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| Figure 2: Expression of T7-INcwt/mut in HIV-1 producing cells significantly inhibited virus infectivity. A: Viruses were produced from 293T cells co-transfected with each T7-INc expressor and HIV-1 provirus HxBru. Produced viruses were used to infect HeLa- b-Gal-CD4-CCR5 cells, and the infection levels were analyzed by MAGI assay. B: Viruses were produced from 293T cells co-transfected with each T7-INc expressor and HIV-1 pNL4.3-GFP. CD4+ MT4 cells were infected with equal amounts of pNL4.3-GFP viruses, and the HIV-1 infection was determined by monitoring the percentage of GFP-positive cells with FACS analysis. C. Each of the wild type and mutated HIV-1 IN CTD was fused in frame to the C-terminus of YFP vector. 293T cells were transfected with YFP vector, YFP-INcwt or YFP-INc 215,9AA and HIV-1 pNL4.3-GFP provirus. The expressions of viral associated GFP and YFP-INc fusion proteins in 293T cells were detected by western blot using anti-GFP anti-body (a). Produced pNL4.3-GFP viruses were used to infect CD4+ C8166 cells and the virus production from C8166 cells was monitored by measuring HIV-1 p24gag antigen in the supernatants with p24gag ELISA assay (b, upper panel). The p24gag expression in the cells was detected by western blot with anti-p24 antibody (b, lower panel). Meanwhile, the HIV-1 infected GFP positive cells were observed under fluorescence microscopy at 48 h post-infection (c). All results are representative of two independent experiments.. |
