The Influence Of Several Key Molecules In The PI3K/Akt Pathway On The Propagation Of Influenza A Virus

Viruses in the host cells can activate intracellular signal transduction to support their efficient replication. Influenza A virus infection can activate the PI3K/Akt pathway to promote host cell survival to prolong its replication. But the downstream mechanism for that PI3K/Akt signaling supports the effective replication of influenza A virus is still not clear. In order to clarify the roles of the key molecules of PI3K downstream signaling such as mdm2, GSK-3βand mTOR in influenza A virus propagation, the human lung adenocarcinoma cell line A549 was infected by Influenza A Puerto Rico/8/34-HIN1. We down-regulated the expressions of mdm2, GSK-3βand mTOR by RNAi with the specific siRNAs. To determine the influences of genes down-regulation to the infected A549 cells, we examined the infected A549 cells' viabilities using the Cell Counting Kit-8 cell proliferation assay kit and examined the influenza virus induced A549 cell apoptosis by measuring the caspase activities with Caspase-Glo 3/7 Kit and flow cytometry. To determine the influences of genes down-regualtion to influenza virus propagation, we performed the TCID 50 assay to measure the viral titers in the A549 supernatants. Then we did an in-depth study about the PI3K/Akt signaling mediated viral protein translation control, we blocked the PI3K/Akt signaling by the specific inhibitor LY294002 and blocked the function of mTOR by its specific substrate-Rapamycin. We detected the influenza A virus induced phosphorylatio of the translation initiation associated proteins such as p70S6K and 4E-BP1 in A549cells with or without specific inhibitors treated by western-blotting. Then we detected the viral protein expression level by western-blotting and immunoflorencent in A549 cells treated with different reagents to determine the influences of different inhibitors to viral protein translation. We also performed the TCID 50 assay to measure the viral titers in the infected A549 cell with different treatments. At last, we expressed the influenza A NS1 protein in A549 cells using a eukaryotic plasmid expression vector. The NS1 expression and 4E-BP1 phoshorylation were detected by western-blotting.We found that mdm2 show the interesting subcellular localization changes during influenza A virus infection; mdm2 gene down-regulation increased the cell viability of infected A549 cell and decreased the cell apoptosis of infected A549 cell, the viral titer was increaed in the mdm2 knockdown A549 cell supernatant; GSK-3βgene down-regulation decreased the cell viability of infected A549 cell and increased the cell apoptosis of infected A549 cell, the viral titer was decreaed in the GSK-3βknockdown A549 cell supernatant; mTOR gene down-regulation decreased the cell viability of infected A549 cell and increased the cell apoptosis of infected A549 cell, the viral titer was decreaed in the mTOR knockdown A549 cell supernatant. Through the studies above, we defined the roles of these three key PI3K/Akt downstream elements in influenza A virus infection caused host cell apoptosis and viral propagation.In the viral protein translation control study, we found that we found that influenza A virus activate PI3K/Akt pathway and lead to 4E-BP1 phosphorylation; LY294002 blocked PI3K/Akt signaling pathway as well as the p70S6Kianse and 4E-BP1 phosphorylation, while Rapamycin blocked the mTORCl function and caused p70S6K phosphorylation inhibition but had no significant effect on 4E-BP1 phosphorylation; LY294002 decreased the influenza virus titer by surpressing the NP expression and the nuclear export of vRNP; Rapamycin had no significant effect on NP expression and vRNP nuclear export, but the viral titer was reduced. Expression of influenza A virus NS1 with the plasmid vector pcDNA3.1 activated PI3K/Akt pathway and induced the 4E-BP1 phosphorylation. Based on the findings above, we suggested that influenza A virus NS1 protein induced 4E-BP1 phosphorylation in a PI3K/Akt dependent manner and played a role in viral protein translation control. According to our knowlege, this is the first report about that PI3K/Akt signaling was involved in the inflenza A virus propagation by reguating the translation initiation. We also discussed the mechanism of this process.Our research clarfied the roles of three key PI3K/Akt downstream substrates (mdm2,GSK-3β,mTOR) in influenza A virus propagation, and discussed the molecular mechanism of PI3K/Akt mediated translation control in influenza A virus infected A549 cells. It is important for the research of virus-host interaction.