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

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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Structural and Functional Role of INI1, a Cellular Cofactor of HIV-1 integrase in the Early Step of HIV Infection

Biophysical Journal ◽

10.1016/j.bpj.2010.12.1968 ◽

2011 ◽

Vol 100 (3) ◽

pp. 324a

Author(s):

Benoit Maillot ◽

Corinne Crucifix ◽

Sylvia Eiler ◽

Karine Pradeau ◽

Nicolas Levy ◽

...

Keyword(s):

Hiv Infection ◽

Functional Role ◽

Early Step ◽

Cellular Cofactor ◽

Hiv 1

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

10.1101/2021.01.16.426924 ◽

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.

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Going beyond Integration: The Emerging Role of HIV-1 Integrase in Virion Morphogenesis

Viruses ◽

10.3390/v12091005 ◽

2020 ◽

Vol 12 (9) ◽

pp. 1005 ◽

Cited By ~ 2

Author(s):

Jennifer L. Elliott ◽

Sebla B. Kutluay

Keyword(s):

Life Cycle ◽

Viral Genome ◽

Critical Role ◽

Viral Rna ◽

Target Cells ◽

Host Chromosome ◽

Virion Morphogenesis ◽

Rna Genome ◽

Hiv 1

The HIV-1 integrase enzyme (IN) plays a critical role in the viral life cycle by integrating the reverse-transcribed viral DNA into the host chromosome. This function of IN has been well studied, and the knowledge gained has informed the design of small molecule inhibitors that now form key components of antiretroviral therapy regimens. Recent discoveries unveiled that IN has an under-studied yet equally vital second function in human immunodeficiency virus type 1 (HIV-1) replication. This involves IN binding to the viral RNA genome in virions, which is necessary for proper virion maturation and morphogenesis. Inhibition of IN binding to the viral RNA genome results in mislocalization of the viral genome inside the virus particle, and its premature exposure and degradation in target cells. The roles of IN in integration and virion morphogenesis share a number of common elements, including interaction with viral nucleic acids and assembly of higher-order IN multimers. Herein we describe these two functions of IN within the context of the HIV-1 life cycle, how IN binding to the viral genome is coordinated by the major structural protein, Gag, and discuss the value of targeting the second role of IN in virion morphogenesis.

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The HIV-1 Pre-Integration Complex: Structural and Functional Role of INI1 and LEDGF, Cellular Cofactors of Viral Integrase

Biophysical Journal ◽

10.1016/j.bpj.2011.11.3484 ◽

2012 ◽

Vol 102 (3) ◽

pp. 640a

Author(s):

Benoit Maillot ◽

Sylvia Eiler ◽

Corinne Crucifix ◽

Nicolas Levy ◽

Karine Pradeau ◽

...

Keyword(s):

Functional Role ◽

Complex Structural ◽

Hiv 1

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Molecular Characterization of Preintegration Latency in Human Immunodeficiency Virus Type 1 Infection

Journal of Virology ◽

10.1128/jvi.76.17.8518-8513.2002 ◽

2002 ◽

Vol 76 (17) ◽

pp. 8518-8531 ◽

Cited By ~ 169

Author(s):

Theodore C. Pierson ◽

Yan Zhou ◽

Tara L. Kieffer ◽

Christian T. Ruff ◽

Christopher Buck ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

T Cells ◽

T Cell ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Reverse Transcription ◽

Viral Genome ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Most current evidence suggests that the infection of resting CD4+ T cells by human immunodeficiency virus type 1 (HIV-1) is not productive due to partial or complete blocks in the viral life cycle at steps prior to integration of the viral genome into the host cell chromosome. However, stimulation of an infected resting T cell by antigen, cytokines, or microenvironmental factors can overcome these blocks and allow for the production of progeny virions. In this study, we sought to understand the structure and fate of the virus in unstimulated resting CD4+ T cells. Using a novel linker-mediated PCR assay designed to detect and characterize linear unintegrated forms of the HIV-1 genome, we demonstrate that reverse transcription can proceed to completion following the infection of resting T cells, generating the substrate for the retroviral integration reaction. However, reverse transcription in resting T cells is far slower than in activated T cells, requiring 2 to 3 days to complete. The delay in completing reverse transcription may make the viral DNA genome more susceptible to competing decay processes. To explore the relationship between the formation of the linear viral genome and the stability of the preintegration state, we employed a recombinant HIV-1 virus expressing the enhanced green fluorescent protein to measure the rate at which HIV-1 decays in the preintegration state. Our results demonstrate that the preintegration state is labile and decays rapidly (half-life = 1 day) following the entry of HIV-1 into a resting T cell, with significant decay occurring during the slow process of reverse transcription.

