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 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 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 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 Host Cell Metabolite Inositol Hexakisphosphate Promotes Efficient Endogenous HIV-1 Reverse Transcription by Stabilizing the Viral Capsid

mBio ◽  
2020 ◽  
Vol 11 (6) ◽  
Author(s):  
Jordan Jennings ◽  
Jiong Shi ◽  
Janani Varadarajan ◽  
Parker J. Jamieson ◽  
Christopher Aiken
Keyword(s):  
Host Cell ◽  
Reverse Transcription ◽  
Full Length ◽  
Viral Capsid ◽  
Inositol Hexakisphosphate ◽  
Antiviral Compound ◽  
Viral Core ◽  
Double Stranded Dna ◽  
Hiv 1

ABSTRACT A defining activity of retroviruses is reverse transcription, the process by which the viral genomic RNA is converted into the double-stranded DNA required for virus replication. Reverse transcriptase (RT), the viral enzyme responsible for this process, was identified in 1970 by assaying permeabilized retrovirus particles for DNA synthesis in vitro. Such reactions are inefficient, with only a small fraction of viral genomes being converted to full-length double-stranded DNA molecules, possibly owing to disruption of the structure of the viral core. Here, we show that reverse transcription in purified HIV-1 cores is enhanced by the addition of the capsid-binding host cell metabolite inositol hexakisphosphate (IP6). IP6 potently enhanced full-length minus-strand synthesis, as did hexacarboxybenzene (HCB), which also stabilizes the HIV-1 capsid. Both IP6 and HCB stabilized the association of the viral CA and RT proteins with HIV-1 cores. In contrast to the wild type, cores isolated from mutant HIV-1 particles containing intrinsically hyperstable capsids exhibited relatively efficient reverse transcription in the absence of IP6, further indicating that the compound promotes reverse transcription by stabilizing the viral capsid. We also observed that the capsid-destabilizing antiviral compound PF74 inhibited endogenous reverse transcription with a potency that mirrors its ability to inhibit reverse transcription during infection. Our results show that the stabilization of the HIV-1 capsid permits efficient reverse transcription in HIV-1 cores, providing a sensitive experimental system for analyzing the functions of viral and host cell molecules and the role of capsid disassembly (uncoating) in the process. IMPORTANCE HIV-1 infection requires reverse transcription of the viral genome. While much is known about the biochemistry of reverse transcription from simplified biochemical reactions, reverse transcription during infection takes place within a viral core. However, endogenous reverse transcription reactions using permeabilized HIV-1 virions or purified viral cores have been inefficient. Using viral cores purified from infectious HIV-1 particles, we show that efficient reverse transcription is achieved in vitro by addition of the capsid-stabilizing metabolite inositol hexakisphosphate. The enhancement of reverse transcription was linked to the capsid-stabilizing effect of the compound, consistent with the known requirement for an intact or semi-intact viral capsid for HIV-1 infection. Our results establish a biologically relevant system for dissecting the function of the viral capsid and its disassembly during reverse transcription. The system should also prove useful for mechanistic studies of capsid-targeting antiviral drugs.

scholarly journals Chemical Stabilization of the HIV-1 Capsid Results in Efficient HIV-1 Reverse Transcription in vitro

2020 ◽  
Author(s):  
Jordan Jennings ◽  
Jiong Shi ◽  
Janani Varadarajan ◽  
Parker J. Jamieson ◽  
Christopher Aiken
Keyword(s):  
Host Cell ◽  
Reverse Transcription ◽  
Experimental System ◽  
Full Length ◽  
Viral Capsid ◽  
Inositol Hexakisphosphate ◽  
Viral Core ◽  
Hiv 1 ◽  
Double Strand Dna

