MEK1/2抑制剂U0126抑制肠道病毒71型的复制

为了揭示肠道病毒71型(enterovirus71,EV71)的复制与宿主细胞Raf/MEK/ERK信号通路(简称ERK通路)的相互关系,本研究应用临床诊断为手足口病的患儿疱疹液,通过易感细胞分离培养、RT-PCR及序列测定,以及Western印迹技术等方法,成功分离到EV71临床株.进一步用该分离株感染易感细胞,通过观察宿主细胞p-ERK1/2蛋白磷酸化水平、病毒特异性衣壳蛋白VP1水平、病毒半数组织培养感染量(50%tissue culture infectious dose,TCID50),以及感染细胞的CPE等指标,以期揭示ERK通路在EV71复制的作用.结果表明,EV71的复制可引起细胞ERK通路的活化;而用MEK1/2特异性的抑制剂U0126预先抑制ERK通路的活化,可显著地降低受染细胞上清液中的病毒的感染滴度(以TCID50表示)、受染细胞中EV71VP1蛋白水平、受染细胞中EV71核酸水平,以及受染细胞的细胞病变效应(cytopathic effect,CPE).提示ERK信号通路的活化对EV71的复制具有重要的作用.本研究为进一步阐明EV71在宿主细胞内的复制机制、寻找新型抗病毒靶标等研究奠定了良好的基础.

Replication of EV71 Was Suppressed by MEK1/2 inhibitor U0126

Enterovirus 71 (EV71) is one of the main etiological agents of hand, foot and mouth disease (HFMD) and has been responsible for severe neurological complications such as poliomyelitis like paralysis, encephalitis and meningitis with high mortality and serious sequelae in recent outb reaks in the Asia Pacific region. In this study, EV71 was isolated from culturedcells inoculated with the vesicular fluid of a pediatric patient diagnosed as HFMD and were identified by RT- PCR, DNA sequencing and Western blot analysis. To investigate whether MEK1/2 and its associated ERK pathway may be involved in EV71 replication, biphasic activation kinetics of ERK were observed in RD cells following infection with EV71 at a multiplicity of infection (MOI) of 1.5 As a consequence, EV71 propagation, which was represented by viral specific capsid protein VP1, infectious viral titers (TCID50), EV71 RT-PCR products and EV71- caused cytopathic effect (CPE), were detected at 24 hours post infection (p.i.). Perturbing the ERK signaling pathway by incubated with U0126, a specific inhibitor of MEK1/2, exhibited an evident anti-EV71 effect. Altogether, our data suggest that MEK1/2 and its associated ERK signaling pathway play a critically positive role in the replication of EV71, indicating that MEK1/2 could be a potential target to develop new anti-EV71 drugs. To our knowledge, this is the first report to demonstrate the effective inhibition of EV71 replication by targeting human MEK 1/2 with U0126.