scholarly journals Cyclophilin A protects HIV-1 from restriction by human TRIM5α

2019 ◽  
Author(s):  
Kyusik Kim ◽  
Ann Dauphin ◽  
Sevnur Komurlu ◽  
Leonid Yurkovetskiy ◽  
William E. Diehl ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Cyclophilin A ◽  
Cellular Protein ◽  
Human Cells ◽  
Sub Saharan Africa ◽  
Restriction Factor ◽  
Target Cells ◽  
Chromosomal Dna ◽  
Sub Saharan ◽  
Hiv 1

The capsid (CA) protein lattice of HIV-1 and other retroviruses encases viral genomic RNA and regulates steps that are essential to retroviral invasion of target cells, including reverse transcription, nuclear trafficking, and integration of viral cDNA into host chromosomal DNA1. Cyclophilin A (CypA), the first cellular protein reported to bind HIV-1 CA2, has interacted with invading lentiviruses related to HIV-1 for millions of years3–7. Disruption of the CA-CypA interaction decreases HIV-1 infectivity in human cells8–12, but stimulates infectivity in non-human primate cells13–15. Genetic and biochemical data suggest that CypA interaction with CA protects HIV-1 from a restriction factor in human cells16–20. Discovery of the CA-specific restriction factor TRIM5α21, and of TRIM5-CypA fusion genes that were independently generated at least four times in phylogeny4,5,15,22–25, pointed to human TRIM5α as the CypA-sensitive restriction factor. However, significant HIV-1 restriction by human TRIM5α21, let alone inhibition of such activity by CypA26, has not been detected. Here, exploiting reverse genetic tools optimized for primary human CD4+T cells, macrophages, and dendritic cells, we demonstrate that disruption of the CA-CypA interaction renders HIV-1 susceptible to restriction by human TRIM5α, with the block occurring before reverse transcription. Identical findings were obtained with single-cycle vectors or with replication-competent HIV-1, including sexually-transmitted clones from sub-Saharan Africa. Endogenous TRIM5α was observed to associate with virion cores as they entered the macrophage cytoplasm, but only when the CA-CypA interaction was disrupted. These experiments resolve the long-standing mystery of the role of CypA in HIV-1 replication by demonstrating that this ubiquitous cellular protein shields HIV-1 from previously inapparent, but potent inhibition, imposed by human TRIM5α. Hopefully this reinvigorates development of CypA-inhibitors for treatment of HIV-1 and other CypA-dependent pathogens27–30.

scholarly journals TRIM5α-Mediated Ubiquitin Chain Conjugation Is Required for Inhibition of HIV-1 Reverse Transcription and Capsid Destabilization

2015 ◽  
Vol 90 (4) ◽  
pp. 1849-1857 ◽  
Author(s):  
Edward M. Campbell ◽  
Jared Weingart ◽  
Paola Sette ◽  
Silvana Opp ◽  
Jaya Sastri ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Molecular Mechanisms ◽  
Catalytic Domain ◽  
Biological Activities ◽  
Second Step ◽  
Restriction Factor ◽  
Target Cells ◽  
Ubiquitin Chain ◽  
Ubiquitin Conjugation ◽  
Hiv 1

