Immunoblotting

Viral and cell lysates were resuspended in sodium dodecyl sulfate (SDS) sample buffer and separated by electrophoresis on Bolt 4–12% Bis-Tris Plus gels (Life Technologies), blotted onto nitrocellulose membranes and probed overnight at 4°C with the following antibodies in Odyssey Blocking Buffer (LI-COR): mouse monoclonal anti-HIV p24 antibody (183-H12-5C, NIH AIDS reagents), mouse monoclonal anti-HIV integrase antibody (Bouyac-Bertoia et al., 2001), rabbit polyclonal anti-HIV integrase antibody raised in-house against Q44-LKGEAMHGQVD-C56 peptide and hence unlikely to be affected by the substitutions introduced into IN in this study, rabbit polyclonal anti-HIV-1 reverse transcriptase antibody (6195, NIH AIDS reagents), rabbit polyclonal anti-Vpr antibody (11836, NIH AIDS Reagents), rabbit polyclonal anti-MA antibody (4811, NIH AIDS Reagents). Membranes were probed with fluorophore-conjugated secondary antibodies (LI-COR) and scanned using an LI-COR Odyssey system. IN and CA levels in virions were quantified using Image Studio software (LI-COR). For analysis of the fates of core components in infected cells, antibody incubations were done using 5% non-fat dry milk. Membranes were probed with HRP-conjugated secondary antibodies and developed using SuperSignal West Femto reagent (Thermo-Fisher).