The Effect On Mammalian Target Of Rapamycin (mTOR) During The Infection Of Influenza A Virus

Purpose:The specific aim of this study is to investigate the influence of mTOR on cellular and viral NP protein expression during the influenza virus infection.Methods:The present study detected Akt, p-AktSer473 and p4E-BP1Ser65 levels by Western-blot in influenza A virus Puerto Rico/8/34-H1N1 infected A549 cells at five hours post infection. A plasimid of pcDNA3.1-NS1, which can express NS1 protein in eukaryotic cells, was constructed with routine molecular cloning techniques. In A549 cells, the effect of NS1 on the host cellular protein expression was indicated by its effect on the expression of firefly luciferase reporting gene (cells were transfected with pGL3 plasmid 24 hours after transfection of pcDNA3.1-NS1 plasmid). PI3K specific inhibitor LY294002 and two mTOR inhibitors (mTORC1 inhibitor Rapamycin and mTORC1/2 inhibitor Torinl) were used to block PI3K or mTOR activity for validation the effect of mTOR on host-influenza virus interaction.Results:Infection of Influenza A virus PR8 led to Akt phosphorylation and increased 4E-BP1 phosphorylation, which indicated that mTOR might have an effect on the infection of influenza A virus. Rapamycin, Torinl and LY294002 inhibited Influenza A virus PR8-induced 4E-BP1 phosphorylation in different degree. Torinl, the inhibitor for both mTORC1 and mTORC2, much more potently inhibited Influenza A virus PR8-induced Akt phosphorylation than mTORC1 inhibitor Rapamycin, which indicated that mTORC2 participated the activation of mTOR in in influenza A viurs infection. In A549 cells expressed NS1, Akt and 4E-BP1 phosporylation were enhanced. In A549 cell line co-transfected with pcDNA3.1-NS1 and pGL3 in turn, the luciferase activity increased significantly, while LY294002,Rapamycin and Torinl inhibited luciferase activity in different degree. At five hours post infection and at given concerntration of each inhibitor, Rapamycin, Torinl and LY294002 had no significant impact on the expression of NP protein.Discussion:NS1 protein can activate mTOR-4E-BP1 pathway through activation of PI3K/Akt. Both mTORC1 and mTORC2 play roles in the activation of this pathway. NS1 may be important for maintaining the cellular basic level of translation in influeza A virus infected cells, and this effect should be due to, at least be partially due to, the activation of mTOR-4E-BP1 pathway.