ABSTRACTRhesus macaque TRIM5α (rhTRIM5α) is a retroviral restriction factor that inhibits HIV-1 infection. Previous studies have revealed that TRIM5α restriction occurs via a two-step process. The first step is restriction factor binding, which is sufficient to inhibit infection. The second step, which is sensitive to proteasome inhibition, prevents the accumulation of reverse transcription products in the target cell. However, because of the pleotropic effects of proteasome inhibitors, the molecular mechanisms underlying the individual steps in the restriction process have remained poorly understood. In this study, we have fused the small catalytic domain of herpes simplex virus UL36 deubiquitinase (DUb) to the N-terminal RING domain of rhTRIM5α, which results in a ubiquitination-resistant protein. Cell lines stably expressing this fusion protein inhibited HIV-1 infection to the same degree as a control fusion to a catalytically inactive DUb. However, reverse transcription products were substantially increased in the DUb-TRIM5α fusion relative to the catalytically inactive control or the wild-type (WT) TRIM5α. Similarly, expression of DUb-rhTRIM5α resulted in the accumulation of viral cores in target cells following infection, while the catalytically inactive control and WT rhTRIM5α induced the abortive disassembly of viral cores, indicating a role for ubiquitin conjugation in rhTRIM5α-mediated destabilization of HIV-1 cores. Finally, DUb-rhTRIM5α failed to activate NF-κB signaling pathways compared to controls, demonstrating that this ubiquitination-dependent activity is separable from the ability to restrict retroviral infection.IMPORTANCEThese studies provide direct evidence that ubiquitin conjugation to rhTRIM5α-containing complexes is required for the second step of HIV-1 restriction. They also provide a novel tool by which the biological activities of TRIM family proteins might be dissected to better understand their function and underlying mechanisms of action.

scholarly journals Fusion of Cyclophilin A to Fv1 Enables Cyclosporine-Sensitive Restriction of Human and Feline Immunodeficiency Viruses

2007 ◽  
Vol 81 (18) ◽  
pp. 10055-10063 ◽  
Author(s):  
Torsten Schaller ◽  
Laura M. J. Ylinen ◽  
Benjamin L. J. Webb ◽  
Shalene Singh ◽  
Greg J. Towers
Keyword(s):  
Reverse Transcription ◽  
Half Life ◽  
Leukemia Virus ◽  
Susceptibility Gene ◽  
Cyclophilin A ◽  
Restriction Factor ◽  
Coding Region ◽  
Immunodeficiency Virus ◽  
Species Specific ◽  
Hiv 1

ABSTRACT TRIM5α is a potent intracellular antiviral restriction factor governing species-specific retroviral replication. In the New World species owl monkey the coding region for the viral binding B30.2 domain of TRIM5α has been replaced by a cyclophilin A (CypA) pseudogene by retrotransposition. The resultant TRIM5-CypA fusion protein restricts human immunodeficiency virus type 1 (HIV-1), as well as feline immunodeficiency virus (FIV), by recruitment of the CypA domain to the incoming viral capsids. Infectivity is rescued by agents such as cyclosporine that disrupt CypA binding to its substrates. Mice encode an antiviral restriction factor called Fv1 (for Friend virus susceptibility gene 1), which is active against murine leukemia virus and related to endogenous gag sequences. Here we show that fusing CypA to Fv1 generates a restriction factor with the antiviral specificity of TRIMCyp but the antiviral properties of Fv1. Like TRIMCyp, Fv1-Cyp restricts HIV-1 and FIV and is sensitive to inhibition by cyclosporine. TRIM5α is known to have a short half-life and block infectivity before viral reverse transcription. We show that Fv1-Cyp has a long half-life and blocks after reverse transcription, suggesting that its longer half-life gives the restricted virus the opportunity to synthesize DNA, leading to a later block to infection. This notion is supported by the observation that infectivity of Fv1-Cyp restricted virus can be rescued by cyclosporine for several hours after infection, whereas virus restricted by TRIMCyp is terminally restricted after around 40 min. Intriguingly, the Fv1-Cyp-restricted HIV-1 generates closed circular viral DNA, suggesting that the restricted virus complex enters the nucleus.

scholarly journals HIV-1 Uncoating and Nuclear Import Precede the Completion of Reverse Transcription in Cell Lines and in Primary Macrophages

Viruses ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 1234
Author(s):  
Ashwanth C. Francis ◽  
Mariana Marin ◽  
Mathew J. Prellberg ◽  
Kristina Palermino-Rowland ◽  
Gregory B. Melikyan
Keyword(s):  
Cell Lines ◽  
Reverse Transcription ◽  
Nuclear Import ◽  
Cyclophilin A ◽  
Target Cells ◽  
Nuclear Pore ◽  
Virus Escape ◽  
Infected Cells ◽  
Kinetics Of ◽  
Hiv 1