ABSTRACTA defining activity of retroviruses is reverse transcription, the process during which the viral genomic RNA is converted into the double strand DNA required for virus replication. Reverse transcriptase (RT), the viral enzyme responsible for this process, was identified in 1970 by assaying permeabilized retrovirus particles for DNA synthesis in vitro. Such reactions are inefficient with only a small fraction of viral genomes being converted to full-length double strand DNA molecules, possibly owing to disruption of the structure of the viral core. Here we show that reverse transcription in purified HIV-1 cores is enhanced by the addition of the capsid-binding host cell metabolite inositol hexakisphosphate (IP6). IP6 potently enhanced full-length minus strand synthesis, as did hexacarboxybenzene (HCB) which also stabilizes the HIV-1 capsid. Both IP6 and HCB stabilized the association of the viral CA and RT proteins with HIV-1 cores. In contrast to the wild type, cores isolated from mutant HIV-1 particles containing intrinsically hyperstable capsids exhibited efficient reverse transcription in the absence of IP6, further indicating that the compound promotes reverse transcription by stabilizing the viral capsid. Our results show that stabilization of the HIV-1 capsid permits efficient reverse transcription in HIV-1 cores, providing a sensitive experimental system for analyzing the functions of viral and host cell molecules and the role of capsid disassembly (uncoating) in the process.IMPORTANCEHIV-1 infection requires reverse transcription of the viral genome. While much is known about the biochemistry of reverse transcription from simplified biochemical reactions, reverse transcription during infection takes place within a viral core. However, endogenous reverse transcription reactions using permeabilized virions or purified viral cores have been inefficient. Using viral cores purified from infectious HIV-1 particles, we show that efficient reverse transcription is achieved in vitro by addition of the capsid-stabilizing metabolite inositol hexakisphosphate. Enhancement of reverse transcription was linked to the capsid-stabilizing effect of the compound, consistent with the known requirement for an intact or semi-intact viral capsid for HIV-1 infection. Our results establish a biologically relevant system for dissecting the function of the viral capsid and its disassembly during reverse transcription. The system may also prove useful for mechanistic studies of emerging capsid-targeting antiviral drugs.

scholarly journals Bicaudal D2 facilitates the cytoplasmic trafficking and nuclear import of HIV-1 genomes during infection

2017 ◽  
Vol 114 (50) ◽  
pp. E10707-E10716 ◽  
Author(s):  
Adarsh Dharan ◽  
Silvana Opp ◽  
Omar Abdel-Rahim ◽  
Sevnur Komurlu Keceli ◽  
Sabrina Imam ◽  
...  
Keyword(s):  
Viral Genome ◽  
Target Cells ◽  
Adapter Protein ◽  
Nuclear Entry ◽  
Microtubule Network ◽  
Monocytic Cell ◽  
Monocytic Cell Line ◽  
Microtubule Motor ◽  
Hiv 1

Numerous viruses, including HIV-1, exploit the microtubule network to traffic toward the nucleus during infection. Although numerous studies have observed a role for the minus-end microtubule motor dynein in HIV-1 infection, the mechanism by which the viral core containing the viral genome associates with dynein and induces its perinuclear trafficking has remained unclear. Here, we report that the dynein adapter protein bicaudal D2 (BICD2) is able to interact with HIV-1 viral cores in target cells. We also observe that BICD2 can bind in vitro-assembled capsid tubes through its CC3 domain. We observe that BICD2 facilitates infection by promoting the trafficking of viral cores to the nucleus, thereby promoting nuclear entry of the viral genome and infection. Finally, we observe that depletion of BICD2 in the monocytic cell line THP-1 results in an induction of IFN-stimulated genes in these cells. Collectively, these results identify a microtubule adapter protein critical for trafficking of HIV-1 in the cytoplasm of target cells and evasion of innate sensing mechanisms in macrophages.

scholarly journals The HIV-1 capsid and reverse transcription

Retrovirology ◽  
2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Christopher Aiken ◽  
Itay Rousso
Keyword(s):  
Small Molecule ◽  
Reverse Transcription ◽  
Viral Capsid ◽  
Biophysical Properties ◽  
Transcription In Vitro ◽  
Hiv 1

AbstractThe viral capsid plays a key role in HIV-1 reverse transcription. Recent studies have demonstrated that the small molecule IP6 dramatically enhances reverse transcription in vitro by stabilizing the viral capsid. Reverse transcription results in marked changes in the biophysical properties of the capsid, ultimately resulting in its breakage and disassembly. Here we review the research leading to these advances and describe hypotheses for capsid-dependent HIV-1 reverse transcription and a model for reverse transcription-primed HIV-1 uncoating.

