HIV-1 CA Inhibitors Are Antagonized by Inositol Phosphate Stabilization of the Viral Capsid in Cells

Abstract

The HIV-1 capsid, composed of the CA protein, is the target of the novel antiretroviral drug lenacapavir (LCV). CA inhibitors block host factor binding and alter capsid stability to prevent nuclear entry and reverse transcription (RTN), respectively. Capsid stability is mediated in vitro by binding to the host cell metabolite inositol hexakisphosphate (IP6). IP6 depletion in target cells has little effect on HIV-1 infection. We hypothesized that capsid-altering concentrations of CA inhibitors might reveal an effect of IP6 depletion on HIV-1 infection in target cells. To test this, we studied the effects of IP6 depletion on inhibition of infection by the CA inhibitors PF74 and LCV. At low doses of either compound that affect HIV-1 nuclear entry, no effect of IP6 depletion on antiviral activity was observed. Increased antiviral activity was observed in IP6-depleted cells at inhibitor concentrations that affect capsid stability, correlating with increased RTN inhibition. Assays of uncoating and endogenous RTN of purified cores in vitro provided additional support. Our results show that inositol phosphates stabilize the HIV-1 capsid in target cells, thereby dampening the antiviral effects of capsid-targeting antiviral compounds. We propose that targeting of the IP6-binding site in conjunction with CA inhibitors will lead to robust antiretroviral therapy (ART). IMPORTANCE HIV-1 infection and subsequent depletion of CD4⁺ T cells result in AIDS. Antiretroviral therapy treatment of infected individuals prevents progression to AIDS. The HIV-1 capsid has recently become an ART target. Capsid inhibitors block HIV-1 infection at multiple steps, offering advantages over current ART. The cellular metabolite inositol hexakisphosphate (IP6) binds the HIV-1 capsid, stabilizing it in vitro. However, the function of this interaction in target cells is unclear. Our results imply that IP6 stabilizes the incoming HIV-1 capsid in cells, thus limiting the antiviral efficiency of capsid-destabilizing antivirals. We present a model of capsid inhibitor function and propose that targeting of the IP6-binding site in conjunction with capsid inhibitors currently in development will lead to more robust ART.

Keywords: CA; GS-6207; HIV; IP6; IPMK; IPPK; PF74; capsid; endogenous reverse transcription; lenacapavir.

FIG 1

Depletion of IP5 and/or IP6 increases HIV-1 susceptibility to PF74. (A and B) Efficiency of HIV-1-GFP_VSVg infection by flow cytometry when the indicated CEM (A) or MT-4 (B) cell lines are used as targets for infection. (C and D) PF74 dose-response curves for HIV-1-GFP_VSVg-infected CEM- (C) or MT-4-derived (D) cell lines as measured by flow cytometry. No HIV-1-infected cells were detected when IPMK KO MT-4 cells were infected in the presence of 7.5 and 10 μM PF74. For IPPK KO MT-4 cells, no infected cells were detected in the presence of 10 μM PF74. Cyan and red asterisks represent significance when comparing HIV-1 infection of IPPK KO_Vector and IPMK KO_Vector cells, respectively, to that of the corresponding complemented cell line. Green asterisks denote significantly different HIV-1 infection of IPPK KO_Vector cells to that of IPMK KO_Vector cells. (E) Target cell HIV-1-GFP_VSVg infection efficiency of the indicated CEM cell lines as measured by flow cytometry. Asterisks signify significance of the designated KO_Vector cell line compared to the corresponding complemented cell line. (F and G) Early (FST) (F) or late (FLM) (G) RTN products at 8 h following infection with HIV-1_VSVg as measured by quantitative PCR (qPCR). The nonnucleoside reverse transcriptase inhibitor Efavirenz (EFV) was used to identify the background of the assay. Black asterisks indicate significantly different susceptibility to HIV-1 infection between WT_Vector cells and the designated KO_Vector cell line. Yellow and green asterisks signify significance between infection of the IPPK KO_Vector cells or IPMK KO_Vector cells, respectively, relative to the corresponding complemented cell line. Each panel displays the average of 3 independent experiments with error bars representing standard deviation. Significance levels are as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 2