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Capsid lattice destabilization leads to premature loss of the viral genome and integrase enzyme during HIV-1 infection

10.1101/2020.03.26.009902 ◽

2020 ◽

Cited By ~ 1

Author(s):

Jenna E. Eschbach ◽

Jennifer L. Elliott ◽

Wen Li ◽

Kaneil K. Zadrozny ◽

Keanu Davis ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Reverse Transcription ◽

Viral Genome ◽

Target Cells ◽

Viral Core ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACTThe human immunodeficiency virus type 1 (HIV-1) capsid (CA) protein forms a conical lattice around the viral ribonucleoprotein complex (vRNP) consisting of a dimeric viral genome and associated proteins, together constituting the viral core. Upon entry into target cells, the viral core undergoes a process termed uncoating, during which CA molecules are shed from the lattice. Although the timing and degree of uncoating are important for reverse transcription and integration, the molecular basis of this phenomenon remains unclear. Using complementary approaches, we assessed the impact of core destabilization on the intrinsic stability of the CA lattice in vitro and fates of viral core components in infected cells. We found that substitutions in CA can impact the intrinsic stability of the CA lattice in vitro in the absence of vRNPs, which mirrored findings from assessment of CA stability in virions. Altering CA stability tended to increase the propensity to form morphologically aberrant particles, in which the vRNPs were mislocalized between the CA lattice and the viral lipid envelope. Importantly, destabilization of the CA lattice led to premature dissociation of CA from vRNPs in target cells, which was accompanied by proteasomal-independent losses of the viral genome and integrase enzyme. Overall, our studies show that the CA lattice protects the vRNP from untimely degradation in target cells and provide the mechanistic basis of how CA stability influences reverse transcription.AUTHOR SUMMARYThe human immunodeficiency virus type 1 (HIV-1) capsid (CA) protein forms a conical lattice around the viral RNA genome and the associated viral enzymes and proteins, together constituting the viral core. Upon infection of a new cell, viral cores are released into the cytoplasm where they undergo a process termed “uncoating”, i.e. shedding of CA molecules from the conical lattice. Although proper and timely uncoating has been shown to be important for reverse transcription, the molecular mechanisms that link these two events remain poorly understood. In this study, we show that destabilization of the CA lattice leads to premature dissociation of CA from viral cores, which exposes the viral genome and the integrase enzyme for degradation in target cells. Thus, our studies demonstrate that the CA lattice protects the viral ribonucleoprotein complexes from untimely degradation in target cells and provide the first causal link between how CA stability affects reverse transcription.

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HIV‐1 Reverse Transcription: Close Encounters Between the Viral Genome and a Cellular tRNA

Advances in Pharmacology - HIV‐1: Molecular Biology and Pathogenesis Viral Mechanisms, Second Edition ◽

10.1016/s1054-3589(07)55003-9 ◽

2007 ◽

pp. 99-135 ◽

Cited By ~ 13

Author(s):

Truus E.M. Abbink ◽

Ben Berkhout

Keyword(s):

Reverse Transcription ◽

Viral Genome ◽

Close Encounters ◽

Hiv 1

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Nuclear Import of the HIV-1 Core Precedes Reverse Transcription and Uncoating

10.1101/2020.03.31.018747 ◽

2020 ◽

Author(s):

Anastasia Selyutina ◽

Mirjana Persaud ◽

Kyeongeun Lee ◽

Vineet KewalRamani ◽

Felipe Diaz-Griffero

Keyword(s):

Reverse Transcription ◽

Nuclear Import ◽

Viral Genome ◽

Cellular Compartment ◽

Nuclear Compartment ◽

Nuclear Compartments ◽

The Core ◽

Biochemical Assays ◽

Infected Cells ◽

Hiv 1

SUMMARYHIV-1 particles contain a core formed by ~1500 capsid protein monomers housing viral RNA. HIV-1 core uncoating---disassembly---is required for infection. HIV-1 reverse transcription (RT) occurs before or during uncoating, but the cellular compartment where RT and uncoating occurs is unknown. Using imaging and biochemical assays to track HIV-1 capsids in nuclei during infection, we demonstrated that higher-order capsid complexes or complete cores containing viral genome are imported into nuclear compartments. Additionally, inhibition of RT that stabilizes the core during infection does not prevent capsid nuclear import; thus, RT may occur in nuclear compartments. We separated infected cells into cytosolic and nuclear fractions to measure RT during infection. Most observable RT intermediates were enriched in nuclear fractions, suggesting that most HIV-1 RT occurs in the nuclear compartment alongside uncoating. Thus, nuclear import precedes RT and uncoating, fundamentally changing our understanding of HIV-1 infection.

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