Enterovirus-A71 exploits RAB11 to recruit chaperones for virus morphogenesis.

IF 9 2区 医学 Q1 CELL BIOLOGY
Qing Yong Ng, Vikneswari Mahendran, Ze Qin Lim, Jasmine Hwee Yee Tan, Joel Jie Feng Wong, Justin Jang Hann Chu, Vincent T K Chow, Newman Siu Kwan Sze, Sylvie Alonso
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引用次数: 0

Abstract

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.

肠病毒-A71 利用 RAB11 招募伴侣,促进病毒形态发生。
背景:肠道病毒71型(EV-A71)会导致儿童手足口病(HFMD),并与神经系统并发症有关。EV-A71致病的分子机制仍未确定:方法:在EV-A71感染的运动神经元中进行了siRNA筛选,靶向112个参与细胞膜内转运的基因,然后使用去卷积siRNA对前4个命中基因进行验证。下游方法包括病毒进入旁路、qPCR 细胞内病毒基因组定量、Western 印迹分析和荧光素酶报告实验,从而确定了候选基因 RAB11A 所参与的感染周期阶段。为了研究病毒成分与 RAB11A 之间的相互作用,研究人员采用了邻近连接试验、共免疫沉淀和多重共焦成像技术。利用显性阴性和组成型活性 RAB11A 构建物来确定该蛋白在 EV-A71 感染过程中 GTPase 活性的重要性。质谱分析和蛋白质相互作用分析用于鉴定 RAB11A 在感染过程中的宿主相互作用伙伴:结果:小GTP酶RAB11A被鉴定为EV-A71感染过程中的新型促病毒宿主因子。RAB11A和RAB11B异构体被主要EV-A71基因组的毒株和手足口病的另一种主要致病病毒柯萨奇病毒A16相互利用。我们发现,RAB11A 不参与病毒进入、IRES 介导的蛋白质翻译、病毒基因组复制和病毒排出。RAB11A 与复制细胞器共定位,在复制细胞器中与病毒的结构性和非结构性成分相互作用。过量表达显性阴性(S25N;GDP结合)和组成型活性(Q70L;GTP结合)RAB11A突变体对EV-A71感染结果没有影响,从而排除了RAB11A参与病毒或宿主成分胞内运输的可能性。相反,在 siRAB11 处理过的细胞中,细胞内成熟病毒颗粒与病毒 RNA 副本的比例降低,VP0:VP2 的比例升高,这证明 RAB11A 在前病毒成熟过程中发挥作用,其特征是 VP0 分裂成 VP2 和 VP4。最后,研究发现在 EV-A71 感染过程中,RAB11A 的首要相互作用伙伴是伴侣蛋白,而不是贩运和转运蛋白。其中,伴侣蛋白复合物TRiC/CCT中的CCT8亚基得到了进一步验证,并被证明能与病毒结构蛋白发生特异性相互作用,是EV-A71感染过程中另一种新型亲病毒宿主因子:本研究描述了 RAB11A 在病毒感染过程中扮演的新颖、非传统的角色,它通过招募必要的伴侣蛋白参与病毒形态发生的复杂过程。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Journal of Biomedical Science
Journal of Biomedical Science 医学-医学:研究与实验
CiteScore
18.50
自引率
0.90%
发文量
95
审稿时长
1 months
期刊介绍: The Journal of Biomedical Science is an open access, peer-reviewed journal that focuses on fundamental and molecular aspects of basic medical sciences. It emphasizes molecular studies of biomedical problems and mechanisms. The National Science and Technology Council (NSTC), Taiwan supports the journal and covers the publication costs for accepted articles. The journal aims to provide an international platform for interdisciplinary discussions and contribute to the advancement of medicine. It benefits both readers and authors by accelerating the dissemination of research information and providing maximum access to scholarly communication. All articles published in the Journal of Biomedical Science are included in various databases such as Biological Abstracts, BIOSIS, CABI, CAS, Citebase, Current contents, DOAJ, Embase, EmBiology, and Global Health, among others.
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