Role of Post-transcriptional Modifications of Primer tRNALys,3in the Fidelity and Efficacy of Plus Strand DNA Transfer during HIV-1 Reverse Transcription

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.

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Determination of the Ex Vivo Rates of Human Immunodeficiency Virus Type 1 Reverse Transcription by Using Novel Strand-Specific Amplification Analysis

Journal of Virology ◽

10.1128/jvi.02471-06 ◽

2007 ◽

Vol 81 (9) ◽

pp. 4798-4807 ◽

Cited By ~ 23

Author(s):

David C. Thomas ◽

Yegor A. Voronin ◽

Galina N. Nikolenko ◽

Jianbo Chen ◽

Wei-Shau Hu ◽

...

Keyword(s):

Dna Synthesis ◽

Reverse Transcription ◽

Ex Vivo ◽

Dna Transfer ◽

Minus Strand ◽

Immunodeficiency Virus ◽

Specific Amplification ◽

Kinetics Of ◽

Hiv 1

ABSTRACT Replication of human immunodeficiency virus type 1 (HIV-1), like all organisms, involves synthesis of a minus-strand and a plus-strand of nucleic acid. Currently available PCR methods cannot distinguish between the two strands of nucleic acids. To carry out detailed analysis of HIV-1 reverse transcription from infected cells, we have developed a novel strand-specific amplification (SSA) assay using single-stranded padlock probes that are specifically hybridized to a target strand, ligated, and quantified for sensitive analysis of the kinetics of HIV-1 reverse transcription in cells. Using SSA, we have determined for the first time the ex vivo rates of HIV-1 minus-strand DNA synthesis in 293T and human primary CD4+ T cells (∼68 to 70 nucleotides/min). We also determined the rates of minus-strand DNA transfer (∼4 min), plus-strand DNA transfer (∼26 min), and initiation of plus-strand DNA synthesis (∼9 min) in 293T cells. Additionally, our results indicate that plus-strand DNA synthesis is initiated at multiple sites and that several reverse transcriptase inhibitors influence the kinetics of minus-strand DNA synthesis differently, providing insights into their mechanism of inhibition. The SSA technology provides a novel approach to analyzing DNA replication processes and should facilitate the development of new antiretroviral drugs that target specific steps in HIV-1 reverse transcription.

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Initiation of HIV-1 reverse transcription and functional role of nucleocapsid-mediated tRNA/viral genome interactions

Virus Research ◽

10.1016/j.virusres.2012.06.006 ◽

2012 ◽

Vol 169 (2) ◽

pp. 324-339 ◽

Cited By ~ 30

Author(s):

Dona Sleiman ◽

Valérie Goldschmidt ◽

Pierre Barraud ◽

Roland Marquet ◽

Jean-Christophe Paillart ◽

...

Keyword(s):

Reverse Transcription ◽

Viral Genome ◽

Functional Role ◽

Genome Interactions ◽

Hiv 1

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Role of Human Immunodeficiency Virus Type 1 Integrase in Uncoating of the Viral Core

Journal of Virology ◽

10.1128/jvi.02382-09 ◽

2010 ◽

Vol 84 (10) ◽

pp. 5181-5190 ◽

Cited By ~ 33

Author(s):

Marisa S. Briones ◽

Charles W. Dobard ◽

Samson A. Chow

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Reverse Transcription ◽

Core Stability ◽

The Core ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT After membrane fusion with a target cell, the core of human immunodeficiency virus type 1 (HIV-1) enters into the cytoplasm, where uncoating occurs. The cone-shaped core is composed of the viral capsid protein (CA), which disassembles during uncoating. The underlying factors and mechanisms governing uncoating are poorly understood. Several CA mutations can cause changes in core stability and a block at reverse transcription, demonstrating the requirement for optimal core stability during viral replication. HIV-1 integrase (IN) catalyzes the insertion of the viral cDNA into the host genome, and certain IN mutations are pleiotropic. Similar to some CA mutants, two IN mutants, one with a complete deletion of IN (NL-ΔIN) and the other with a Cys-to-Ser substitution (NL-C130S), were noninfectious, with a replication block at reverse transcription. Compared to the wild type (WT), the cytoplasmic CA levels of the IN mutants in infected cells were reduced, suggesting accelerated uncoating. The role of IN during uncoating was examined by isolating and characterizing cores from NL-ΔIN and NL-C130S. Both IN mutants could form functional cores, but the core yield and stability were decreased. Also, virion incorporation of cyclophilin A (CypA), a cellular peptidyl-prolyl isomerase that binds specifically to CA, was decreased in the IN mutants. Cores isolated from WT virus depleted of CypA had an unstable-core phenotype, confirming a role of CypA in promoting optimal core stability. Taken together, our results indicate that IN is required during uncoating for maintaining CypA-CA interaction, which promotes optimal stability of the viral core.

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Insights on the role of nucleic acid/protein interactions in chaperoned nucleic acid rearrangements of HIV-1 reverse transcription

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0700166104 ◽

2007 ◽

Vol 104 (13) ◽

pp. 5261-5267 ◽

Cited By ~ 31

Author(s):

H.-W. Liu ◽

Y. Zeng ◽

C. F. Landes ◽

Y. J. Kim ◽

Y. Zhu ◽

...

