The host proteins transportin SR2/TNPO3 and cyclophilin A exert opposing effects on HIV-1 uncoating

Abstract

Following entry of the HIV-1 core into target cells, productive infection depends on the proper disassembly of the viral capsid (uncoating). Although much is known regarding HIV-1 entry, the actions of host cell proteins that HIV-1 utilizes during early postentry steps are poorly understood. One such factor, transportin SR2 (TRN-SR2)/transportin 3 (TNPO3), promotes infection by HIV-1 and some other lentiviruses, and recent studies have genetically linked TNPO3 dependence of infection to the viral capsid protein (CA). Here we report that purified recombinant TNPO3 stimulates the uncoating of HIV-1 cores in vitro. The stimulatory effect was reduced by RanGTP, a known ligand for transportin family members. Depletion of TNPO3 in target cells rendered HIV-1 less susceptible to inhibition by PF74, a small-molecule HIV-1 inhibitor that induces premature uncoating. In contrast to the case for TNPO3, addition of the CA-binding host protein cyclophilin A (CypA) inhibited HIV-1 uncoating and reduced the stimulatory effect of TNPO3 on uncoating in vitro. In cells in which TNPO3 was depleted, HIV-1 infection was enhanced 4-fold by addition of cyclosporine, indicating that the requirement for TNPO3 in HIV-1 infection is modulated by CypA-CA interactions. Although TNPO3 was localized primarily to the cytoplasm, depletion of TNPO3 from target cells inhibited HIV-1 infection without reducing the accumulation of nuclear proviral DNA, suggesting that TNPO3 facilitates a stage of the virus life cycle subsequent to nuclear entry. Our results suggest that TNPO3 and cyclophilin A facilitate HIV-1 infection by coordinating proper uncoating of the core in target cells.

Fig 1

Effects of CA substitutions on TNPO3 dependency of HIV-1 infection. (A) Quantitative immunoblot analysis of TNPO3-depleted HeLa cells. Cell extracts were immunoblotted with anti-TNPO3 and anti-GAPDH antibodies. Shown under each lane is the ratio of TNPO3 intensity to GAPDH intensity. (B) GFP reporter viruses were titrated on HeLa, sh-empty, and sh-TNPO3 cells. The percentage of GFP-positive cells is shown for each input viral dose (ng of p24). The data are representative of two independent experiments. (C) Control and TNPO3-depleted cells were challenged with CA mutant GFP reporter viruses. At 48 h, the percentage of GFP-positive cells was determined as a measure of infectivity. The ratio of infection of TNPO3-depleted relative to control cells is shown. Error bars represent the range of values from two independent experiments. Unfilled bars represent TNPO3-dependent viruses, black bars represent TNPO3-independent mutants, and gray bars represent partially TNPO3-dependent mutants. (D) Location of CA amino acid residues important for TNPO3 dependence of HIV-1 on the structural model of CA monomer extracted from the hexameric structure (Protein Data Bank [PDB] no. 3H4E). CA substitutions that do not alter TNPO3 dependence are shown in yellow, those that partially alter it are in orange, and those that completely alter it are in red.

Fig 2

TNPO3 stimulates HIV-1 uncoating in vitro. (A) Purified HIV-1 cores were incubated at 37°C for 30 min in the absence or presence of the indicated concentrations of recombinant TNPO3. After incubation, the extent of uncoating was determined as the fraction of the total CA in the supernatant. Shown are the means ± standard errors of the means (SEM) from three independent experiments performed in duplicate. Asterisks above the bars indicate statistically significant differences in uncoating values in the absence or presence of the indicated TNPO3 concentrations. *, P < 0.05; **, P < 0.01. (B) The N74D substitution does not alter the intrinsic stability of the HIV-1 capsid. Cores from wild-type and N74D mutant HIV-1 particles were isolated by sucrose gradient centrifugation. The level of core-associated CA was determined from the CA contents of core-containing fractions as a percentage of total CA protein in the gradient. Shown are the means ± SEM from three separate experiments. (C) The N74D substitution does not affect HIV-1 uncoating in vitro. Wild-type and N74D cores were incubated at 37°C, and the extent of uncoating was quantified at each time point. Shown are the means ± SEM from two independent experiments performed in duplicate. (D) TNPO3 potently stimulates the uncoating of wild-type HIV-1 cores. Wild-type and N74D mutant cores were incubated at 37°C for 35 min in the absence or presence of the indicated concentrations of TNPO3, and the extent of uncoating was determined. Shown are the means ± SD from five independent experiments performed in duplicate. (E) RanQ69LGTP inhibits the ability of TNPO3 to stimulate uncoating of wild-type cores. Purified cores were incubated at 37°C for 45 min in the absence or presence of the indicated recombinant proteins, and the extent of uncoating was determined. Shown are means ± SEM from three independent experiments performed in duplicate. **, P < 0.01.

