FIG 5.

Inhibition of autophagy with specific shRNA targeting ATG5, ATG7, or BECN1 reduces RSV replication. (A to C) HEp-2 cells stably expressing specific shRNA against ATG5 (HEp-2-sh-ATG5), ATG7 (HEp-2-sh-ATG7), BECN1 (HEp-2-sh-BECN1), or luciferase gene (HEp-2-sh-luciferase) were established as described in Materials and Methods. The silencing efficiency of ATG5 shRNA (A), ATG7 shRNA (B), and BECN1 shRNA (C) was validated by immunoblotting with anti-ATG5, anti-ATG7, and anti-BECN1 antibodies, respectively, as well as with anti-GAPDH. (D, G, and J) HEp-2-sh-ATG5 (D), HEp-2-sh-ATG7 (G), HEp-2-sh-BECN1 (J), or HEp-2-sh-luciferase cells were infected with RSV (MOI = 5) for 24 h. Relative expressions of RSV targeted genes were determined by fluorescence quantitative PCR. (E, H, and K) HEp-2-sh-ATG5 (E), HEp-2-sh-ATG7 (H), HEp-2-sh-BECN1 (K) and HEp-2-sh-luciferase (E, H, K) cells were infected with RSV (MOI = 5) for 48 h. The expression of LC3II and RSV F was analyzed by immunoblotting with specific antibodies. (F, I, and L) HEp-2-sh-ATG5 (F), HEp-2-sh-ATG7 (I), and HEp-2-sh-BECN1 (L) cells were infected with RSV for indicated times, and total virus titers (intracellular + extracellular) were determined as described in Materials and Methods. Data represent means ± SD for 3 independent experiments. *, P < 0.05; #, P > 0.05.