Synthesis of enterovirus positive-strand RNAs: discovery of viral and host determinants of RNP complex formation

肠道病毒属小核糖核酸病毒的成员可引起多种疾病，包括 心肌炎、麻痹性脊髓灰质炎、普通感冒和脑炎。在感染细胞的细胞质中，这些 病毒(如柯萨奇病毒、脊髓灰质炎病毒、EV71、人鼻病毒)通过 这是一个两步走的过程。首先，以正向基因组RNA为模板，合成互补的 负核糖核糖核酸链。然后，该中间负性RNA链用作模板以产生多个正性 RNA链既可以作为信使核糖核酸，也可以作为新病毒颗粒的基因组RNA。到目前为止，大多数研究 对肠道病毒RNA复制的研究主要集中在第一步：合成负链RNA。实验 在本申请中提出的将重点放在第二个步骤，也可以说是更核心的步骤：多个 正的RNA链需要形成新的病毒颗粒。具体地说，这些研究将集中在预测的 在负链的3‘末端形成的RNA三叶式结构，即所谓的3’-三叶草(3‘-CL)。 3‘-CL被认为形成了一个平台，作为不同病毒和宿主的关键玩家和结合部位 共同启动第二步RNA复制的蛋白质。该提案的第一个目标将实施 最先进的方法，如Chirp-MS和RNA亲和方法与质谱学相结合来鉴定 与柯萨奇病毒B3(CVB3)的3‘-CL相互作用的宿主和病毒蛋白。对于第二个目标，三个- 将解决预测的CVB3RNA的3‘-CL的空间结构，提供第一个高分辨率图像 这一关键的复制平台。这将通过最先进的核磁共振/小角X射线组合完成 散射(SAXS)方法。在最终目标上，CLIP-SEQ分析和基于核磁共振的化学位移微扰将 用于将已鉴定蛋白质的结合映射到3‘-CL RNA结构上。这些研究将显著地 提高对肠道病毒RNA复制关键第二步的认识，寻找新的治疗方法 可通过阻断病毒组装来提供多种抗击肠道病毒感染机会的靶点 RNA复制复合体。

Members of the Enterovirus genus of picornaviruses are responsible for a wide range of diseases, including myocarditis, paralytic poliomyelitis, the common cold, and encephalitis. In the cytoplasm of infected cells, these viruses (e.g., coxsackievirus, poliovirus, EV71, human rhinovirus) replicate their positive-sense RNA genome via a two-step process. First, the positive-sense genomic RNA is used as a template to synthesize a complementary negative RNA strand. This intermediate negative RNA strand then serves as a template to make multiple positive RNA strands that function both as mRNA and as genomic RNAs for new virus particles. To date, most studies of enterovirus RNA replication have focused on the first step: synthesis of the negative-strand RNA. Experiments proposed in this application will focus on the second, and arguably more central, step: synthesis of the multiple positive RNA strands needed to form new virus particles. Specifically, these studies will focus on a predicted RNA cloverleaf-like structure that forms at the 3’ end of the negative strand, the so-called 3’-cloverleaf (3’-CL). The 3’-CL is thought to form a platform that serves as a crucial player and binding site for different virus and host proteins that together initiate the 2nd RNA replication step. The first aim of the proposal will implement state-of- the-art approaches such as ChIRP-MS and RNA affinity methods coupled with mass spectrometry to identify host and virus proteins that interact with the 3’-CL of coxsackievirus B3 (CVB3). For the second aim, the three- dimensional structure of the predicted 3’-CL of CVB3 RNA will be solved, providing the first high resolution image of this critical replication platform. This will be done via a state-of-the-art combined NMR/Small Angle X-ray Scattering (SAXS) approach. In the final aim, CLIP-seq analysis and NMR-based chemical shift perturbation will be used to map the binding of the identified proteins onto the 3’-CL RNA structure. These studies will significantly advance our understanding of the key 2nd step of enterovirus RNA replication and identify novel therapeutic targets that may provide multiple opportunities to combat enterovirus infections by blocking the assembly of viral RNA replication complexes.