FIG 3.

Detection of viral proteins and biotinylation in cells. ΔMA-BirA* or MA-BirA* proviral constructs were transfected into 293T cells alone or along with WT (+WT) or PR⁻ (+PR−) HIV-1 proviral constructs. At 3 days posttransfection, protein samples from transfected cells and mock-transfected cells (M; lanes A and H) were electrophoretically separated, and subjected to detection of viral proteins (anti-CA; lanes A to G) or biotinylated proteins (streptavidin; lanes H to N). Molecular size markers, as indicated, were run in parallel lanes. (Left panel) In lanes B to D and E to G, the anti-CA antibody detected the precursor PrΔMA-BirA* and PrMA-BirA* proteins and capsid proteins, respectively. Also detected were PrGag proteins in samples cotransfected with WT and PR⁻ HIV-1 constructs (lanes C, D, F, and G). In addition to the PrΔMA-BirA* band, the ΔMA-BirA* construct yielded CA-reactive bands 1 and 2: band 1 migrates at approximately 60-kDa and potentially corresponds to a BirA*-CA processing intermediate, while band 2, at about 41 kDa, potentially corresponds to a CA-NC-p6 intermediate. Similarly, band 3, detected in transfections containing the MA-BirA* construct, migrates at about 74 kDa, which corresponds to the predicted size of a MA-BirA*-CA processing intermediate. (Right panel) Bands in the mock lane (H) near the 114-kDa (band 4) and 74-kDa (band 5) markers correspond to the endogenously biotinylated pyruvate carboxylase (130-kDa) protein and to the alpha chains of proprionyl-CoA carboxylase (80 kDa) and methylcrotonyl-CoA carboxylase (80 kDa). All the transfected cell sample lanes show numerous biotinylated protein bands, including the PrMABirA* band (lanes L to M). Note that the 82-kDa biotinylated PrΔMA-BirA* band (lanes I to K) comigrates with the endogenously biotinylated carboxylase alpha chains. Note that these results are representative of nine separate experiments.