Figure 4.

EnvCT W757 is required for HIV-1 infectivity and budding. (A) Infectivity of virus particles released 48 hpi from Jurkat and primary CD4⁺ T cells was measured by titrating virus on HeLa TZM-bl reporter cells and RLU normalised per RT unit to calculate relative particle infectivity. (B) Western blot of cell lysate and purified virus from infected primary CD4⁺ T cells at 48 hpi probed with anti-Env (gp120/gp160) and anti-Gag (p55/p24). (C) Quantification of Env (gp120) incorporation into virions normalised to Gag (p24) density, produced in Jurkat and primary CD4⁺ T cells. (D) Quantification of virus release as measured by Gag (p24) density normalised to total Gag (p24 + p55) produced in infected Jurkat cells. (E) Virus budding from infected Jurkat T cells and primary CD4⁺ T cells at 48 hpi measured by quantifying RT activity in viral supernatants by SG-PERT qPCR from infections shown in (A). (F) Quantification of infected Jurkat T cells at 24 hpi measured by flow cytometry analysis for the % Gag+ cells (left) and Gag MFI of the Gag+ population (middle). The percentage live cells for WT and W757A is shown (right). (G) Virus release from infected Jurkat T cells (from (F)) at 16 hpi (left) and 24 hpi (middle) measured by quantifying RT activity in viral supernatants by SG-PERT qPCR. Right panel shows combined data from both time points. Data show the mean and SEM from at least three independent experiments compared using a two-tailed unpaired t-test (ns, not significant; ** p < 0.01, *** p < 0.001 **** p < 0.0001).