Colletodiol降低聚合酶活性抑制流感病毒(WSN/H1N1)复制

Colletodiol inhibits the replication of influenza A virus WSN/H1N1 by reducing the activity of viral RNA polymerase

摘要:

摘要:【目的】利用流感病毒启动子报告细胞株(HeLa-IAV-Luc)，从微生物代谢产物化合物库中筛选具有抑制流感病毒(WSN/H1N1)效果的化合物，并初步探索其抑制流感病毒的机理。【方法】首先用化合物处理感染流感病毒的HeLa-IAV-Luc细胞，通过荧光素酶实验来筛选出能够抑制流感病毒的化合物。其次通过在流感病毒感染的不同时间点加入化合物并检测病毒蛋白的表达水平，以此来研究化合物在流感病毒复制的何时发挥作用，并用假病毒实验进一步验证。同时通过流感病毒启动子报告实验来检测化合物对流感病毒RNA 聚合酶(viral RNA polymerase，vRNP)活性的影响，探究化合物抑制流感病毒的机理。最后通过实时荧光定量PCR来检测化合物对流感病毒RNA 合成的影响。【结果】通过检测流感病毒感染的HeLa-IAV-Luc细胞中荧光素酶活性从化合物库中筛选出了具有抑制流感病毒活性的化合物Colletodiol。通过在流感病毒感染的不同时间点加药以及假病毒实验证明了Colletodiol在流感病毒进入细胞后发挥作用，对血凝素(hemagglutinin，HA)蛋白功能没有影响。通过启动子报告系统证明了Colletodiol能显著抑制流感病毒vRNP的活性，流感病毒RNA的合成也因此受到抑制。【结论】通过HeLa-IAV-Luc细胞筛选出了具有抑制流感病毒活性的化合物Colletodiol，其抑制流感病毒的机理是能够显著降低流感病毒vRNP 复合物的活性。

Abstract:

Abstract:［Objective］To screen for compounds against influenza A virus by using the viral promoter reporter cell line HeLa-IAV-Lucand investigate their anti-viral mechanism.［Methods］We screened the inhibitors of influenza A virus by infecting HeLa-IAV-Luc cells with influenza A virus WSN /H1N1 and treating it with the compounds isolated from microbial metabolites．The cell lysates were then subjected to the luciferase assay． We conducted the pesudovirus assay to analyze whether the compound affected the function of hemagglutinin (HA)．We carried out the influenza viral promoter reporter assay to examine whether the compound could inhibitinfluenza viral RNA polymerase (vRNP) activity． The effect of antiviral compoundon influenza viral RNA synthesis was measured by real-time fluorescence quantitative PCR．［Results］Colletodiol inhibited the replication of influenza A virus in HeLa-IAV-Luc cells by luciferase assay．It did not inhibit the function of HA protein based on the results of the time-of-addition experiment and pseudovirus assay．The influenza viral polymerase promoter reporter assay indicated that Colletodiol could inhibit the activity of vRNP dramatically．Due to the inhibition of vRNP，the influenza viral RNA synthesis decreased significantly.［Conclusion］Compound Colletodiol was an inhibitor of influenza A virus． It blocks the replication of influenza A virus by reducing the activity of vRNP.