Figure 2

CA proteins expression and sensitivity of chimeric viruses to Debio-025. (A) Western blotting. The particle-associated proteins of HIV-1 NL4-3 (lane 2), HIV-1 expressing the capsid proteins from the two laboratory-adapted HIV-2 GL-AN (lane 2) and ROD10 (lane 3), from two subtype A clinical isolates HIV-2 CA-A4 (lane 5), A8 (lane 6), and from a subtype B clinical isolate HIV-2 B1.2 (lane 7) were analyzed. The viral supernatants of 293T transfected cells were concentrated by ultracentrifugation, through 20% sucrose cushion, resuspended and normalized by RT ELISA (0.3 ng) before western blot analysis using a mixture of serum derived from HIV-2 subtype A or B-infected patients. Lane 1: supernatant of untransfected 293T cells. The location of the CA proteins is shown on the left. The molecular mass marker (in kilodaltons) is indicated on the right. Results are representative of three independent experiments. (B) Effect of Debio-025 on viral infectivity. U373-X4 cells overexpressing hTRIM5γ were pretreated for 20 h with 100 U/ml IFN-α, and infected with equal amounts (0.2 ng RT) of the indicated VSV-G-pseudotyped chimeric viruses in the presence of the indicated concentrations of Debio-025. Forty hours after infection, medium was removed, the cells were lysed, and luciferase activity was determined by luminometry. The results, which are expressed relative to the infectivity of that of untreated cultures, are the mean ± SEM for 3 experiments. Asterisks indicate the results of statistical analyses comparing infectivity of chimeric virus to that of NL4-3, as determined using the non-parametric Mann–Whitney test: **, p = 0.0014; ***, p = 0.0001.