FIGURE 3.

Validation of the modulative effect of candidate proteins in vitro. A, ORF of the four candidate E3 ubiquitin ligases chosen by the screen assay were cloned into the pGEX-2T bacterial expression vector, expressed as GST fusion proteins in E. coli, and purified by glutathione-Sepharose beads, followed by size exclusion chromatography. Purified proteins were checked by SDS-PAGE and CBB staining analysis. The first lane shows a molecular weight marker. B, effect of E. coli-produced candidate proteins on HIV-1 PIC integration activity. Two different concentrations (1 and 10 nm) of GST fusion proteins and a control protein (GST) were incubated with HIV-1 PICs, and the reactions were subjected to the microtiter plate-based integration assay. The amount of integrated products was quantified by qPCR. The level of integration was expressed as the mean value of viral DNA copies of three independent experiments, with error bars indicating S.D. The p value versus GST control protein at the respective concentration was determined by Student's t test (*, p < 0.05).