FIG 7.

Site-directed mutagenesis of Vpu amino acid 76: effects on CD4 and BST2 downregulation and enhancement of virion release. (A to D) Function and expression of Vpu proteins with site-directed mutation of W76. (E to H) Test of whether mutation of amino acid 76 to W restores function of impaired alleles. (A and E) Relative levels of CD4 downregulation by site-directed Vpu mutants. Data are expressed relative to NL4-3 Vpu as described for Fig. 2D. The values shown are means of the results of two experiments performed in duplicate. Error bars are the standard deviations. (B and F) Relative levels of BST2 downregulation by site-directed Vpu mutants. Data are expressed relative to NL4-3 Vpu as described for Fig. 2E. The values shown are means of the results of two experiments performed in duplicate. Error bars are the standard deviations. (C and G) Virion release enhancement by the site-directed Vpu mutants. Pelletable p24 antigen was measured by ELISA in supernatants from cultures of cells transfected with the Vpu expression plasmid and NL4-3 ΔVpu as described for Fig. 5D. Error bars represent the standard deviations of the results of quadruplicate experiments. (D) Immunoblot analysis of the expression of site-directed Vpu mutants and controls. Lysates from cells transfected for FACS analysis as described for panels A and B were analyzed for actin and Vpu (FLAG) by immunoblotting. (H) Immunoblot analysis of the expression of site-directed mutants of functionally impaired Vpu proteins. Lysates from cells transfected for virion release analysis as described for panel G were analyzed for Gag/p55, actin, and FLAG (Vpu) by immunoblotting.