. TNPO3 is well established to regulate HIV-1 nuclear import and viral replication. Here, we report that the type I interferon inducible miR-128 directly targets two sites in the TNPO3 mRNA, significantly down-regulating TNPO3 mRNA and protein expression levels. Manipulation of miR-128 levels in HIV target cell lines and in primary human CD4 T-cells by over-expression or knockdown showed that modulation of TNPO3 by miR-128 affects HIV-1 replication but not MLV infection. In addition, we found that miR-128 modulation of HIV-1 replication is reduced with TNPO3-independent HIV-1 virus and in cells depleted of CPSF6, suggesting that miR-128-indued TNPO3 repression is partly required for miR-128-induced inhibition of HIV-1 replication. Finally, challenging miR-modulated Jurkat cells or primary CD4 T-cells with wildtype, replication-competent HIV-1 shows that miR-128 significantly delays spreading infection. Thus, we have established a novel role of miR-128 in anti-viral defense in human cells, inhibiting HIV-1 replication partly by targeting TNPO3.

scholarly journals Kinetics of Human Immunodeficiency Virus Type 1 Decay following Entry into Resting CD4+ T Cells

2005 ◽  
Vol 79 (4) ◽  
pp. 2199-2210 ◽  
Author(s):  
Yan Zhou ◽  
Haili Zhang ◽  
Janet D. Siliciano ◽  
Robert F. Siliciano
Keyword(s):  
Gene Expression ◽  
Human Immunodeficiency Virus ◽  
T Cells ◽  
Human Immunodeficiency Virus Type ◽  
Reverse Transcription ◽  
Half Life ◽  
Immunodeficiency Virus ◽  
Kinetics Of ◽  
Hiv 1

ABSTRACT In untreated human immunodeficiency virus type 1 (HIV-1) infection, most viral genomes in resting CD4+ T cells are not integrated into host chromosomes. This unintegrated virus provides an inducible latent reservoir because cellular activation permits integration, virus gene expression, and virus production. It remains controversial whether HIV-1 is stable in this preintegration state. Here, we monitored the fate of HIV-1 in resting CD4+ cells by using a green fluorescent protein (GFP) reporter virus carrying an X4 envelope. After virus entry into resting CD4+ T cells, both rescuable virus gene expression, visualized with GFP, and rescuable virion production, assessed by p24 release, decayed with a half-life of 2 days. In these cells, reverse transcription goes to completion over 2 to 3 days, and 50% of the viruses that have entered undergo functional decay before reverse transcription is complete. We distinguished two distinct but closely related factors contributing to loss of rescuable virus. First, some host cells undergo virus-induced apoptosis upon viral entry, thereby reducing the amount of rescuable virus. Second, decay processes directly affecting the virus both before and after the completion of reverse transcription contribute to the loss of rescuable virus. The functional half-life of full-length, integration-competent reverse transcripts is only 1 day. We propose that rapid intracellular decay processes compete with early steps in viral replication in infected CD4+ T cells. Decay processes dominate in resting CD4+ T cells as a result of the slow kinetics of reverse transcription and blocks at subsequent steps. Therefore, the reservoir of unintegrated HIV-1 in recently infected resting CD4+ T cells is highly labile.

scholarly journals PF74 Reinforces the HIV-1 Capsid To Impair Reverse Transcription-Induced Uncoating

2018 ◽  
Vol 92 (20) ◽  
Author(s):  
Sanela Rankovic ◽  
Ruben Ramalho ◽  
Christopher Aiken ◽  
Itay Rousso
Keyword(s):  
Atomic Force Microscopy ◽  
Small Molecule ◽  
Reverse Transcription ◽  
Direct Evidence ◽  
Main Body ◽  
Force Microscopy ◽  
The Core ◽  
Atomic Force ◽  
Capsid Stability ◽  
Hiv 1

