Permeability of the HIV-1 capsid to metabolites modulates viral DNA synthesis

Xu, Chaoyi; Fisher, Douglas A.; Rankovic, Sanela; Li, Wen; Dick, R L; Runge, Brent; Zadorozhnyi, Roman; Ahn, Jin-Woo; Aiken, Chris; Polenova, Tatyana; Engelman, Alan; Ambrose, Zandrea; Rousso, Itay; Perilla, Juan R.

doi:10.1101/2020.04.30.071217

Cited by 13 publications

(10 citation statements)

References 55 publications

(62 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Together, these observations support a capsid stabilization mechanism for enhancement of ERT by IP6. In addition, studies involving computational modeling have suggested that IP6 binding to the HIV-1 capsid also facilitates the import of dNTPs into the viral core, an effect that may contribute to the enhancement of ERT observed in our experiments (34,35).…”

Section: Discussionmentioning

confidence: 63%

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

Jennings

Shi

Varadarajan

et al. 2020

mBio

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Section: Discussionmentioning

confidence: 63%

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

Jennings

Shi

Varadarajan

et al. 2020

mBio

Self Cite

View full text Add to dashboard Cite

show abstract

“…Subsequently, we observed that treatment of HIV-1 cores with IP6 appeared to accelerate the process, resulting in more rapid uncoating and possibly also reverse transcription (18). Under these conditions, the previously observed single stiffness peak was not apparent, perhaps owing to the low temporal resolution of the averaged stiffness measurements (~2 h) employed in that study, which may have obscured the more rapid aspects of the reactions.…”

Section: Analysis Of the Stiffness And Morphology Of Isolated Cores Umentioning

confidence: 71%

“…Recently, we used AFM to observe the physical changes in the HIV-1 core occurring during uncoating in vitro. We found that addition of capsid-stabilizing ligands, including the host protein cyclophilin A, the small molecule antiviral compound PF74, and the cell metabolite IP6, increased the stiffness of cores (18)(19)(20).…”

Section: Introductionmentioning

confidence: 95%

See 1 more Smart Citation

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

Rankovic

Deshpande

Harel

et al. 2021

Preprint

Self Cite

View full text Add to dashboard Cite

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. 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.

show abstract

“…IP6 also enhances mature capsid assembly and core stability leading to increased efficiency of encapsidated reverse transcription. Binding of IP6 to capsids is mediated by an electropositive pore that binds and facilitates the transport of deoxynucleotide triphosphates (dNTPs) into the capsid [ 51 , 52 ]. These data suggest a model in which IP6 facilitates assembly of the immature Gag lattice by stabilizing the six-helix bundle and is effectively packaged into virions.…”

Section: Introductionmentioning

confidence: 99%

HIV-1 Maturation: Lessons Learned from Inhibitors

Kleinpeter

Freed

2020

Viruses

View full text Add to dashboard Cite

Since the emergence of HIV and AIDS in the early 1980s, the development of safe and effective therapies has accompanied a massive increase in our understanding of the fundamental processes that drive HIV biology. As basic HIV research has informed the development of novel therapies, HIV inhibitors have been used as probes for investigating basic mechanisms of HIV-1 replication, transmission, and pathogenesis. This positive feedback cycle has led to the development of highly effective combination antiretroviral therapy (cART), which has helped stall the progression to AIDS, prolong lives, and reduce transmission of the virus. However, to combat the growing rates of virologic failure and toxicity associated with long-term therapy, it is important to diversify our repertoire of HIV-1 treatments by identifying compounds that block additional steps not targeted by current drugs. Most of the available therapeutics disrupt early events in the replication cycle, with the exception of the protease (PR) inhibitors, which act at the virus maturation step. HIV-1 maturation consists of a series of biochemical changes that facilitate the conversion of an immature, noninfectious particle to a mature infectious virion. These changes include proteolytic processing of the Gag polyprotein by the viral protease (PR), structural rearrangement of the capsid (CA) protein, and assembly of individual CA monomers into hexamers and pentamers that ultimately form the capsid. Here, we review the development and therapeutic potential of maturation inhibitors (MIs), an experimental class of anti-HIV-1 compounds with mechanisms of action distinct from those of the PR inhibitors. We emphasize the key insights into HIV-1 biology and structure that the study of MIs has provided. We will focus on three distinct groups of inhibitors that block HIV-1 maturation: (1) compounds that block the processing of the CA-spacer peptide 1 (SP1) cleavage intermediate, the original class of compounds to which the term MI was applied; (2) CA-binding inhibitors that disrupt capsid condensation; and (3) allosteric integrase inhibitors (ALLINIs) that block the packaging of the viral RNA genome into the condensing capsid during maturation. Although these three classes of compounds have distinct structures and mechanisms of action, they share the ability to block the formation of the condensed conical capsid, thereby blocking particle infectivity.

show abstract

Permeability of the HIV-1 capsid to metabolites modulates viral DNA synthesis

Cited by 13 publications

References 55 publications

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

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

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

HIV-1 Maturation: Lessons Learned from Inhibitors

Contact Info

Product

Resources

About