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

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.

Keywords: HIV-1; IP6; capsid; reverse transcription; uncoating.

FIG 1

Characterization of purified HIV-1 cores. HIV-1 cores were isolated from concentrated virions by sucrose density gradient sedimentation. Gradient fractions were assayed for CA protein (A) and RT activity (B). (C) Electron micrographs from negatively stained samples of the three gradient fractions containing HIV-1 cores.

FIG 2

Representative results from an ERT experiment. HIV-1 cores were incubated for 16 h in a preliminary ERT reaction buffer containing the indicated concentrations of PEG3350. DNA was purified in the reactions and analyzed by qPCR for the indicated products of reverse transcription. This experiment was one of many early attempts to improve the efficiency of ERT.

FIG 3

IP6 markedly stimulates ERT in vitro by enhancing minus-strand synthesis. ERT reaction mixtures containing the indicated concentrations of IP6 were incubated at 37°C for 16 h, and DNA products were purified and quantified.

FIG 4

Optimization of the ERT reaction. (A to C) ERT reaction mixtures containing 10 μM IP6 under the indicated conditions were incubated and analyzed for HIV-1 DNA products. The variables were NaCl concentration (A), pH in reaction mixtures containing 150 mM NaCl (B), and MgCl₂ concentration in reaction mixtures at pH 7.6 with 150 mM NaCl (C). (D) Titration of IP6 in ERT reaction mixtures under optimized conditions. Duplicate reaction mixtures were incubated for 16 h at 37°C in 50-μl reaction mixtures containing HIV-1 cores in a buffer containing 10 mM Tris-HCl, pH 7.6, 150 mM NaCl, 2 mM MgCl₂, 0.5 mM DTT, 1 mg/ml BSA.

FIG 5

Time course of ERT in the presence and absence of IP6. ERT reaction mixtures containing or lacking 10 μM IP6 were incubated for the indicated times and subsequently analyzed for various HIV-1 DNA products by qPCR. (A) Reaction mixtures containing IP6; (B) reaction mixtures lacking IP6. Values represent averages from duplicate ERT reactions. (C) Quantification of products in 16-h reactions for sequences spanning the viral genome in reaction mixtures containing or lacking IP6. The blue and green symbols represent values from pairs of ERT reactions from two different experiments. Dashed lines connect values from ERT reaction mixtures lacking IP6. Results shown are from one of two independent experiments.

FIG 6

Capsid-stabilizing compound HCB also stimulates ERT. Reaction mixtures containing the indicated concentrations of HCB were incubated for 16 h at 37°C, and the products were analyzed by qPCR. Results shown are representative of two independent experiments.

FIG 7

IP6 and HCB stabilize viral cores during reverse transcription. Shown are ERT reaction mixtures containing no additive, 10 μM IP6, or 100 μM HCB, not incubated or incubated at 37°C for 6 h. Reaction mixtures were diluted 10-fold with reaction buffer, the cores were pelleted by ultracentrifugation, and the pellets and supernatants were analyzed for CA and RT activity. Shown is the fraction of the total CA and RT activity in the pellets. The values shown are the average values of duplicate reactions from one of two independent experiments, which showed similar outcomes.

FIG 8

Cores from the E45A HIV-1 mutant, which contains a hyperstable capsid, are less dependent on IP6 or HCB for ERT. Cores were purified from HIV-1 particles that had been produced by transfection of 293T cells. ERT reactions were performed with and without added IP6 or HCB. Results shown are representative of two independent experiments.

FIG 9

Capsid-targeting antiviral compound PF74 inhibits ERT. Optimized ERT reaction mixtures containing the indicated concentrations of PF74 were incubated for 16 h. DNA products were purified and analyzed for HIV-1 sequences by qPCR. These results are representative of 3 independent experiments.