权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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

肠道病毒正链RNA的合成：RNP复合物形成的病毒和宿主决定因素的发现

基本信息

批准号：
10092097
负责人：
Steven Pascal
金额：
$ 19万
依托单位：
UNIVERSITY OF CALIFORNIA-IRVINE
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-01-30 至 2023-09-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10092097
关键词：
5&apos Untranslated Regions Address Affinity Antiviral Agents Binding Binding Proteins Binding Sites Biological Assay Biological Models Cardiomyopathies Cells Chemicals Chronic Common Cold Complex Coupled Coxsackie B Viruses Coxsackie Viruses Cytoplasm DNA-Directed RNA Polymerase Disease Encephalitis Enterovirus Enterovirus Infections Family Family Picornaviridae Genetic Materials Genome Geometry Goals Heart Transplantation Human Human poliovirus Infection Lead Maps Mass Spectrum Analysis Membrane Messenger RNA Methods Modeling Myocarditis Nucleotides Paralysed Peptide Hydrolases Poliomyelitis Process Proteins RNA RNA Viruses RNA chemical synthesis RNA replication RNA-Protein Interaction Rhinovirus Roentgen Rays Role Site Structure Tertiary Protein Structure Testing Therapeutic Viral Viral Proteins Virion Virus Virus Assembly Virus Diseases Work base combat crosslinking and immunoprecipitation sequencing experimental study fighting genomic RNA high resolution imaging insight member new therapeutic target novel prevent stem three dimensional structure viral RNA

项目摘要

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.

小核糖核酸病毒的肠病毒属成员引起广泛的疾病，包括心肌炎、麻痹性脊髓灰质炎、普通感冒和脑炎。在受感染细胞的细胞质中，病毒（例如，柯萨奇病毒、脊髓灰质炎病毒、EV 71、人鼻病毒）通过以下途径复制其正义RNA基因组：一个两步的过程。首先，使用正义基因组RNA作为模板来合成互补的负RNA链。然后，该中间负RNA链作为模板， RNA链既作为mRNA又作为新病毒颗粒的基因组RNA。迄今为止，大多数研究肠道病毒RNA复制的研究集中在第一步：负链RNA的合成。实验在本申请中提出的方法将集中于第二个，并且可以说是更中心的步骤：形成新病毒颗粒所需的正RNA链。具体来说，这些研究将集中在预测的 RNA负链3'端形成的三叶草样结构，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参与者和结合位点这些蛋白共同启动第二步RNA复制。该提案的第一个目标是实现国家- 最先进的方法，如ChIRP-MS和RNA亲和方法与质谱联用，与柯萨奇病毒B3（CVB 3）的3 '-CL相互作用的宿主和病毒蛋白。对于第二个目标，三个- 将解析预测的CVB 3 RNA的3 '-CL的三维结构，提供第一个高分辨率图像这个关键的复制平台。这将通过最先进的核磁共振/小角度X射线组合进行散射（SAXS）方法。在最终目标中，CLIP-seq分析和基于NMR的化学位移扰动将用于将鉴定的蛋白质结合到3 '-CL RNA结构上。这些研究将大大推进我们对肠道病毒RNA复制关键第二步的理解，并确定新的治疗方法这些靶点可以通过阻断病毒的组装来提供多种机会来对抗肠道病毒感染， RNA复制复合物。