| This study explored the role of eukaryotic translation initiation factor 4B (eIF4B) in influenza A virus replication by in vitro and in vivo experiments. Human A549 cell line, A549 cells silencing or overexpressing eIF4B, and the transgenic mice with eIF4B knockdown were used in this project.We firstly examined eIF4B protein and mRNA expression levels by Western blot and RT-PCR assays in A549 cells infected with A/WSN/33 (multiplicity of infection [MOI] of 0.1) for 4h,8h,12h,16h,20h, and 24h respectively. Then the protein levels of eIF4B in the lungs, spleens, and livers of Balb/c mice, which was inoculated intranasally with A/WSN/33 (8×106 PFU/ml), were also analysed by Western blot. The viral titers in infected A549 cells having eIF4B knocked down or over-expressing eIF4B and in infected Mouse Embryonic Fibroblasts (MEFs) derived from transgenic mice that express shRNA targeting eIF4B were determined by plaque assay. Lastly, we analyzed the resistance of transgenic mice to influenza A virus infection through investigating their survival time, body weight changes, and organ pathology. The results are shown as follows:(1) With lastingness of infection, the protein expression levels of eIF4B in A549 cells decreased gradually. But the changes of their mRNA expression levels were not significant.(2) The eIF4B protein level reduced obviously in the lungs and spleens of Balb/c mice after influenza A virus infection.(3) The results of plaque assay showed that the ability of influenza A virus replication was increased significantly in A549 cells silencing eIF4B, and the replication was inhibited when eIF4B was ectopically expressed.(4) The viral titer in infected MEFs silencing eIF4B was much higher than that of control group. Interestingly, the transgenic mice expressing shRNA targeting eIF4B were more susceptible to influenza A virus, exhibiting greatly reduced survival time, faster weight loss, and more severe organ pathology. |
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