Keyword(s):

Nucleic Acid ◽

Protein Interactions ◽

Reverse Transcription ◽

Acid Protein ◽

Hiv 1

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The Role of Capsid in the Early Steps of HIV-1 Infection: New Insights into the Core of the Matter

Viruses ◽

10.3390/v13061161 ◽

2021 ◽

Vol 13 (6) ◽

pp. 1161

Author(s):

Nawal Al Burtamani ◽

Alwin Paul ◽

Ariberto Fassati

Keyword(s):

Life Cycle ◽

Small Molecules ◽

Reverse Transcription ◽

Nuclear Import ◽

Protein A ◽

Host Factors ◽

Virus Life Cycle ◽

Experimental Approaches ◽

Hiv 1

In recent years, major advances in research and experimental approaches have significantly increased our knowledge on the role of the HIV-1 capsid in the virus life cycle, from reverse transcription to integration and gene expression. This makes the capsid protein a good pharmacological target to inhibit HIV-1 replication. This review covers our current understanding of the role of the viral capsid in the HIV-1 life cycle and its interaction with different host factors that enable reverse transcription, trafficking towards the nucleus, nuclear import and integration into host chromosomes. It also describes different promising small molecules, some of them in clinical trials, as potential targets for HIV-1 therapy.

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Subunit-Specific Analysis of the Human Immunodeficiency Virus Type 1 Reverse Transcriptase In Vivo

Journal of Virology ◽

10.1128/jvi.78.13.7089-7096.2004 ◽

2004 ◽

Vol 78 (13) ◽

pp. 7089-7096 ◽

Cited By ~ 17

Author(s):

Alok Mulky ◽

Stefan G. Sarafianos ◽

Edward Arnold ◽

Xiaoyun Wu ◽

John C. Kappes

Keyword(s):

Human Immunodeficiency Virus ◽

Reverse Transcriptase ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Reverse Transcription ◽

Coding Region ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is a heterodimer comprised of two structurally distinct subunits (p51 and p66). Since p51 and p66 are derived from the same coding region, subunit-specific structure-function studies of RT have been conducted exclusively by in vitro biochemical approaches. To study RT subunit function in the context of infectious virus, we constructed an LTR-vpr-p51-IRES-p66 expression cassette in which the HIV-1 vpr gene was fused in frame with p51, followed by an internal ribosome entry site (IRES) sequence and the p66 coding region. By coexpression with RT-deficient proviral DNA, we demonstrated that the p66 subunit is specifically and selectively packaged into virions as a Vpr-p51/p66 complex. Our analysis showed that cleavage by the viral protease liberates Vpr and generates functional heterodimeric RT (p51/p66) that supports HIV-1 reverse transcription and virus infection. By exploiting this novel trans-complementation approach, we demonstrated, for the first time with infectious virions, that the YMDD aspartates of p66 are both required and sufficient for RT polymerase function. Mutational analyses of the p51 YMDD aspartates indicated that they play an important structural role in p51 folding and subunit interactions that are required for the formation of an active RT heterodimer within infected cells. Understanding the role of the individual RT subunits in RNA- and DNA-dependent DNA synthesis is integral to our understanding of RT function. Our findings will lead to important new insights into the role of the p51 and p66 subunits in HIV-1 reverse transcription.

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K63-Linked Ubiquitin Is Required for Restriction of HIV-1 Reverse Transcription and Capsid Destabilization by Rhesus TRIM5α

Journal of Virology ◽

10.1128/jvi.00558-19 ◽

2019 ◽

Vol 93 (14) ◽

Cited By ~ 3

Author(s):

Sabrina Imam ◽

Sevnur Kömürlü ◽

Jessica Mattick ◽

Anastasia Selyutina ◽

Sarah Talley ◽

...

Keyword(s):

Reverse Transcription ◽

Adapter Protein ◽

Deubiquitinating Enzymes ◽

Retroviral Infection ◽

Autophagic Degradation ◽

Species Specific ◽

Autophagic Proteins ◽

Hiv 1 ◽

Viral Reverse Transcription

ABSTRACTTRIM5α is an antiviral restriction factor that inhibits retroviral infection in a species-specific fashion. TRIM5α binds to and forms assemblies around the retroviral capsid. Following binding, poorly understood, ubiquitin-dependent events lead to the disassembly of the viral core, prior to the accumulation of viral reverse transcription products in the target cell. It is also known that assemblies of TRIM5α and other TRIM family proteins can be targets of autophagic degradation. The goal of this study was to define the role of specific ubiquitin linkages in the retroviral restriction and autophagic degradation of TRIM5α and delineate any connection between these two processes. To this end, we generated fusion proteins in which the catalytic domains of different deubiquitinase (DUB) enzymes, with different specificities for polyubiquitinated linkages, were fused to the N-terminal RING domain of Rhesus macaque TRIM5α. We assessed the role of ubiquitination in restriction and the degree to which specific types of ubiquitination are required for the association of TRIM5α with autophagic proteins. We determined that K63-linked ubiquitination by TRIM5α is required to induce capsid disassembly and to inhibit reverse transcription of HIV, while the ability to inhibit HIV-1 infection was not dependent on K63-linked ubiquitination. We also observed that K63-linked ubiquitination is required for the association of TRIM5α with autophagosomal membranes and the autophagic adapter protein p62.IMPORTANCEAlthough the mechanisms by which TRIM5α can induce the abortive disassembly of retroviral capsids have remained obscure, numerous studies have suggested a role for ubiquitination and cellular degradative pathways. These studies have typically relied on global perturbation of cellular degradative pathways. Here, through the use of linkage-specific deubiquitinating enzymes tethered to TRIM5α, we delineate the ubiquitin linkages which drive specific steps in restriction and degradation by TRIM5α, providing evidence for a noncanonical role for K63-linked ubiquitin in the process of retroviral restriction by TRIM5α and potentially providing insight into the mechanism of action of other TRIM family proteins.

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