Mutagenesis at SP1 residues 6-8 results in varying degrees of CA-SP1 processing in the presence of BVM. (A) Mutagenesis of SP1 residues 6-8. HIV-1 Gag is represented at the top. The MA, CA, NC and p6 domains and the SP1 and SP2 spacer peptides are indicated. The alignment shows the pNL4-3 wild type (WT) amino acid sequence at the CA-SP1 junction in Gag and the panel of SP1 mutant derivatives examined in this study. The residues to which BVM resistance was previously mapped in vitro are shaded in grey. (B and C) Quantitative CA-SP1 processing assay. HeLa cells were transfected with WT pNL4-3 and the panel of SP1 mutant derivatives and cultured either without BVM or in 1 μg/ml BVM. Cells were metabolically labeled for 2 h with [35S]Met/Cys, and released virions were pelleted by ultracentrifugation. Cell and virus lysates were immunoprecipitated with HIV-Ig, and processing of CA-SP1 to CA was analyzed by SDS-PAGE and fluorography (B) and by phosphorimager analysis to quantify the percentage of CA-SP1 relative to total CA-SP1 plus CA (C). Error bars indicate standard deviations (n = 3-5).