FIG 4.

Induction of autophagy promotes the early stages of the viral life cycle or viral gene expression. (A and B) The culture medium of A549 cells was exchanged with Ham’s F-12 medium without FBS for 6 h or pretreated with rapamycin for 12 h and then incubated with the HM/06 virus at an MOI of 10 for 30 min (at 37°C). Cells were washed with PBS-HCl (pH 1.3) to remove all attached virions from the surface of A549 cells. Cell lysates were collected for Western blot analysis (A), and cell culture supernatants (noninfectious virus particles) were collected and determined by TCID₅₀ assay (B). (C) A549 cells were treated as described for panels A and B and then were mock infected or infected with the HM/06 virus for 4 h. Lysates were harvested and analyzed by Western blotting. (D) A549 cells were treated as described for panels A and B and infected with the HM/06 virus for 4 h. The levels of M mRNA, vRNA, and cRNA were determined by RT-PCR. GAPDH was used as a control for the normalization of cellular mRNA and intracellular viral RNA. Means and SD (error bars) were determined for triplicates of three independent experiments and were standardized to the levels of control-infected cells at 4 hpi (*, P < 0.05; ns, nonsignificant).