FIG. 2.

Effect of RSK2 knockdown on influenza virus replication. (A) Knockdown of human RSK2. 293 cells were transduced with retroviral vectors for shRNAs to human RSK2 or GFP (as a control), resulting in shRSK2 and shGFP cells. RSK2 expression levels were assessed by Western blotting with an antibody to this protein. β-Actin expression levels served as an internal control. (B) Viral polymerase activity in shRSK2 and shGFP cells. Cells were transfected with plasmids that directed synthesis of PB2-627E, PB1, PA, NP, and pPolI-luc. After incubation at 33°C for 24 h, cell lysates were prepared and subjected to luciferase assays. Knockdown of RSK2 resulted in more-efficient replication of the virus-like RNA, suggesting that RSK2 suppresses influenza virus replication. The error bars represent standard deviations from three independent experiments. The statistical significance of the difference between the control and test samples is shown by the P value, which was determined by Student's t test (*, P < 0.05). (C) Viral protein production in shRSK2 cells and shGFP cells. shRSK2 and shGFP cells were infected with virus possessing PB2-627E and incubated at 33°C. At the indicated time points postinfection, Western blot analysis was carried out with antibodies against M1 and β-actin. (D) Influenza A virus growth in shRSK2 cells. shRSK2 and control shGFP cells were infected at an MOI of 0.05 with PB2-627E or PB2-627K virus, which possesses glutamic acid or lysine at position 627 in the PB2 protein, respectively. Cells were incubated at 33°C or 37°C for the indicated time periods. Virus titers in MDCK cells were determined. The error bars represent standard deviations from three independent experiments. The statistical significance of the difference between the control and test samples is shown by P values, which were determined by Student's t test (**, P < 0.01; *, P < 0.05; black asterisks indicate the PB2-627K virus, and gray asterisks indicate the PB2-627E virus). (E) Influenza B virus and Sendai virus growth in shRSK2 cells. shRSK2 and control shGFP cells were infected at an MOI of 0.05 with influenza B virus or Sendai virus. At the indicated times after infection, cells were harvested and the virus titers in MDCK cells were determined. The error bars represent standard deviations from three independent experiments. The statistical significance of the difference between the control and test samples is shown by the P value, which was determined by Student's t test (**, P < 0.01; *, P < 0.05).