An assembly of capsid proteins (CA) form the mature viral core enclosing the HIV-1 ribonucleoprotein complex. Discrepant findings have been reported regarding the cellular sites and the extent of core disassembly (uncoating) in infected cells. Here, we combined single-virus imaging and time-of-drug-addition assays to elucidate the kinetic relationship between uncoating, reverse transcription, and nuclear import of HIV-1 complexes in cell lines and monocyte-derived macrophages (MDMs). By using cyclophilin A-DsRed (CDR) as a marker for CA, we show that, in contrast to TZM-bl cells, early cytoplasmic uncoating (loss of CDR) is limited in MDMs and is correlated with the efficiency of reverse transcription. However, we find that reverse transcription is dispensable for HIV-1 nuclear import, which progressed through an uncoating step at the nuclear pore. Comparison of the kinetics of nuclear import and the virus escape from inhibitors targeting distinct steps of infection, as well as direct quantification of viral DNA synthesis, revealed that reverse transcription is completed after nuclear import of HIV-1 complexes. Collectively, these results suggest that reverse transcription is dispensable for the uncoating step at the nuclear pore and that vDNA synthesis is completed in the nucleus of unrelated target cells.

scholarly journals Active Components from Cassia abbreviata Prevent HIV-1 Entry by Distinct Mechanisms of Action

2021 ◽  
Vol 22 (9) ◽  
pp. 5052
Author(s):  
Yue Zheng ◽  
Xian-Wen Yang ◽  
Dominique Schols ◽  
Mattia Mori ◽  
Bruno Botta ◽  
...  
Keyword(s):  
Crude Extract ◽  
Female Genital Tract ◽  
Binding Activity ◽  
Sub Saharan Africa ◽  
Active Components ◽  
Chemical Structures ◽  
3D Space ◽  
In Silico Study ◽  
Sub Saharan ◽  
Hiv 1

Cassia abbreviata is widely used in Sub-Saharan Africa for treating many diseases, including HIV-1 infection. We have recently described the chemical structures of 28 compounds isolated from an alcoholic crude extract of barks and roots ofC. abbreviata, and showed that six bioactive compounds inhibit HIV-1 infection. In the present study, we demonstrate that the six compounds block HIV-1 entry into cells: oleanolic acid, palmitic acid, taxifolin, piceatannol, guibourtinidol-(4α®8)-epiafzelechin, and a novel compound named as cassiabrevone. We report, for the first time, that guibourtinidol-(4α®8)-epiafzelechin and cassiabrevone inhibit HIV-1 entry (IC50 of 42.47 µM and 30.96 µM, respectively), as well as that piceatannol interacts with cellular membranes. Piceatannol inhibits HIV-1 infection in a dual-chamber assay mimicking the female genital tract, as well as HSV infection, emphasizing its potential as a microbicide. Structure-activity relationships (SAR) showed that pharmacophoric groups of piceatannol are strictly required to inhibit HIV-1 entry. By a ligand-based in silico study, we speculated that piceatannol and norartocarpetin may have a very similar mechanism of action and efficacy because of the highly comparable pharmacophoric and 3D space, while guibourtinidol-(4α®8)-epiafzelechin and cassiabrevone may display a different mechanism. We finally show that cassiabrevone plays a major role of the crude extract of CA by blocking the binding activity of HIV-1 gp120 and CD4.

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 HIV-1 uncoating occurs via a series of rapid biomechanical changes in the core related to individual stages of reverse transcription

2021 ◽  
Author(s):  
Sanela Rankovic ◽  
Akshay Deshpande ◽  
Shimon Harel ◽  
Christopher Aiken ◽  
Itay Rousso
Keyword(s):  
Reverse Transcription ◽  
Viral Genome ◽  
Morphological Changes ◽  
Viral Capsid ◽  
Target Cells ◽  
Viral Core ◽  
Successful Infection ◽  
Capsid Shell ◽  
Hiv 1

