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 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 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 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 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 HIV-1 Uncoating and Reverse Transcription Require eEF1A Binding to Surface-Exposed Acidic Residues of the Reverse Transcriptase Thumb Domain

mBio ◽  
2018 ◽  
Vol 9 (2) ◽  
pp. e00316-18 ◽  
Author(s):  
Daniel J. Rawle ◽  
Dongsheng Li ◽  
Joakim E. Swedberg ◽  
Lu Wang ◽  
Dinesh C. Soares ◽  
...  
Keyword(s):  
T Cells ◽  
Reverse Transcriptase ◽  
Host Cell ◽  
Reverse Transcription ◽  
Genomic Rna ◽  
Viral Core ◽  
Thumb Domain ◽  
Acidic Residues ◽  
Host Cell Factors ◽  
Hiv 1

ABSTRACTOnce HIV-1 enters a cell, the viral core is uncoated by a poorly understood mechanism and the HIV-1 genomic RNA is reverse transcribed into DNA. Host cell factors are essential for these processes, although very few reverse transcription complex binding host cell factors have been convincingly shown to affect uncoating or reverse transcription. We previously reported that cellular eukaryotic translation elongation factor 1A (eEF1A) interacts tightly and directly with HIV-1 reverse transcriptase (RT) for more efficient reverse transcription. Here we report that the surface-exposed acidic residues in the HIV-1 RT thumb domain alpha-J helix and flanking regions are important for interaction with eEF1A. Mutation of surface-exposed acidic thumb domain residues D250, E297, E298, and E300 to arginine resulted in various levels of impairment of the interaction between RT and eEF1A. This indicates that this negatively charged region in the RT thumb domain is important for interaction with the positively charged eEF1A protein. The impairment of RT and eEF1A interaction by the RT mutations correlated with the efficiency of reverse transcription, uncoating, and infectivity. The best example of this is the strictly conserved E300 residue, where mutation significantly impaired the interaction of RT with eEF1A and virus replication in CD4+T cells without affectingin vitroRT catalytic activity, RT heterodimerization, or RNase H activity. This study demonstrated that the interaction between surface-exposed acidic residues of the RT thumb domain and eEF1A is important for HIV-1 uncoating, reverse transcription, and replication.IMPORTANCEHIV-1, like all viruses, requires host cell proteins for its replication. Understanding the mechanisms behind virus-host interactions can lay the foundation for future novel therapeutic developments. Our lab has identified eEF1A as a key HIV-1 RT binding host protein that is important for the reverse transcription of HIV-1 genomic RNA into DNA. Here we identify the first surface-exposed RT residues that underpin interactions with eEF1A. Mutation of one strictly conserved RT residue (E300R) delayed reverse transcription and viral core uncoating and strongly inhibited HIV-1 replication in CD4+T cells. This study advances the structural and mechanistic detail of the key RT-eEF1A interaction in HIV-1 infection and indicates its importance in uncoating for the first time. This provides a further basis for the development of an RT-eEF1A interaction-inhibiting anti-HIV-1 drug and suggests that the surface-exposed acidic patch of the RT thumb domain may be an attractive drug target.

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 Extended Interactions between HIV-1 Viral RNA and tRNALys3 Are Important to Maintain Viral RNA Integrity

2020 ◽  
Vol 22 (1) ◽  
pp. 58
Author(s):  
Thomas Gremminger ◽  
Zhenwei Song ◽  
Juan Ji ◽  
Avery Foster ◽  
Kexin Weng ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Potential Interaction ◽  
Viral Rna ◽  
Primer Binding Site ◽  
Rna Integrity ◽  
Immunodeficiency Virus ◽  
Infected Cells ◽  
Extended Interaction ◽  
Hiv 1

The reverse transcription of the human immunodeficiency virus 1 (HIV-1) initiates upon annealing of the 3′-18-nt of tRNALys3 onto the primer binding site (PBS) in viral RNA (vRNA). Additional intermolecular interactions between tRNALys3 and vRNA have been reported, but their functions remain unclear. Here, we show that abolishing one potential interaction, the A-rich loop: tRNALys3 anticodon interaction in the HIV-1 MAL strain, led to a decrease in viral infectivity and reduced the synthesis of reverse transcription products in newly infected cells. In vitro biophysical and functional experiments revealed that disruption of the extended interaction resulted in an increased affinity for reverse transcriptase (RT) and enhanced primer extension efficiency. In the absence of deoxyribose nucleoside triphosphates (dNTPs), vRNA was degraded by the RNaseH activity of RT, and the degradation rate was slower in the complex with the extended interaction. Consistently, the loss of vRNA integrity was detected in virions containing A-rich loop mutations. Similar results were observed in the HIV-1 NL4.3 strain, and we show that the nucleocapsid (NC) protein is necessary to promote the extended vRNA: tRNALys3 interactions in vitro. In summary, our data revealed that the additional intermolecular interaction between tRNALys3 and vRNA is likely a conserved mechanism among various HIV-1 strains and protects the vRNA from RNaseH degradation in mature virions.

