Figure 3.

ERI1 silencing alters viral life cycle. (A) A549 cells were treated with non-targeting (NT) or ERI1 siRNAs and infected 48hpt with WSN at an moi of 3 pfu/cell. Total cell extracts were prepared at the indicated times post-infection and analyzed by immunoblot using antibodies directed against the indicated proteins. Results representative of three independent experiments are shown. (B, D, E) A549 cells treated with non-targeting (NT, black bars) or ERI1 (gray bars) siRNAs were infected with WSN at an moi of 3 pfu/cell in the absence (B) or presence (D, E) of cycloheximide (CHX) (100μg/ml) that blocks translation. Total RNAs were extracted at the indicated time points post infection and the levels of NP or NA mRNAs, vRNAs and cRNAs were determined by strand specific RT-qPCRs. The results are expressed as the mean ± SEM determined in three independent experiments. The significance was tested by two-way ANOVA with Sidak's multiple correction test in GraphPad Prism Software (****P < 0.0001; *P < 0.05). (C) HEK-293T cells were transfected with Strep-ERI1 or Strep-empty (as a control) and infected with WSN at an moi of 3 pfu/cell. At 3 hpi total RNAs were extracted and the levels of NP mRNAs and vRNAs were determined by strand specific RT-qPCRs. The results are expressed as the mean ratios of mRNA/vRNA ± SEM normalized to empty control, determined in three independent experiments. The significance was tested with an unpaired t-test using GraphPad Prism Software (*P < 0.05). (E) Total extracts from infected cells treated with non-targeting (NT) or ERI1 siRNAs, and treated with CHX or not, were prepared at 6hpi and analyzed by immunoblot using antibodies directed against the indicated proteins. No viral protein was detectable at 6hpi upon CHX treatment demonstrating that CHX effectively blocked de novo translation.