Recovery of HIV-1 particle release by patient-derived Vpus.

<p>TZM-bl cells were transiently transfected with HIV-1NL4.3 Δvpu provirus plasmids and the indicated Vpu allele or controls. 48 h post transfection, the resulting viral supernatants were assayed for infectivity on TZM-bl indicator cells to determine the amount of infectious virions produced. TZM-bl cell lysates from one replicate of the assay were subjected to Western blot detection, and TZM-bl cells from the same replicate were harvested and assayed for cell-surface level of CD317/tetherin. <b>A</b>) The yield of infectious HIV-1 in the supernatant and cell-associated levels of GFP, p24CA, and p55Gag were analyzed. HIV-1 particle release in the supernatant was assessed by measuring the induction of Luciferase units (top) in infected TZM-bl cells. Values (y axis) are normalized to that of control NL4.3Δ<i>vpu</i>, which was set to 1, error bars represent the standard error of the mean (SEM) for three independent experiments. Western blot results show the expression level of GFP, p24 and p55 in one representative experiment. <b>B</b>) Comparison of enhancement of virion release mediated by EC and CP Vpu alleles. Statistical significance was assessed using two-tailed Mann-Whitney U-Test (p = 0.07), bars represent median and interquartile ranges. <b>C</b>) CD317/tetherin cell surface downregulation by patient Vpu. TZM-bl cells transfected with HIV-1 NL4.3 Δvpu and the indicated VpuIRESGFP plasmids were harvested 48 h post transfection and CD317/tetherin cell surface levels quantified by flow cytometry. The y-axis represents the relative CD317/tetherin cell surface levels remaining normalized to control cells transfected with NL4.3Δ<i>vpu</i> and IRESGFP (set to 100%). <b>D</b>) Comparison of CD317/tetherin cell surface levels in TZM-bl cells producing viral particles and expressing EC or CP Vpu alleles. Statistical significance was assessed using two-tailed Mann-Whitney U-Test (p = 0.29), bars represent median and interquartile ranges.