Cyclophilin A-dependent restriction of human immunodeficiency virus type 1 capsid mutants for infection of nondividing cells

Abstract

Among retroviruses, lentiviruses are unusual in their ability to efficiently infect both dividing and nondividing cells, such as activated T cells and macrophages, respectively. Recent studies implicate the viral capsid protein (CA) as a key determinant of cell-cycle-independent infection by human immunodeficiency virus type 1 (HIV-1). We investigated the effects of the host cell protein cyclophilin A (CypA), which binds to HIV-1 CA, on HIV-1 infection of nondividing cells. The HIV-1 CA mutants A92E, T54A, and R132K were impaired for infection of aphidicolin-arrested HeLa cells, but not HOS cells. The mutants synthesized normal quantities of two-long-terminal-repeat circles in arrested HeLa cells, indicating that the mutant preintegration complexes can enter the nuclei of both dividing and nondividing cells. The impaired infectivity of the CA mutants on both dividing and nondividing HeLa cells was relieved by either pharmacological or genetic disruption of the CypA-CA interaction or by RNA interference-mediated depletion of CypA expression in target cells. A second-site suppressor of the CypA-restricted phenotype also restored the ability of CypA-restricted HIV-1 mutants to infect growth-arrested HeLa cells. These results indicate that CypA-restricted mutants are specifically impaired at a step between nuclear import and integration in nondividing HeLa cells. This study reveals a novel target cell-specific restriction of HIV-1 CA mutants in nondividing cells that is dependent on CypA-CA interactions.

FIG. 1.

CsA-dependent HIV-1 mutants are impaired for infection of arrested HeLa-P4 cells. (A) Infectivities of CA viruses on dividing HeLa-P4 cells. CsA was used in these assays at 10 μM. (B) HIV-1 infectivity on aphidicolin (APC)-arrested HeLa-P4 cells. Shown are the mean values of triplicate infections, with error bars representing 1 standard deviation. The results shown are from one representative of three independent experiments. WT, wild type. (C and D) HeLa-P4 cells were arrested by gamma irradiation and challenged with the indicated VSV-G-pseudotyped viruses. For HIV-1, infectivity was determined as the number of infected cells per nanogram of p24 in the inoculum. For MLV, the infectivity was determined as the number of GFP-positive cells per microliter of viral supernatant. The results shown are the mean values of three independent experiments, with error bars representing 1 standard deviation. (D) Assays were performed in cultures containing CsA (5 μM). The results shown in panels C and D are representative of two independent experiments.

FIG. 2.

The CypA-CA interaction is necessary for the cell-cycle-dependent infectivity of CsA-resistant/dependent HIV-1 mutants. (A) Infectivities of the CsA-dependent HIV-1 mutants in arrested HeLa-P4 cells. CsA (10 μM) was added during inoculation and maintained throughout the 2-day assay period. WT, wild type. APC, aphidicolin. (B) Infectivities of HIV-1 mutants on aphidicolin-arrested HeLa-P4 cells depleted for CypA expression. (C) Infectivities of the indicated viruses as determined on CsA-treated or aphidicolin-arrested cells. The results shown are the mean values of triplicate determinations, with error bars representing 1 standard deviation. The data are from one representative of three independent experiments.

FIG. 3.

The second-site suppressor mutation A105T relieves the impaired infectivity of CsA-resistant/dependent mutants on dividing and nondividing cells. Control cells were inoculated in the presence and absence of CsA (10 μM). Shown are the mean values of triplicate infections, with error bars representing 1 standard deviation. The results shown are from one representative of three independent experiments. WT, wild type; APC, aphidicolin.

FIG. 4.

CsA-dependent mutants are not impaired for entry into the nuclei of nondividing cells. Viral two-LTR circles were assayed by quantitative PCR. Shown are the mean values of triplicate infections, with error bars representing 1 standard deviation. The results shown are from one representative of three independent experiments. WT, wild type; APC, aphidicolin.

FIG. 5.

The infectivity impairment of CsA-dependent mutants in arrested HeLa cells can be rescued 20 h after inoculation (p.i.). Viral stocks were inoculated in control (A) and aphidicolin-arrested (B) cells. Two hours later, the inocula were removed, the cells were washed, and the media were replenished. At various times, CsA (10 μM) was added to the cultures and maintained for another 24 h. Infected cells were quantified 2 days after inoculation. Shown are the mean infectivity values of triplicate infections, with error bars representing 1 standard deviation. The results shown are from one representative of three independent experiments. WT, wild type.

FIG. 6.

The cell-cycle dependence of CsA-dependent mutants varies with the cell type. VSV-G-pseudotyped viruses were used to infect HOS cells (A), as well as activated CD4⁺ T cells and monocyte-derived macrophages (MDM), from two individual donors (B and C). Two days later, the extent of infection was quantified by flow cytometry and was calculated as the percentage of GFP-expressing cells per nanogram of input p24 antigen in the inoculum. WT, wild type; APC, aphidicolin.