LCV more potently inhibits HIV-1 RTN upon IPMK or IPPK KO. (A and B) Target cell HIV-1-GFP_VSVg infection in the presence of LCV measured by flow cytometry. (A) Zero infected IPMK KO cells were detected when cells were infected in the presence of 3.1 nM LCV. (C and D) Quantification of early (FST) (C) or late (FLM) (D) RTN products. Black asterisks signify significance of the denoted bar from WT_Vector cells. Yellow and green asterisks above a bar indicate significance of that bar from the corresponding complemented cell line. (E to H) Target cell HIV-1-GFP_VSVg infection of the indicated cell lines when dosed with BI-2 (E), EFV (F), Raltegravir (G), or cyclosporine A (H). An average of 3 independent experiments is shown with error bars for standard deviation. Significance levels are as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 3

IP5 and IP6 depletion can partially sensitize PF74-resistant HIV-1 mutants to LCV. (A and B) Effect of CA 4-mut and CA 5-mut on HIV-1-GFP_VSVg infection of the indicated target cells. (C) LCV dose-response curves for HIV-1 CA 4-mut and CA 5-mut. Zero HIV-1_{CA WT}-infected IPMK KO_Vector CEM cells were detected in cultures containing 1.56 nM LCV. Black asterisks denote significance between HIV-1-infected- WT_Vector cells and IPMK KO_Vector cells. Differences between HIV-1 infection of IPMK-Flag complemented cells and IPMK KO_Vector cells is indicated by colored asterisks. Data shown is the average of 3 or greater independent experiments. Error bars show the standard deviation. Significance levels are as follows: *, P < 0.05; **, P < 0.01.

FIG 4

IP6 abrogates the PF74 and LCV effects on premature HIV-1 core uncoating in vitro. (A, B, D, and E) Uncoating of purified HIV-1 cores measured by HIV-1 p24 ELISA (A and D) or exogenous RTN of pelleted cores (B and E). Controls lacking inhibitors are the same in panels A and D (and B and E). Brackets show statistical significance in the indicated comparison. Statistical comparisons lacking brackets compare the indicated condition to the same condition lacking CA inhibitor. (D and E) Statistical comparisons for cores lacking LCV are not shown, but P values are the same as those shown in panels A and B, respectively. (C) Quantification of ERT by qPCR. Significant comparisons in the absence of PF74 or LCV are shown using brackets. For comparisons lacking brackets, black asterisks indicate significance differences in the indicated RTN product between the marked bar and the corresponding vehicle-treated bar containing the same amount of IP6. (A, B, and E) Each bar consists of the average of 3 independent experiments with corresponding error bars for standard deviation. Panels D and E represent 2 independent experiments along with error calculated using standard deviation. Significance levels are as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 5

The interplay of IP6 and CA inhibitors. Model of HIV-1 uncoating in the presence of IP6, CA inhibitors, and/or dNTPs. (I) An intact HIV-1 core is released into the cell or added to a reaction. (II) When no IP6 is present in ERT reactions, the core quickly uncoats. The core is proficient to generate early RTN products in vitro. (III) HIV-1 cores in the cell and in vitro are stabilized by IP6 and polymerize full-length HIV-1 cDNA. (IV) The balance of IP6 and CA inhibitor concentrations alters the flux between HIV-1 being RTN proficient with a stable capsid (promoted by IP6) and inhibiting HIV-1 RTN via capsid destabilization (CA inhibitor). (V) PF74 addition to the core immediately elicits RT loss from the core. Upon incubation the core disassembles. (VI) LCV binding to the core causes abrupt uncoating.