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.
Highlights
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
In an effort to improve the efficiency of the HIV-1 endogenous reverse transcription (ERT) reaction, we incubated samples of purified HIV-1 cores with deoxynucleoside triphosphates (dNTPs) in vitro and analyzed the products by quantitative PCR
In reaction mixtures lacking IP6, E45A cores produced approximately 20% of the late-stage products relative to parallel reaction mixtures containing IP6. This is in stark contrast to reactions with wild-type cores, in which synthesis of late products was less than 0.1% of that observed in reaction mixtures containing IP6. These results indicate that E45A mutant cores are capable of synthesizing substantial quantities of late reverse transcripts in the absence of capsid-stabilizing agents, further indicating that the ERT-stimulating activity of IP6 results from stabilization of the viral capsid
Summary
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. 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. Premature capsid disruption in cells expressing restrictive TRIM5 proteins is associated with impaired reverse transcription [5, 6] These studies have established that the integrity of the viral capsid is important for efficient HIV-1 reverse transcription in target cells. A role of IP6 in reverse transcription itself has not been established
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