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：
9904920
负责人：
Steven Pascal
金额：
$ 24.32万
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 2021-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9904920
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 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

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 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

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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.

picornaviruses肠病毒属的成员负责各种疾病，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（例如Coxsackievirus，Poliovirus，EV71，人类鼻病毒）通过通过一个两步的过程。首先，阳性基因组RNA用作合成完整性的模板负RNA链。然后，此中间负RNA链充当模板以使多个阳性新病毒颗粒的RNA链作为mRNA和基因组RNA。迄今为止，大多数研究肠病毒RNA复制的重点是第一步：负链RNA的合成。实验在本应用程序中提出的将重点关注第二个，可以说是更中心的步骤：综合倍数形成新病毒颗粒所需的正RNA链。具体而言，这些研究将集中于预测在负链的3'末端形成的RNA三叶草状结构，即所谓的3'-Cloverleaf（3'-Cl）。人们认为3'-CL形成了一个平台，该平台是不同病毒和宿主的关键参与者和具有约束力的站点共同启动第二个RNA复制步骤的蛋白质。该提案的第一个目标将实施 ART方法，例如Chirp-Ms和RNA亲和力方法，结合质谱法以识别与Coxsackievivirus B3（CVB3）相互作用的宿主和病毒蛋白。为了第二个目标，三将求解预测的3'-CL CVB3 RNA的尺寸结构，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NMR/小角度X射线来完成散射（SAXS）方法。在最终目的中，剪辑序列分析和基于NMR的化学移动扰动将用于将鉴定蛋白的结合映射到3'-CL RNA结构上。这些研究将大大促进我们对肠病毒RNA复制的关键第二步的理解并确定新疗法可以通过阻止病毒的组装来打击肠内病毒感染的多种机会来打击肠病毒感染的目标 RNA复制复合物。