三种小干扰RNA干扰VP16 mRNA对单纯疱疹病毒-1复制的影响研究

Analysis of biological roles of VP16 in cultured cells infected with HSV-1 by RNA interference

目的探讨体外合成小干扰RNA(siRNA),在RNA干扰(RNAi)技术条件下,单纯疱疹病毒-1(HSV-1)感染Vero细胞过程中,病毒间层蛋白VP16的生物学功能。方法用T7RNA聚合酶体外合成针对VP16mRNA的三种siRNA。用脂质体2000转染Vero细胞后接种HSV-1,感染后不同时相点(12,24,36,48,60,72hpi)分别采取实验组(R)和对照组(C)标本,检测病毒滴度值,绘制子代病毒一步生长曲线。用同位素标记新合成的VP16进行免疫沉淀法检测病毒蛋白表达的变化。结果病毒子代一步生长曲线显示,转染siRNA的实验组Vero细胞接种HSV-1后24h(24hpi),病毒滴度明显低于对照组;12hpi也观察到病毒滴度低于对照组的现象;多位点的siRNA具有协同作用;实验组病毒滴度高峰出现时间后移大约12h。siRNA影响了病毒的蛋白表达。结论T7RNA聚合酶体外合成的siRNA可以用于RNAi技术研究;HSV-1间层蛋白VP16对病毒复制和组装、成熟起重要的生物学作用。

Objective To determine the roles of tegument protein VP16 of HSV-1 during HSV- 1 viral replication and assembly/maturation of infection virion by RNA interference. Methods Short interfering RNAs （siRNA）were transeripted in vitro using TTRNA polymerase. The Veto cells were transfected with siRNA using lipofeetamine 2000 followed by HSV-1 infecting and was monitored at 12,24,36,48,60 and 72h post infeetion（pi） by titers using single-step growth curve assay. Newly synthesized VP16 using 35S met-MEM of HSV-1 in infected cells was analyzed by immunoprecipitation （IP）. Results Pre-treatment of Vero cells with siRNA homologous to VP16, followed by HSV-1 infecting showed a markedly reduces the titer of virus progeny. One-step growth curves indicated that cells treated with siRNA produced only 10% of the virus titer observed in control-treated cells with lipofeetamine 2000 alone at 24h pi, the results also suggest that siRNA have a synergistic effect of targeting multiple sites,and the peak value of HSV-1 infection titer in siRNA-treated was delayed about 12h. The newly synthesized VP16 was reduced. Conclusion siRNA transeripted in vitro using T7 RNA polymerase can be used in researches based on the technology of RNAi. These results demonstrate that HSV-1 VP16 has important biological functions for HSV-1 lytic replication and correlates with HSV-1 virion assembly/maturation.