AbstractThe HIV core consists of the viral genome and associated proteins encased by a cone-shaped protein shell termed the capsid. Successful infection requires reverse transcription of the viral genome and disassembly of the capsid shell within a cell in a process known as uncoating. The integrity of the viral capsid is critical for reverse transcription, yet the viral capsid must be breached to release the nascent viral DNA prior to integration. We employed atomic force microscopy to study the stiffness changes in HIV-1 cores during reverse transcription in vitro in reactions containing the capsid-stabilizing host metabolite IP6. Cores exhibited a series of stiffness spikes, with up to three spikes typically occurring between 10-30, 40-80, and 120-160 minutes after initiation of reverse transcription. Addition of the reverse transcriptase (RT) inhibitor efavirenz eliminated the appearance of these spikes and the subsequent disassembly of the capsid, thus establishing that both result from reverse transcription. Using timed addition of efavirenz, and analysis of an RNAseH-defective RT mutant, we established that the first stiffness spike requires minus-strand strong stop DNA synthesis, with subsequent spikes requiring later stages of reverse transcription. Additional rapid AFM imaging experiments revealed repeated morphological changes in cores that were temporally correlated with the observed stiffness spikes. Our study reveals discrete mechanical changes in the viral core that are likely related to specific stages of reverse transcription. Our results suggest that reverse-transcription-induced changes in the capsid progressively remodel the viral core to prime it for temporally accurate uncoating in target cells.

scholarly journals A Randomized Switch From Nevirapine-Based Antiretroviral Therapy to Single Tablet Rilpivirine/Emtricitabine/Tenofovir Disoproxil Fumarate in Virologically Suppressed Human Immunodeficiency Virus-1-Infected Rwandans

2016 ◽  
Vol 3 (3) ◽  
Author(s):  
Sean E. Collins ◽  
Philip M. Grant ◽  
Francois Uwinkindi ◽  
Annie Talbot ◽  
Eric Seruyange ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Antiretroviral Therapy ◽  
Tenofovir Disoproxil Fumarate ◽  
Sub Saharan Africa ◽  
Current Therapy ◽  
Virologic Suppression ◽  
Open Label ◽  
Immunodeficiency Virus ◽  
Sub Saharan ◽  
Hiv 1

Abstract Background.  Many human immunodeficiency virus (HIV)-infected patients remain on nevirapine-based antiretroviral therapy (ART) despite safety and efficacy concerns. Switching to a rilpivirine-based regimen is an alternative, but there is little experience with rilpivirine in sub-Saharan Africa where induction of rilpivirine metabolism by nevirapine, HIV subtype, and dietary differences could potentially impact efficacy. Methods.  We conducted an open-label noninferiority study of virologically suppressed (HIV-1 ribonucleic acid [RNA] < 50 copies/mL) HIV-1-infected Rwandan adults taking nevirapine plus 2 nucleos(t)ide reverse-transcriptase inhibitors. One hundred fifty participants were randomized 2:1 to switch to coformulated rilpivirine-emtricitabine-tenofovir disoproxil fumarate (referenced as the Switch Arm) or continue current therapy. The primary efficacy endpoint was HIV-1 RNA < 200 copies/mL at week 24 assessed by the US Food and Drug Administration Snapshot algorithm with a noninferiority margin of 12%. Results.  Between April and September 2014, 184 patients were screened, and 150 patients were enrolled; 99 patients switched to rilpivirine-emtricitabine-tenofovir, and 51 patients continued their nevirapine-based ART. The mean age was 42 years and 43% of participants were women. At week 24, virologic suppression (HIV-1 RNA level <200 copies/mL) was maintained in 93% and 92% in the Switch Arm versus the continuation arm, respectively. The Switch Arm was noninferior to continued nevirapine-based ART (efficacy difference 0.8%; 95% confidence interval, −7.5% to +12.0%). Both regimens were generally safe and well tolerated, although 2 deaths, neither attributed to study medications, occurred in participants in the Switch Arm. Conclusions.  A switch from nevirapine-based ART to rilpivirine-emtricitabine-tenofovir disoproxil fumarate had similar virologic efficacy to continued nevirapine-based ART after 24 weeks with few adverse events.

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