Fig 3

TNPO3 sensitizes HIV-1 to inhibition by PF74. (A) Cells were inoculated with wild-type or N74D mutant HIV-GFP reporter particles in the presence of the indicated concentrations of PF74. The extent of infection was determined 48 h later by quantifying GFP-positive cells. The results were normalized to the corresponding infections performed in the absence of PF74. Normalized values are represented as means ± SEM from four independent experiments performed in duplicate. Asterisks above each point represent the difference between sh-empty and sh-TNPO3 values at that particular concentration of PF74. *, P < 0.05; **, P < 0.01. (B) Analysis of additional HIV-1 CA mutants for the effects of TNPO3 depletion on sensitivity to inhibition by PF74. Data represent means ± SEM from three independent experiments performed in duplicate.

Fig 4

TNPO3 potentiates HIV-1 uncoating induced by PF74 in vitro. HIV-1 cores were incubated at 37°C for 45 min in the presence or absence of TNPO3 and PF74, and the extent of uncoating was quantified. Shown are the means ± SEM from three independent experiments performed in duplicate. The dashed lines represent basal uncoating in the absence of PF74 and TNPO3.

Fig 5

TNPO3 depletion affects a step in HIV-1 infection after nuclear entry. (A and B) Cells were challenged with wild-type or N74D virus in the presence or absence of raltegravir. DNA was isolated from these cells at 7 and 24 h postinfection, followed by quantification of total viral DNA (A) and 2-LTR circles (B) by qPCR. The data represent means ± SEM from three independent experiments. (C) Immunoblot analysis was performed to ensure the integrity of subcellular fractionation by using a cytoplasmic protein, GAPDH, and a nuclear protein, lamin B1. (D) Infection was performed as for panels A and B, and cells were lysed and fractionated into cytoplasmic and nuclear fractions at 7 h and 24 h postinfection. Total viral DNA in the nuclear fractions was quantified by qPCR. Data represents means ± SEM from two separate experiments. (E) Whole-cell (Total), cytoplasmic (cyto), and nuclear (Nuc) lysates were resolved by SDS-PAGE and immunoblotted for TNPO3. GAPDH and lamin B1 were used as markers to ensure the integrity of subcellular fractionation. The right panel shows the quantitative distribution of TNPO3 in cytoplasmic and nuclear fractions of sh-empty and sh-TNPO3 cells. Data represents means ± standard deviations (SD) from three independent experiments.

Fig 6

Cyclophilin A stabilizes HIV-1 cores and inhibits TNPO3-enhanced uncoating. (A) Wild-type and G89V cores were incubated at 37°C for 60 min in the presence or absence of the indicated concentrations of cyclophilin A. Following incubation, the extent of uncoating was determined. Shown are the means ± SEM from two independent experiments performed in duplicate. Asterisks above the bars indicate differences in uncoating in the absence or presence of the indicated concentration of cyclophilin A. *, P < 0.05; **, P < 0.01. (B) Uncoating of HIV-1 cores was assayed in the presence or absence of the indicated recombinant proteins (TNPO3 [1 μM] or CypA [20 μM]). Shown are the means ± SEM from three independent experiments performed in duplicate. (C and D) Cells were infected with wild-type (C and D), N74D mutant (C), or G89V mutant (D) particles, and the extent of infection was quantified by flow cytometry for GFP expression. Results are expressed as means ± SEM from three independent experiments. Asterisks indicate differences between the fold changes in infectivity in control versus sh-TNPO3 cells. *, P < 0.05.