Vpr-IN transcomplementation experiments

A class I IN mutant virus (HIV-1_NL4-3 IN_D116N) was trans-complemented with class II mutant IN proteins as described previously (Liu et al., 1997). Briefly, HEK293T cells grown in 24-well plates were co-transfected with a derivative of the full-length HIV-1_NL4-3 proviral plasmid bearing a class I IN substitution (pNL4-3_D116N), VSV-G, and derivatives of the pLR2P-vprIN plasmid bearing class II IN mutations at a ratio of 6:1:3. Two days post-transfection cell-free virions were collected from cell culture supernatants. Integration capability of the trans-complemented class II IN mutants was tested by infecting MT-4 cells and measuring the yield of progeny virions in cell culture supernatants over a 6 d period as described previously (Liu et al., 1997). In brief, MT-4 cells were incubated with virus inoculum in 96 V-bottom well plates for 4 hr at 37°C after which the virus inoculum was washed away and replaced with fresh media. Immediately following removal of the virus inoculum and during the 6 subsequent days, the number of virions present in the culture supernatant was quantified by measuring RT activity using a Q-PCR-based assay (Pizzato et al., 2009).