ABSTRACTThe RNA genome of human immunodeficiency virus type 1 (HIV-1) is enclosed in a cone-shaped capsid shell that disassembles following cell entry via a process known as uncoating. During HIV-1 infection, the capsid is important for reverse transcription and entry of the virus into the target cell nucleus. The small molecule PF74 inhibits HIV-1 infection at early stages by binding to the capsid and perturbing uncoating. However, the mechanism by which PF74 alters capsid stability and reduces viral infection is presently unknown. Here, we show, using atomic force microscopy (AFM), that binding of PF74 to recombinant capsid-like assemblies and to HIV-1 isolated cores stabilizes the capsid in a concentration-dependent manner. At a PF74 concentration of 10 μM, the mechanical stability of the core is increased to a level similar to that of the intrinsically hyperstable capsid mutant E45A. PF74 also prevented the complete disassembly of HIV-1 cores normally observed during 24 h of reverse transcription. Specifically, cores treated with PF74 only partially disassembled: the main body of the capsid remained intact and stiff, and a cap-like structure dissociated from the narrow end of the core. Moreover, the internal coiled structure that was observed to form during reverse transcriptionin vitropersisted throughout the duration of the measurement (∼24 h). Our results provide direct evidence that PF74 directly stabilizes the HIV-1 capsid lattice, thereby permitting reverse transcription while interfering with a late step in uncoating.IMPORTANCEThe capsid-binding small molecule PF74 inhibits HIV-1 infection at early stages and perturbs uncoating. However, the mechanism by which PF74 alters capsid stability and reduces viral infection is presently unknown. We recently introduced time-lapse atomic force microscopy to study the morphology and physical properties of HIV-1 cores during the course of reverse transcription. Here, we apply this AFM methodology to show that PF74 prevented the complete disassembly of HIV-1 cores normally observed during 24 h of reverse transcription. Specifically, cores with PF74 only partially disassembled: the main body of the capsid remained intact and stiff, but a cap-like structure dissociated from the narrow end of the core HIV-1. Our result provides direct evidence that PF74 directly stabilizes the HIV-1 capsid lattice.

scholarly journals Cell Type-Dependent Escape of Capsid Inhibitors by Simian Immunodeficiency Virus SIVcpz

2020 ◽  
Vol 94 (23) ◽  
Author(s):  
Augustin Penda Twizerimana ◽  
Rachel Scheck ◽  
Daniel Becker ◽  
Zeli Zhang ◽  
Marianne Wammers ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Simian Immunodeficiency Virus ◽  
Pan Troglodytes ◽  
Hela Cells ◽  
Cyclophilin A ◽  
Human Pbmcs ◽  
Cell Type ◽  
Content Type ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Pandemic human immunodeficiency virus type 1 (HIV-1) is the result of the zoonotic transmission of simian immunodeficiency virus (SIV) from the chimpanzee subspecies Pan troglodytes troglodytes (SIVcpzPtt). The related subspecies Pan troglodytes schweinfurthii is the host of a similar virus, SIVcpzPts, which did not spread to humans. We tested these viruses with small-molecule capsid inhibitors (PF57, PF74, and GS-CA1) that interact with a binding groove in the capsid that is also used by CPSF6. While HIV-1 was sensitive to capsid inhibitors in cell lines, human macrophages, and peripheral blood mononuclear cells (PBMCs), SIVcpzPtt was resistant in rhesus FRhL-2 cells and human PBMCs but was sensitive to PF74 in human HOS and HeLa cells. SIVcpzPts was insensitive to PF74 in FRhL-2 cells, HeLa cells, PBMCs, and macrophages but was inhibited by PF74 in HOS cells. A truncated version of CPSF6 (CPSF6-358) inhibited SIVcpzPtt and HIV-1, while in contrast, SIVcpzPts was resistant to CPSF6-358. Homology modeling of HIV-1, SIVcpzPtt, and SIVcpzPts capsids and binding energy estimates suggest that these three viruses bind similarly to the host proteins cyclophilin A (CYPA) and CPSF6 as well as the capsid inhibitor PF74. Cyclosporine treatment, mutation of the CYPA-binding loop in the capsid, or CYPA knockout eliminated the resistance of SIVcpzPts to PF74 in HeLa cells. These experiments revealed that the antiviral capacity of PF74 is controlled by CYPA in a virus- and cell type-specific manner. Our data indicate that SIVcpz viruses can use infection pathways that escape the antiviral activity of PF74. We further suggest that the antiviral activity of PF74 capsid inhibitors depends on cellular cofactors. IMPORTANCE HIV-1 originated from SIVcpzPtt but not from the related virus SIVcpzPts, and thus, it is important to describe molecular infection by SIVcpzPts in human cells to understand the zoonosis of SIVs. Pharmacological HIV-1 capsid inhibitors (e.g., PF74) bind a capsid groove that is also a binding site for the cellular protein CPSF6. SIVcpzPts was resistant to PF74 in HeLa cells but sensitive in HOS cells, thus indicating cell line-specific resistance. Both SIVcpz viruses showed resistance to PF74 in human PBMCs. Modulating the presence of cyclophilin A or its binding to capsid in HeLa cells overcame SIVcpzPts resistance to PF74. These results indicate that early cytoplasmic infection events of SIVcpzPts may differ between cell types and affect, in an unknown manner, the antiviral activity of capsid inhibitors. Thus, capsid inhibitors depend on the activity or interaction of currently uncharacterized cellular factors.

