蛋白激酶抑制剂Flavopiridol对流感病毒复制的体外抑制作用

Protein kinase inhibitor flavopiridol inhibits the replication of influenza virus in vitro

摘要:

摘要:【目的】在细胞水平上研究黄酮类化合物flavopiridol的抗流感病毒效果，初步探索了其抗流感病毒的机制。【方法】首先用Western blot 初步检测了在蛋白激酶抑制剂flavopiridol 处理下流感病毒NP 和M1 蛋白的水平，然后通过免疫荧光实验观察了宿主细胞中流感病毒vRNP 的合成，又利用噬斑实验检测了flavopiridol对病毒复制的影响，最后通过检测flavopiridol处理的宿主细胞内RNA 聚合酶Ⅱ的磷酸化状态和病毒各种RNA 的合成量，探究了flavopiridol抑制流感病毒复制的机理。【结果】结果表明，flavopiridol在细胞水平上可以显著抑制流感病毒蛋白质和vRNP 的合成及病毒的复制，同时flavopiridol也可以抑制宿主RNA 聚合酶Ⅱ大亚基CTD 结构域七肽重复序列中的2 位丝氨酸的磷酸化来抑制聚合酶的转录延伸活性，显著地减少病毒vRNA 的合成。【结论】Flavopiridol可以通过抑制宿主细胞RNA 聚合酶Ⅱ的转录延伸活性有效地抑制流感病毒的复制。

Abstract:

Abstract:［Objective］To investigate the antiviral effect of the flavonoid compound flavopiridol on influenza A virus and explore its antiviral mechanism． ［Methods］The A549 or Madin-Darby canine kidney (MDCK) cells were infected with influenza A virus A/WSN/33 and treated with flavopiridol． The viral proteins were determined by immunolotting and immunofluorescence． The virus titer was measured by plaque assay． To verify whether the activity of host RNA polymerase Ⅱ was affected by flavopiridol，the phosphorylation status of RNA polymerase Ⅱ CTD domain was analyzed by immunoblotting with phosphor-specific antibody． The amount of viral mRNA，vRNA and cRNA was measured by reverse transcription and PCR． ［Results］The amount of viral proteins was significantly decreased and the titer of virus was greatly reduced in cells treated with flavopiridol． Further analysis showed that the phosphorylation of Ser-2 in the heptad repeat of the CTD domain in RNA polymeraseⅡ was decreased in falvopiridol treated cell． This result indicated that the transcription elongation activity of RNA pol II was impaired upon treatment with flavopiridol． Then we found that the amount of viral vRNA was significantly decreased in flavopiridol treated cells while only moderate decrease of mRNA was observed and almost no reduction of cRNA was detected． ［Conclusion］Flavopiridol can greatly suppress the replication of influenza virus． We propose that the inhibition of the transcription elongation activity of host RNA polymeraseⅡ would cause the decrease of viral mRNA transcription．