Analysis of N-Vpu function in the context of HIV-1 proviral constructs.

<p>(<b>A</b>) Location of modifications introduced into a replication competent HIV-1 group N (DJO0131) proviral clone. Asterisks indicate the positions of mutations in the <i>vpu</i>, <i>env</i> and <i>nef</i> genes, respectively. (<b>B</b>) 293T cells were cotransfected with the indicated proviral constructs and different quantities of tetherin expression constructs. The levels of p24 released in the culture supernatant were determined as described in the methods section. The results were confirmed in three independent experiments and by measuring the reverse transcriptase activity in the cell culture supernatants. (<b>C</b>) PHA-activated PBMCs were transduced with VSV-G pseudotyped wild-type or <i>vpu</i>-defective HIV-1 M NL4-3 or N DJO0131 constructs and examined for intracellular p24 capsid antigen and tetherin surface expression five days later. The results in panels C and D show average levels (±SD) of tetherin surface expression in virally infected (p24+) cells relative to uninfected cells (100%) and were derived from three independent experiments with two different PBMC donors in each experiment. (<b>D</b>) PHA-stimulated PBMCs were transduced with VSV-G pseudotyped NL4-3 IRES <i>env</i> constructs expressing group M (NL4-3, JR-CSF, YU-2), N (YBF30) or SIVcpz (GAB1, MP7) <i>vpu</i> alleles and analyzed by flow cytometric analysis as described in panel C.