CAML蛋白对流感病毒复制调控机制的初步研究

Study on the mechanism of CAML protein regulating influenza virus replication

为探究宿主因子钙离子信号调节亲环素配体(CAML)调控流感病毒复制的机制,本研究通过siRNA干扰技术下调CAML的表达后,利用病毒蚀斑试验检测CAML对流感病毒复制的影响,利用激光共聚焦显微镜观察CAML对流感病毒NP蛋白入核的影响,通过流式细胞仪检测CAML对流感病毒诱导的细胞凋亡的影响。瞬时过表达CAML后,利用双荧光素酶报告基因试验检测CAML对流感病毒聚合酶活性的影响。通过qRT-PCR检测流感病毒感染后对CAML mRNA转录水平的影响。结果显示:下调表达CAML可显著抑制流感病毒的复制水平,但对流感病毒NP蛋白的入核无影响;瞬时过表达CAML对流感病毒聚合酶活性无影响;下调表达CAML能够促进流感病毒诱导的细胞凋亡过程,并且流感病毒感染能够导致CAML mRNA转录水平的逐渐降低。以上结果表明:CAML可能通过抑制细胞凋亡的方式促进流感病毒复制,而不是作用于流感病毒的侵入阶段以及转录复制阶段,并且流感病毒感染对CAML的表达具有下调作用。本研究初步探究了宿主因子CAML参与流感病毒复制的分子机制,为其的深入研究奠定了基础。

To investigate the mechanism of calcium signal-modulating cyclophilin ligand(CAML)regulating influenza virus replication,the virus titer was determined by plaque assay,the nuclear accumulation of influenza virus NP protein was observed by laser confocal microscope and the cell apoptosis induced by influenza virus was analyzed by flow cytometer after the expression of CAML was down-regulated by siRNA interference.Influenza virus polymerase activity was detected by dual luciferase reporter assay after the expression level of CAML was up-regulated by transient overexpression.And qRT-PCR was used to detect the effect of influenza virus infection on the transcription level of CAML mRNA.The results showed that down-regulated expression of CAML could significantly inhibit the replication of influenza virus,while down-regulated expression of CAML had no effect on the nuclear accumulation of influenza virus NP protein,and up-regulated expression of CAML did not affect influenza virus polymerase activity.However,knockdown of CAML could promote the cell apoptosis rate induced by influenza virus,and influenza virus infection could lead to a gradual reduction in the transcription level of CAML mRNA.These results suggest that CAML may promote influenza virus replication by inhibiting apoptosis,rather than by acting on the entry stage or transcriptional replication stage,and influenza virus infection inhibits CAML expression.This study explored the molecular mechanism of host factor CAML participating in influenza virus replication,offering basis for further in-depth study.