Figure 7. The effects of the mutations on viral replication and Gag processing.

(A) pNL4-3 plasmids with mutations in pol, env-nef or both were constructed (marked with asterisks in Fig. 5F and G). Jurkat cells were transfected with the resulting plasmids or pNL4-3 (“C”). The amount of p24 produced at 24 h after transfection was analyzed. The amount of p24 produced by the pNL4-3-transfected cells at 24 h post-transfection was set at 100%, and the p24 production in each tested line was expressed as the mean ± S.D. percent of this amount. The bar patterns correspond to the mutational patterns shown in the bottom right and in Fig. 5. (B) Mutant viruses or NL4-3 (“C”) were obtained by transfecting 293T cells with each plasmid, and the resulting viruses were normalized based on the amount of p24. Jurkat cells were infected with 100 ng of p24-normalized virus. The amount of p24 produced by NL4-3-infected cells at 6 days post-infection was set at 100%, and each p24 amount was expressed as the mean ± S.D. percent of this amount. (C) M4C8 cells were infected with 10 ng of p24-normalized virus and analyzed as described in (B). (D) TZM-bl cells were infected with 10 ng of p24-normalized virus. The firefly luciferase activities were assessed at 40 h post-infection and the luciferase activity by NL4-3-infected cells was set at 100%. The (−) indicates the activity of the mock-treated cells. (E) M4C8 cells were infected with 10 ng of p24-normalized virus. The amount and processing of Gag in the infected cells were analyzed at 5 days post-infection by western blot using the HIV-1 p24 Gag monoclonal antibody. The membrane was then stripped and reprobed with an anti-ß actin antibody as a loading control.