scholarly journals TRIM5α Disrupts the Structure of Assembled HIV-1 Capsid Complexes In Vitro

2010 ◽  
Vol 84 (13) ◽  
pp. 6564-6569 ◽  
Author(s):  
Lesa R. Black ◽  
Christopher Aiken
Keyword(s):  
Mammalian Cells ◽  
Restriction Factor ◽  
Viral Capsid ◽  
Target Cells ◽  
Host Restriction ◽  
Host Restriction Factor ◽  
Retroviral Infections ◽  
Insight Into ◽  
Hiv 1

ABSTRACT The host restriction factor TRIM5α provides intrinsic defense against retroviral infections in mammalian cells. TRIM5α blocks infection by targeting the viral capsid after entry but prior to completion of reverse transcription, but whether this interaction directly alters the structure of the viral capsid is unknown. A previous study reported that rhesus macaque TRIM5α protein stably associates with cylindrical complexes formed by assembly of recombinant HIV-1 CA-NC protein in vitro and that restriction leads to accelerated HIV-1 uncoating in target cells. To gain further insight into the mechanism of TRIM5α-dependent restriction, we examined the structural effects of TRIM5 proteins on preassembled CA-NC complexes by electron microscopy. Incubation of assembled complexes with lysate of cells expressing the restrictive rhesus TRIM5α protein resulted in marked disruption of the normal cylindrical structure of the complexes. In contrast, incubation with lysate of control cells or cells expressing comparable levels of the nonrestrictive human TRIM5α protein had little effect on the complexes. Incubation with lysate of cells expressing the TRIMCyp restriction factor also disrupted the cylinders. The effect of TRIMCyp was prevented by the addition of cyclosporine, which inhibits binding of TRIMCyp to the HIV-1 capsid. Thus, disruption of CA-NC cylinders by TRIM5α and TRIMCyp was correlated with the specificity of restriction. Collectively, these results suggest that TRIM5α-dependent restriction of HIV-1 infection results from structural perturbation of the viral capsid leading to aberrant HIV-1 uncoating in target cells.

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 Reverse Transcription Mechanically Initiates HIV-1 Capsid Disassembly

2017 ◽  
Vol 91 (12) ◽  
Author(s):  
Sanela Rankovic ◽  
Janani Varadarajan ◽  
Ruben Ramalho ◽  
Christopher Aiken ◽  
Itay Rousso
Keyword(s):  
Atomic Force Microscopy ◽  
Reverse Transcription ◽  
Target Cells ◽  
Filamentous Structure ◽  
Force Microscopy ◽  
The Core ◽  
Atomic Force ◽  
Capsid Shell ◽  
Hiv 1 ◽  
Capsid Disassembly

ABSTRACT The HIV-1 core consists of the viral genomic RNA and several viral proteins encased within a conical capsid. After cell entry, the core disassembles in a process termed uncoating. Although HIV-1 uncoating has been linked to reverse transcription of the viral genome in target cells, the mechanism by which uncoating is initiated is unknown. Using time-lapse atomic force microscopy, we analyzed the morphology and physical properties of isolated HIV-1 cores during the course of reverse transcription in vitro. We found that, during an early stage of reverse transcription the pressure inside the capsid increases, reaching a maximum after 7 h. High-resolution mechanical mapping reveals the formation of a stiff coiled filamentous structure underneath the capsid surface. Subsequently, this coiled structure disappears, the stiffness of the capsid drops precipitously to a value below that of a pre-reverse transcription core, and the capsid undergoes partial or complete rupture near the narrow end of the conical structure. We propose that the transcription of the relatively flexible single-stranded RNA into a more rigid filamentous structure elevates the pressure within the core, which triggers the initiation of capsid disassembly. IMPORTANCE For successful infection, the HIV-1 genome, which is in the form of a single-stranded RNA 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 that triggers uncoating is a pivotal question of long standing. By using atomic force microscopy, we found that during reverse transcription the pressure inside the capsid increases until the internal stress exceeds the strength of the capsid structure and the capsid breaks open. The application of AFM technologies to study purified HIV-1 cores represents a new experimental platform for elucidating additional aspects of capsid disassembly and HIV-1 uncoating.

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