The Mauriceville plasmid of Neurospora crassa: characterization of a novel reverse transcriptase that begins cDNA synthesis at the 3' end of template RNA

1992 ◽  
Vol 12 (11) ◽  
pp. 5131-5144
Author(s):  
H Wang ◽  
J C Kennell ◽  
M T Kuiper ◽  
J R Sabourin ◽  
R Saldanha ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Reverse Transcription ◽  
Full Length ◽  
Minus Strand ◽  
Ribonucleoprotein Particle ◽  
Cdna Synthesis ◽  
Reverse Transcriptases ◽  
In Vitro Transcripts

The Mauriceville and Varkud plasmids are retroid elements that propagate in the mitochondria of some Neurospora spp. strains. Previous studies of endogenous reactions in ribonucleoprotein particle preparations suggested that the plasmids use a novel mechanism of reverse transcription that involves synthesis of a full-length minus-strand DNA beginning at the 3' end of the plasmid transcript, which has a 3' tRNA-like structure (M. T. R. Kuiper and A. M. Lambowitz, Cell 55:693-704, 1988). In this study, we developed procedures for releasing the Mauriceville plasmid reverse transcriptase from mitochondrial ribonucleoprotein particles and partially purifying it by heparin-Sepharose chromatography. By using these soluble preparations, we show directly that the Mauriceville plasmid reverse transcriptase synthesizes full-length cDNA copies of in vitro transcripts beginning at the 3' end and has a preference for transcripts having the 3' tRNA-like structure. Further, unlike retroviral reverse transcriptases, the Mauriceville plasmid reverse transcriptase begins cDNA synthesis directly opposite the 3'-terminal nucleotide of the template RNA. The ability to initiate cDNA synthesis directly at the 3' end of template RNAs may also be relevant to the mechanisms of reverse transcription used by LINEs, group II introns, and other non-long terminal repeat retroid elements.

scholarly journals A Novel Leu92 Mutant of HIV-1 Reverse Transcriptase with a Selective Deficiency in Strand Transfer Causes a Loss of Viral Replication

2015 ◽  
Vol 89 (16) ◽  
pp. 8119-8129 ◽  
Author(s):  
Eytan Herzig ◽  
Nickolay Voronin ◽  
Nataly Kucherenko ◽  
Amnon Hizi
Keyword(s):  
Life Cycle ◽  
Viral Replication ◽  
Reverse Transcriptase ◽  
Dna Polymerase ◽  
Reverse Transcription ◽  
Polymerase Activity ◽  
Rnase H ◽  
Strand Transfer ◽  
Hiv 1

ABSTRACTThe process of reverse transcription (RTN) in retroviruses is essential to the viral life cycle. This key process is catalyzed exclusively by the viral reverse transcriptase (RT) that copies the viral RNA into DNA by its DNA polymerase activity, while concomitantly removing the original RNA template by its RNase H activity. During RTN, the combination between DNA synthesis and RNA hydrolysis leads to strand transfers (or template switches) that are critical for the completion of RTN. The balance between these RT-driven activities was considered to be the sole reason for strand transfers. Nevertheless, we show here that a specific mutation in HIV-1 RT (L92P) that does not affect the DNA polymerase and RNase H activities abolishes strand transfer. There is also a good correlation between this complete loss of the RT's strand transfer to the loss of the DNA clamp activity of the RT, discovered recently by us. This finding indicates a mechanistic linkage between these two functions and that they are both direct and unique functions of the RT (apart from DNA synthesis and RNA degradation). Furthermore, when the RT's L92P mutant was introduced into an infectious HIV-1 clone, it lost viral replication, due to inefficient intracellular strand transfers during RTN, thus supporting thein vitrodata. As far as we know, this is the first report on RT mutants that specifically and directly impair RT-associated strand transfers. Therefore, targeting residue Leu92 may be helpful in selectively blocking this RT activity and consequently HIV-1 infectivity and pathogenesis.IMPORTANCEReverse transcription in retroviruses is essential for the viral life cycle. This multistep process is catalyzed by viral reverse transcriptase, which copies the viral RNA into DNA by its DNA polymerase activity (while concomitantly removing the RNA template by its RNase H activity). The combination and balance between synthesis and hydrolysis lead to strand transfers that are critical for reverse transcription completion. We show here for the first time that a single mutation in HIV-1 reverse transcriptase (L92P) selectively abolishes strand transfers without affecting the enzyme's DNA polymerase and RNase H functions. When this mutation was introduced into an infectious HIV-1 clone, viral replication was lost due to an impaired intracellular strand transfer, thus supporting thein vitrodata. Therefore, finding novel drugs that target HIV-1 reverse transcriptase Leu92 may be beneficial for developing new potent and selective inhibitors of retroviral reverse transcription that will obstruct HIV-1 infectivity.

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