ADP-核糖基化因子在肠道病毒71型复制中的作用

Hand, foot, and mouth disease, which is caused by the enterovirus genus of the Picornavirus family, is a common viral illness in infants and children. Most hand, foot, and mouth disease infections do not result in serious complications; however, when the pathogen is enterovirus 71 (EV71), the disease can present with serious neurological symptoms such as aseptic meningitis, encephalitis, and acute flaccid paralysis, and may even lead to death. Growing hand, foot and mouth disease outbreaks were observed worldwide in recent years and caused devastating losses both economically and politically. However, vaccines or effective drugs are unavailable to date. All RNA viruses with a positive-strand genome undergo RNA replication in association with membranes of infected cells. However, Picornaviruses, including EV71, generally do not use native organelle membranes for replication but induce the formation of novel cytoplasmic vesicular compartments in infected cells. It is well documented that COPI- and COPII-mediated vesicle budding from the endoplasmic reticulum (ER) and Golgi apparatus are associated with Picornavirus-induced vesicle formation. The fungal metabolite brefeldin A (BFA) inhibits enteroviral RNA replication is another important clue towards the involvement of cellular secretory pathway. BFA specifically inhibits the activation of small cellular GTPases, which comprise the ADP-ribosylation factor (Arf) family. The family consists of six members, which are divided into three classes based on their primary structures. The proteins participate in formation of coated membranous vesicles originating from different organelles and are key regulators of the cellular secretory pathway. To have a better understanding of EV71 replication and to study the mechanism for the involvement of secretory pathway in viral replication, we intend to knock-down the six members one by one and thus identify the key cellular factors required for viral replication. Furthermore, identification of cellular factors required for viral replication will provide potential targets for the development of antiviral drugs.

肠道病毒71 型（EV71）感染通常引起婴幼儿手足口病，部分重症可导致严重的神经系统疾病，致残及病死率较高。EV71已在世界范围内引起十多次爆发与流行。在我国,手足口病也曾经在许多省市发生过数次大规模流行，成为严重威胁人民健康的重大传染病之一。目前尚无有效的药物和疫苗。肠道病毒感染细胞后，需要诱导产生新的囊泡结构，并在此囊泡结构上进行基因组复制。这一过程须要细胞分泌信号通路的参与。ADP-核糖基化因子属于小GTPase，参与胞内囊泡结构的形成，是分泌信号通路的重要调节因子，在多种病毒的复制中扮演重要角色。前期实验结果表明EV71的复制须要ADP-核糖基化因子的参与。本文将对已知的ADP-核糖基化因子的六个成员进行鉴定，确定EV71复制所必须的细胞因子，并对其参与病毒复制的机理进行研究。对这一细胞因子的确定不仅能够对EV71的复制机理有进一步的了解，也为开发抗病毒药物提供潜在的靶标。

肠道病毒71 型（EV71）感染通常引起婴幼儿手足口病，部分重症可导致严重的神经系统疾病，致残及病死率较高。EV71已在世界范围内引起十多次爆发与流行。在我国,手足口病也曾经在许多省市发生过数次大规模流行，成为严重威胁人民健康的重大传染病之一。目前尚无有效的药物和疫苗。肠道病毒感染细胞后，需要诱导产生新的囊泡结构，并在此囊泡结构上进行基因组复制。这一过程须要细胞分泌信号通路的参与。ADP-核糖基化因子属于小GTPase，参与胞内囊泡结构的形成，是分泌信号通路的重要调节因子，在多种病毒的复制中扮演重要角色。前期实验结果表明EV71的复制须要ADP-核糖基化因子的参与。本文将对已知的ADP-核糖基化因子的五个成员进行鉴定，确定EV71复制所必须的细胞因子，并对其参与病毒复制的机理进行研究。我们利用小干扰RNA对五个ADP-核糖基化因子逐一进行基因敲减。 结果显示对单一ADP-核糖基化因子进行基因敲减并不会影响肠道病毒71的复制；然而同时对ADP-核糖基化因子1和3进行基因敲减，则会显著抑制细胞内病毒的复制。基于以上结果，我们得到如下结论：肠病毒71 RNA复制依赖于I类ADP核糖基化因子的活性。同时，本项目中我们还利用实时荧光定量PCR和Westernblot等技术证明了布雷菲德菌素A（brefeldin A)能够抑制细胞内核苷酸交换因子（GBF1）的活性，进而抑制I类ADP核糖基化因子的活性。从而达到抑制肠道病毒71的复制。综上所述，我们的研究结果表明肠道病毒71复制依赖于GBF1介导的I类ADP核糖基化因子的活性。 这一结果不但揭示了肠道病毒71复制和细胞分泌途径之间的联系，同时也为抗病毒治疗提供了新靶点。