背景:肠道病毒71(EV-A71)导致手,儿童口蹄疫(HFMD)与神经系统并发症有关。涉及EV-A71发病机制的分子机制仍然难以捉摸。
方法:在EV-A71感染的运动神经元中进行siRNA筛选,靶向涉及细胞内膜运输的112个基因,然后使用去卷积siRNA验证前四个命中。下游方法,包括病毒进入旁路,通过qPCR进行细胞内病毒基因组定量,蛋白质印迹分析,和荧光素酶报告基因测定允许确定感染周期的阶段,RAB11A参与其中。邻近连接测定,采用免疫共沉淀和多重共聚焦成像来研究病毒成分与RAB11A之间的相互作用。显性阴性和组成型活性的RAB11A构建体用于确定在EV-A71感染期间蛋白质的GTP酶活性的重要性。质谱和蛋白质相互作用分析用于鉴定感染期间RAB11A的宿主相互作用伴侣。
结果:在EV-A71感染期间,小GTP酶RAB11A被鉴定为一种新的前病毒宿主因子。来自主要EV-A71基因组的菌株和手足口病的另一种主要病原体柯萨奇病毒A16可互换地利用RAB11A和RAB11B亚型。我们发现RAB11A不参与病毒进入,IRES介导的蛋白质翻译,病毒基因组复制,病毒退出。RAB11A与复制细胞器共定位,与结构和非结构病毒成分相互作用。显性阴性(S25N;GDP结合)和组成型活性(Q70L;GTP结合)RAB11A突变体的过表达对EV-A71感染结果没有影响,排除RAB11A参与病毒或宿主成分的细胞内运输。相反,在siRAB11处理的细胞中,细胞内成熟病毒颗粒与病毒RNA拷贝的比率降低,VP0:VP2比率增加,这支持了VP0裂解为VP2和VP4所标志的原病毒成熟的作用。最后,监护人,不是贩运和转运蛋白,被发现是EV-A71感染期间RAB11A的主要相互作用伙伴。其中,来自分子伴侣复合物TRiC/CCT的CCT8亚基被进一步验证,并显示与病毒结构蛋白特异性相互作用,在EV-A71感染期间代表另一种新的前病毒宿主因子。
结论:这项研究描述了一种新颖的,RAB11A在病毒感染过程中的非常规作用,它通过招募必需的伴侣蛋白参与病毒形态发生的复杂过程。
BACKGROUND: Enterovirus 71 (EV-A71) causes Hand, Foot and Mouth Disease (HFMD) in children and has been associated with neurological complications. The molecular mechanisms involved in EV-A71 pathogenesis have remained elusive.
METHODS: A siRNA screen in EV-A71 infected-motor neurons was performed targeting 112 genes involved in intracellular membrane trafficking, followed by validation of the top four hits using deconvoluted siRNA. Downstream approaches including viral entry by-pass, intracellular viral genome quantification by qPCR, Western blot analyses, and Luciferase reporter assays allowed determine the stage of the infection cycle the top candidate, RAB11A was involved in. Proximity ligation assay, co-immunoprecipitation and multiplex confocal imaging were employed to study interactions between viral components and RAB11A. Dominant negative and constitutively active RAB11A constructs were used to determine the importance of the protein\'s GTPase activity during EV-A71 infection. Mass spectrometry and protein interaction analyses were employed for the identification of RAB11A\'s host interacting partners during infection.
RESULTS: Small GTPase RAB11A was identified as a novel pro-viral host factor during EV-A71 infection. RAB11A and RAB11B isoforms were interchangeably exploited by strains from major EV-A71 genogroups and by Coxsackievirus A16, another major causative agent of HFMD. We showed that RAB11A was not involved in viral entry, IRES-mediated protein translation, viral genome replication, and virus exit. RAB11A co-localized with replication organelles where it interacted with structural and non-structural viral components. Over-expression of dominant negative (S25N; GDP-bound) and constitutively active (Q70L; GTP-bound) RAB11A mutants had no effect on EV-A71 infection outcome, ruling out RAB11A\'s involvement in intracellular trafficking of viral or host components. Instead, decreased ratio of intracellular mature viral particles to viral RNA copies and increased VP0:VP2 ratio in siRAB11-treated cells supported a role in provirion maturation hallmarked by VP0 cleavage into VP2 and VP4. Finally, chaperones, not trafficking and transporter proteins, were found to be RAB11A\'s top interacting partners during EV-A71 infection. Among which, CCT8 subunit from the chaperone complex TRiC/CCT was further validated and shown to interact with viral structural proteins specifically, representing yet another novel pro-viral host factor during EV-A71 infection.
CONCLUSIONS: This study describes a novel, unconventional role for RAB11A during viral infection where it participates in the complex process of virus morphogenesis by recruiting essential chaperone proteins.