Investigating HIV-Human Interaction Networks to Unravel Pathogenic Mechanism for Drug Discovery: A Systems Biology Approach

2018 ◽  
Vol 16 (1) ◽  
pp. 77-95 ◽  
Author(s):  
Cheng-Wei Li ◽  
Bor-Sen Chen
Keyword(s):  
T Cells ◽  
Reverse Transcription ◽  
Late Stage ◽  
Time Course ◽  
System Modeling ◽  
Human Interaction ◽  
Flufenamic Acid ◽  
Replication Stage ◽  
Core Proteins ◽  
Hiv 1

Background: Two big issues in the study of pathogens are determining how pathogens infect hosts and how the host defends itself against infection. Therefore, investigating host-pathogen interactions is important for understanding pathogenicity and host defensive mechanisms and treating infections.Methods: In this study, we used omics data, including time-course data from high-throughput sequencing, real-time polymerase chain reaction, and human microRNA (miRNA) and protein-protein interaction to construct an interspecies protein-protein and miRNA interaction (PPMI) network of human CD4+ T cells during HIV-1 infection through system modeling and identification.Results: By applying a functional annotation tool to the identified PPMI network at each stage of HIV infection, we found that repressions of three miRNAs, miR-140-5p, miR-320a, and miR-941, are involved in the development of autoimmune disorders, tumor proliferation, and the pathogenesis of T cells at the reverse transcription stage. Repressions of miR-331-3p and miR-320a are involved in HIV-1 replication, replicative spread, anti-apoptosis, cell proliferation, and dysregulation of cell cycle control at the integration/replication stage. Repression of miR-341-5p is involved in carcinogenesis at the late stage of HIV-1 infection.Conclusion: By investigating the common core proteins and changes in specific proteins in the PPMI network between the stages of HIV-1 infection, we obtained pathogenic insights into the functional core modules and identified potential drug combinations for treating patients with HIV-1 infection, including thalidomide, oxaprozin, and metformin, at the reverse transcription stage; quercetin, nifedipine, and fenbendazole, at the integration/replication stage; and staurosporine, quercetin, prednisolone, and flufenamic acid, at the late stage.

scholarly journals Cytokine Signals Are Sufficient for HIV-1 Infection of Resting Human T Lymphocytes

1999 ◽  
Vol 189 (11) ◽  
pp. 1735-1746 ◽  
Author(s):  
Derya Unutmaz ◽  
Vineet N. KewalRamani ◽  
Shana Marmon ◽  
Dan R. Littman
Keyword(s):  
T Cells ◽  
T Lymphocytes ◽  
Structure Function ◽  
Human Lymphocytes ◽  
Stable Gene ◽  
Mutant Form ◽  
Primary Cells ◽  
Human T Cells ◽  
Transformed Cell Lines ◽  
Hiv 1

