Inhibition or induction of MLC phosphorylation inhibits or enhances influenza proliferation, respectively.

<p>Inhibition of MLCK leads to inhibition of influenza replication. MDCK cells were treated with serial dilutions of the MLC kinase inhibitor ML7 for 16 h (A) or were transfected with a DN mutant of MLCK for 24 h (B). The cells were infected with influenza virus (MOI, 0.01), and virus yield was measured 24 h later by RT-PCR. Inhibitors of Rho kinase and phospholipase C inhibitors inhibit influenza proliferation. MDCK cells were treated with the indicated concentrations of Fasudil (C) or the phospholipase C inhibitor, U73122 (D) and 16 h later were infected with influenza virus (MOI, 0.01). Treatment of HUVECs with PMA and TNF-α leads to phosphorylation of MLC. (E) HUVECs treated for 15 min with 5 nM PMA or 20 ng/ml TNF-α. Treatment of HUVECs with PMA, calyculin A or TNF-α enhances influenza proliferation. (F–H) HUVECs were treated with the indicated concentrations of PMA (F), calyculin A (G) or TNF-α (H) for 30 minutes and then infected with influenza (MOI, 0.01). After 24 h, virus yield was measured by RT-PCR. Influenza-induced MLC phosphorylation is inhibited by inhibitors of MEK/ERK, Raf kinase and phospholipase C. (I) HUVECs were pretreated with PD98059(5 Mm, 2 h), GW 5074 or U73112 (10 µM each for 2 h), infected with 10 MOI of influenza virus, and collected 8 h later. *P<0.01; N = 4 for each experiments.