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| Figure 1: Impact of expression of HIV-1 IN C-terminal domain (205-288) on VSV-G pseudotyped HIV-1 infection. A: Upper panel: Schematic representation of T7-INcWT, T7-INc215, 9AA and T7-INc240, 4AE constructs. Each of the wild type and mutated HIV-1 IN CTDs (encompassing from amino acid 205-288 of IN) was fused in frame to the C-terminus of T7 tag. Lower panel: The western blot result showing the expression of T7-INcwt/mut in 293T cells. 293T cells were transfected with CMVin T7 vector or each of CMVin T7-INc expressors. After 48 h, cell lysates were immunoprecipitated with anti-T7 antibodies followed by western blot with anti- T7-HRP antibody. B: Expression of T7-INcwt/mut in 293T cells affected the incoming HIV-1 infection. 293T cells were transfected with T7-INcWT or T7-INc215, 9AA, and after 48 h, equal numbers of transfected cells were infected with P24 normalized VSV-G pseudotyped NL4.3-BruDBgl/luc+ (a) or integration-defective mutant (D64E) virus (b). At 48 h post-infection, equal amounts of 293T cells were collected and the luciferase (luc) activity was measured. C: Expression of T7- INcwt/mut in virus producing cells affected the late stage of single-cycle HIV-1 replication. Viruses were produced from 293T cells co-transfected with each T7-INc expressor (as indicated), HIV-1 provirus NL4.3-BruDBgl/luc+ and a VSV-G expressor. Then, equal amounts of produced viruses (as adjusted by Gag-p24 level) were used to infect C8166 T cells. After 48 h, the infection levels were measured by detecting the luc activity from the same amount of infected cells. Means and standard errors are representative of the results of duplicate samples from a typical experiment and were confirmed in two independent experiments. |
