FIG. 7.

Effect of caffeine on the late steps of HIV-1 replication. (a) Caffeine induces hyperphosphorylation of the ATM kinase substrate Chk2, following hydroxyurea treatment. P4.2 cells were pretreated with 4 mM caffeine for 1 h and then treated with 5 mM hydroxyurea (HU) in the presence or absence of 4 mM caffeine for 3 h. Western blotting of the cellular lysates was performed with anti-Chk2 antibody or anti-phospho-Chk2 (Thr68) antibody (P-T68). (b) Caffeine enhances HIV-1 replication in P4.2 cells. P4.2 cells (2 × 10⁵ cells) were pretreated with the indicated concentration of caffeine for 1 h and infected with HIV-1 (X4) at an MOI of 0.5. p24 levels in the supernatants were measured by ELISA 6 days later. (c) ATM knockdown (ATMi) or control (C) P4.2 cells (2 × 10⁵ cells) were pretreated with 1 mM caffeine for 1 h and infected with HIV-1 (X4) at an MOI of 0.5 in the presence or absence of caffeine. p24 levels in the supernatants were measured at 6 days. (d) Caffeine enhances Rev function in P4.2 cells. P4.2 cells (2 × 10⁴ cells) treated with 1 mM caffeine were transfected with the Rev-dependent luciferase-based reporter gene pDM628 (100 ng) with or without the Rev-expressing plasmid pcRev (100 ng). Twenty-four hours later, luciferase activity in the cellular lysates was determined. Results are from three independent experiments.