FIGURE 4.

Domain of RFPL3 responsible for the enhancement of in vitro HIV-1 PIC activity. A, schematic of FL RFPL3 and its N-terminal truncation mutants (Δ36, Δ98, and Δ148) used for the assay. All proteins were expressed as GST fusion proteins by the wheat germ cell-free system and affinity-purified. B, SDS-PAGE and CBB staining analysis of purified proteins. A negative control protein, GST-DHFR, was also produced. The first lane shows a molecular weight marker. C, effect of RFPL3 mutants on the HIV-1 PIC integration activity. A microtiter plate-based PIC integration assay was performed in the presence of 10 nm recombinant proteins. GST-RFPL3, BAF, and a control GST protein produced by E. coli were also included in the assay as control reactions. The experiment was done in triplicates, and data are expressed as mean value of viral DNA copies, with error bars indicating S.D. Statistical significance was determined by Student's t test. *, p < 0.05; **, p < 0.01, ***, p < 0.001; ns, no significance.