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

2007 ◽  
pp. 99-135 ◽  
Author(s):  
Truus E.M. Abbink ◽  
Ben Berkhout
Keyword(s):  
Reverse Transcription ◽  
Viral Genome ◽  
Close Encounters ◽  
Hiv 1

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

2002 ◽  
Vol 76 (17) ◽  
pp. 8518-8531 ◽  
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.

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

2020 ◽  
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.

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

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.

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

2012 ◽  
Vol 169 (2) ◽  
pp. 324-339 ◽  
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

scholarly journals The HIV-1 Capsid: From Structural Component to Key Factor for Host Nuclear Invasion

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 273
Author(s):  
Viviana Scoca ◽  
Francesca Di Nunzio
Keyword(s):  
Reverse Transcription ◽  
Viral Genome ◽  
Nuclear Translocation ◽  
Viral Capsid ◽  
Structural Protein ◽  
Nuclear Compartment ◽  
Transcription Process ◽  
Key Factor ◽  
Transcription Complexes ◽  
Hiv 1

Since the discovery of HIV-1, the viral capsid has been recognized to have an important role as a structural protein that holds the viral genome, together with viral proteins essential for viral life cycle, such as the reverse transcriptase (RT) and the integrase (IN). The reverse transcription process takes place between the cytoplasm and the nucleus of the host cell, thus the Reverse Transcription Complexes (RTCs)/Pre-integration Complexes (PICs) are hosted in intact or partial cores. Early biochemical assays failed to identify the viral CA associated to the RTC/PIC, possibly due to the stringent detergent conditions used to fractionate the cells or to isolate the viral complexes. More recently, it has been observed that some host partners of capsid, such as Nup153 and CPSF6, can only bind multimeric CA proteins organized in hexamers. Those host factors are mainly located in the nuclear compartment, suggesting the entrance of the viral CA as multimeric structure inside the nucleus. Recent data show CA complexes within the nucleus having a different morphology from the cytoplasmic ones, clearly highlighting the remodeling of the viral cores during nuclear translocation. Thus, the multimeric CA complexes lead the viral genome into the host nuclear compartment, piloting the intranuclear journey of HIV-1 in order to successfully replicate. The aim of this review is to discuss and analyze the main discoveries to date that uncover the viral capsid as a key player in the reverse transcription and PIC maturation until the viral DNA integration into the host genome.

scholarly journals Capsid Lattice Destabilization Leads to Premature Loss of the Viral Genome and Integrase Enzyme during HIV-1 Infection

2020 ◽  
Vol 95 (2) ◽  
pp. e00984-20
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 an 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.IMPORTANCE The 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.

scholarly journals The Annealing Mechanism of HIV-1 Reverse Transcription Primer onto the Viral Genome

2003 ◽  
Vol 279 (5) ◽  
pp. 3588-3595 ◽  
Author(s):  
Carine Tisné ◽  
Bernard P. Roques ◽  
Frédéric Dardel
Keyword(s):  
Reverse Transcription ◽  
Viral Genome ◽  
Reverse Transcription Primer ◽  
Hiv 1

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 ◽  
Viral Capsid ◽  
Target Cells ◽  
Force Microscopy ◽  
The Core ◽  
Atomic Force ◽  
Successful Infection ◽  
Capsid Shell ◽  
Hiv 1

The 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. These reverse-transcription-induced changes in the capsid progressively remodel the viral core to prime it for temporally accurate uncoating in target cells. Importance For successful infection, the HIV-1 genome, which is enclosed inside a capsid shell, must be reverse transcribed into double-stranded DNA and released from the capsid (in a process known as uncoating) before it can be integrated into the target cell genome. The mechanism of HIV-1 uncoating is a pivotal question of long standing. Using atomic force microscopy to analyze individual HIV-1 cores during reverse transcription, we observe a reproducible pattern of stiffness spikes. These spikes were shown to be associated with distinct stages of the reverse transcription reaction. Our findings suggest that these reverse-transcription-induced alterations gradually prepared the core for uncoating at the right time and location in target cells.

scholarly journals SUMOylation of SAMHD1 at Lysine 595 is required for HIV-1 restriction in non-cycling cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Charlotte Martinat ◽  
Arthur Cormier ◽  
Joëlle Tobaly-Tapiero ◽  
Noé Palmic ◽  
Nicoletta Casartelli ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Reverse Transcription ◽  
Immune Cells ◽  
Viral Restriction ◽  
Antiviral Function ◽  
Hiv 1 ◽  
Constitutively Active

AbstractSAMHD1 is a cellular triphosphohydrolase (dNTPase) proposed to inhibit HIV-1 reverse transcription in non-cycling immune cells by limiting the supply of the dNTP substrates. Yet, phosphorylation of T592 downregulates SAMHD1 antiviral activity, but not its dNTPase function, implying that additional mechanisms contribute to viral restriction. Here, we show that SAMHD1 is SUMOylated on residue K595, a modification that relies on the presence of a proximal SUMO-interacting motif (SIM). Loss of K595 SUMOylation suppresses the restriction activity of SAMHD1, even in the context of the constitutively active phospho-ablative T592A mutant but has no impact on dNTP depletion. Conversely, the artificial fusion of SUMO2 to a non-SUMOylatable inactive SAMHD1 variant restores its antiviral function, a phenotype that is reversed by the phosphomimetic T592E mutation. Collectively, our observations clearly establish that lack of T592 phosphorylation cannot fully account for the restriction activity of SAMHD1. We find that SUMOylation of K595 is required to stimulate a dNTPase-independent antiviral activity in non-cycling immune cells, an effect that is antagonized by cyclin/CDK-dependent phosphorylation of T592 in cycling cells.

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