Fig 1.
Binding of CypA to the viral capsid inhibits HIV-1 infection of nondividing cells by HIV-1 mutants with inelastic cores.
Infection of wild type (A-E, F) and CypA KO Hela (E) cells with HIV-1 reporter viruses expressing GFP was quantified by flow cytometry. Cells were seeded in the presence and absence of aphidicolin (APC) to induce cell cycle arrest. In panel B, cells were infected in the presence of the CypA inhibitor CsA. Graphs in A and B show results from one representative of three experiments, with average values from duplicate infections. Error bars show the range of the two values. Panel C shows the infection ratios for dividing vs. nondividing cells in the presence and absence of CsA. Panel D shows the infection ratios with and without CsA in dividing and arrested cells from the same results shown in panel C. Mean values from four independent experiments are shown, with error bars representing standard deviations. Symbols represent results of statistical analysis of the effect of CsA (panel C) and cell cycle arrest (panel D) for each virus. *: p < 0.05; **: p < 0.01; ***: p < 0.001; n.s.: not significant. Panel E shows results of cell cycle arrest on infection of wild type and CypA KO cells. Statistical symbols represent comparisons between values from mutant and wild type viruses. Results shown in panel F show the effects of cell cycle arrest on infection by viruses containing the P90A substitution that prevents binding of CypA to the viral core in target cells. Values shown are means from three independent experiments, with error bars representing standard deviations. Significance of the effect of the P90A substitution was evaluated for each of the four mutants by paired ratio T test in GraphPad Prism.
Fig 2.
CypA inhibits the nuclear entry of HIV-1 mutants with inelastic capsids.
Nuclear entry was monitored in APC-treated Hela cells by (A) quantifying the ratio of 2-LTR circles to late reverse transcripts (second strand transfer DNA) by qPCR, or (B) confocal imaging of INmNG-labeled fluorescently HIV-1 core (fHIV-1) in the nucleus of arrested cells at 8 hours post infection. (C) The nuclear entry of inelastic capsids bearing the P90A substitution was determined in emiRFP-670 tagged lamin-B1 expressing TZM-bl cells. Images of fHIV-1 nuclear entry in aphidicolin-arrested Hela CypA-KO or P90A-mutant infections in WT-TZM-bl cells are shown. Results were compiled from four independent experiments: mean values and standard errors of the mean are shown. Statistical significance was determined using Mann-Whitney Rank Sum test: p > 0.5 not significant (ns); p < 0.0001 (highly significant) is marked by ****. In (A), asterisks placed directly above each bar indicates the significance level relative to the corresponding wild type virus in the indicated cell line. Asterisks above each pair of bars represents analysis of the difference of the indicated virus in Hela vs. CypA KO cells.
Fig 3.
Effects of CypA on the elasticity of purified HIV-1 cores.
Viral cores were purified from detergent-treated virions in the presence of IP6 and immobilized on glass slides under native (unfixed and unstained) conditions. Cores were first identified by AFM scanning. A series of cores was then individually subjected to imaging by scanning, compressed by application of the probe at high force, allowed to recover, and rescanned. Broken cores were identified from the images as clearly damaged (broken capsid or altered shape); representative images are provided in S2 Fig. The analysis was subsequently performed on a separate set of attached cores treated with the indicated concentrations of CypA. Results shown are the percentage of cores that underwent breakage upon CypA addition, with error bars depicting the standard error. The number of cores analyzed for each condition is shown within the corresponding bar. (A): wild type and P90A cores; (B): E45A and E45A/R132T cores; (C): Q63A/Q67A and Q63A/Q67E cores; (D): A92E and A92E/A105T cores. The error of the mean was determined via bootstrap analysis. Significance of the effect of CypA (relative to the respective no-CypA value) was evaluated using a Chi-squared test for independence. n.s.: p > 0.05; **: p < 0.01; ***: p < 0.001. Data from measurements of WT, E45A, E45A/R132T, Q63A/Q67A and Q63A/Q67E without CypA were taken from Deshpande et al. [12].
Fig 4.
CypA affinity for HIV-1 cores in vitro is not affected by the E45A substitution, but the A92E substitution leads to a 2-fold increase in the dissociation constant.
(A): Structural model for the CypA binding loop of HIV-1 CA bound to CypA. (B): Diagram illustrating the experimental approach. Virions were immobilized onto glass, permeabilized with Streptolysin O, and incubated with various concentrations of fluorescently labeled CypA. Binding of CypA to the viral capsid is detected and by TIRF microscopy and quantified by image analysis. (C-E): Median CypA signal (filled circles) bound at equilibrium to cores in permeabilized HIV-1 virions containing wild type (C), E45A (D), and A92E (E) CA. CypA binding was measured by TIRF microscopy as a function of CypA concentration and fit of an equilibrium binding model (black line).
Fig 5.
Reciprocal rescue of nondividing cell infection by suppressors of inelastic and CsA-dependent mutants.
Infection of dividing and arrested Hela cells by wild type and mutant HIV-GFP reporter viruses was quantified by flow cytometry. (A): Infectivity of the viruses on dividing cells, normalized by the p24 content of the respective inocula. (B): Ratio of infection on dividing and arrested cells. Asterisks shown in panel B indicate significant differences between the infection ratio for each double point mutant relative to the corresponding single mutant. Shown are the mean values of results from three independent experiments with error bars representing standard error values. Significance was determined by paired ratio T test comparing each double mutant relative to its corresponding single mutant.