Journal: PLoS ONE

Article Title: Proteolysis of mature HIV-1 p6 Gag protein by the insulin-degrading enzyme (IDE) regulates virus replication in an Env-dependent manner

doi: 10.1371/journal.pone.0174254

Figure Lengend Snippet: (A) HEK293T cells were transfected with pΔR plasmids encoding for either wt Gag, or the p6 mutants 2xPTAPPA or 3xPTAPPA. Cells were lysed and VLPs were purified and subsequently analyzed by Western blot. Noteworthy, Gag processing and virus release of 2x and 3x PTAPPA mutants were comparable to that of the wt , only the apparent molecular weight of p6 and the NCp6 processing intermediate increased by PTAPPA multiplication. (B) The rate of Gag processing was estimated by calculating the ratio of p24 vs. Pr55 detected in released VLPs. Bars represent mean values of three independent experiments ± SD. (C) Efficiency of virus release was calculated as the ratio of Gag (Pr55 and p24) present in the virus pellet relative to the total amount of Gag detected in cells and released VLPs. Bars represent mean values of three independent experiments ± SD. Both Gag processing and virus release for the wt were set to 1. (D) HAP1 wt cells and HAP1 IDE knock out cells were infected with VSV-G-pseudotyped wt HIV-1 particles. 2 days post-infection, cell and virus-fractions were harvested and analyzed by Western blot for viral proteins. Band intensities of virus-associated p6 (E) and Vpr (F) were quantified and normalized for p24 signals. Bars represent mean values of three independent experiments ± SD. (G) HeLa TZM-bl wt and IDE KO cells were transiently transfected with pNLenv1 and virus and cell fractions were analyzed by Western blot. (H) Band intensities of virus-associated p6 were quantified as described in (E).

Article Snippet: HAP1 wt and IDE knock out clones 10 and 12 were obtained from Haplogen and were cultivated in IMDM containing antibiotics and FCS.

Techniques: Transfection, Purification, Western Blot, Virus, Molecular Weight, Knock-Out, Infection