Figure 4: ASK1 restricts HIV-1 replication via A3G reactivation.

(a–c) ASK1 expression in virus-producing cells promotes A3G incorporation into virions and reduces infectivity. HEK293 cells were cotransfected with an HIV-1 molecular clone carrying a GFP reporter gene (pNL4-3ΔEnv-GFP) or its Vif-deficient mutant (pNL4-3ΔEnvΔVif-GFP) together with a plasmid encoding VSV-G, XP-ASK1 and HA-A3G. (a,b) Forty-eight hours after transfection, cell lysates and supernatants were harvested and analysed by western blotting against the indicated antibodies. The bar chart in b indicates the amounts of HA-A3G normalized by p24 levels in virions, as determined by densitometric analysis of western blots (n=3, mean±s.d.). (c) The CD4⁺ T-cell line (M8166) was infected with harvested and normalized virus for 2 days and infected (GFP-positive) cells were then measured by flow cytometry (n=3, mean±s.d.). *P<0.05; **P<0.01, two-tailed unpaired t-test. (d) Expression levels of ASK1 in DOX-treated or untreated CEM-TetON-ASK1 and CEMSS-TetON-ASK1 cells. 293 cells transfected with ASK1 or treated with PMA are shown as positive controls. (e–g) CEM-TetON-ASK1 and CEMSS-TetON-ASK1 cells and their parent cell lines (CEM and CEMSS) were infected with HIV-1. (e) Culture supernatants were harvested at the indicated time-points and subjected to p24 ELISA (n=3, mean±s.d.). *P<0.05, two-tailed unpaired t-test. (f) The incorporation of A3G into virions from indicated cells (at 8 d.p.i.) was detected by western blot. (g) The infected cells (CEM-TetON-ASK1) were harvested at 8 d.p.i. and subjected to G-to-A hypermutation analysis (n=8, mean±s.d.). **P<0.01, two-tailed unpaired t-test. Full images for all western blots analysis are shown in Supplementary Fig. 6. d.p.i., days post infection; ELISA, enzyme-linked immunosorbent assay.