Lentiviral vectors have been advocated to be effective vehicles for the delivery and stable expression of genes in nondividing primary cells. However, certain cell types, such as resting T lymphocytes, are resistant to infection with HIV-1. Establishing parameters for stable gene delivery into primary human lymphocytes and approaches to overcome the resistance of resting T cells to HIV infection may permit potential gene therapy applications, genetic studies of primary cells in vitro, and a better understanding of the stages of the lentiviral life cycle. Here we demonstrate that an HIV-1–derived vector can be used for stable delivery of genes into activated human T cells as well as natural killer and dendritic cells. Remarkably, a sizeable fraction of resting T cells was stably transduced with the HIV-1 vector when cultured with the cytokine interleukin (IL)-2, IL-4, IL-7, or IL-15, or, at a lower level, with IL-6, in the absence of any other stimuli. Resting T cells stimulated with these cytokines could also be infected with replication-competent HIV-1. To test the utility of this system for performing structure–function analysis in primary T cells, we introduced wild-type as well as a mutant form of murine CD28 into human T cells and showed a requirement for the CD28 cytoplasmic domain in costimulatory signaling. The ability to stably express genes of interest in primary T cells will be a valuable tool for genetic and structure–function studies that previously have been limited to transformed cell lines. In addition, the finding that cytokine signals are sufficient to permit transduction of resting T cells with HIV may be relevant for understanding mechanism of HIV-1 transmission and pathogenesis.

scholarly journals CD36-specific antibodies block release of HIV-1 from infected primary macrophages and its transmission to T cells

2013 ◽  
Vol 210 (12) ◽  
pp. 2523-2538 ◽  
Author(s):  
Stefano Berre ◽  
Raphaël Gaudin ◽  
Bruna Cunha de Alencar ◽  
Marion Desdouits ◽  
Mélanie Chabaud ◽  
...  
Keyword(s):  
T Cells ◽  
Scavenger Receptor ◽  
Ectopic Expression ◽  
Inhibitory Effect ◽  
Viral Reservoirs ◽  
Viral Dissemination ◽  
Human Macrophages ◽  
Specific Antibodies ◽  
Natural Ligands ◽  
Hiv 1

HIV-1–infected macrophages likely represent viral reservoirs, as they accumulate newly formed virions in internal virus-containing compartments (VCCs). However, the nature and biogenesis of VCCs remain poorly defined. We show that upon HIV-1 infection of primary human macrophages, Gag is recruited to preexisting compartments containing the scavenger receptor CD36, which then become VCCs. Silencing of CD36 in HIV-1–infected macrophages decreases the amount of virions released. Strikingly, soluble anti-CD36 antibodies, but not the natural ligands of CD36, inhibit release of virions from HIV-1–infected macrophages and the transmission of virus to CD4+ T cells. The effect of the antibodies is potent, rapid, and induces the retention of virions within VCCs. Ectopic expression of CD36 in HeLa cells renders them susceptible to the inhibitory effect of the anti-CD36 mAb upon HIV-1 infection. We show that the anti-CD36 mAb inhibits HIV-1 release by clustering newly formed virions at their site of budding, and that signaling via CD36 is not required. Thus, HIV-1 reservoirs in macrophages may be tackled therapeutically using anti-CD36 antibodies to prevent viral dissemination.

scholarly journals Treatment of Human Immunodeficiency Virus Type 1 Virions Depleted of Cyclophilin A by Natural Endogenous Reverse Transcription Restores Infectivity

2003 ◽  
Vol 77 (7) ◽  
pp. 4431-4434 ◽  
Author(s):  
Mahfuz Khan ◽  
Minerva Garcia-Barrio ◽  
Michael D. Powell
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Cyclosporine A ◽  
Reverse Transcription ◽  
Cyclophilin A ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Binding Loop ◽  
Cypa Binding

ABSTRACT We have previously shown that virions with nef deleted can be restored to wild-type infectivity by treatment to induce natural endogenous reverse transcription (NERT). Since Nef and cyclophilin A (CyPA) appear to act in similar ways on postentry events, we determined whether NERT treatment would restore infectivity to virions depleted of CyPA. Our results show that the infectivity of virions depleted of CyPA by treatment with cyclosporine A could be restored by NERT treatment, while mutants in the CyPA binding loop of capsid could only be partially restored. These results suggest that CyPA is involved in some aspect of the uncoating process.

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