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

Watching conformational rearrangements in picornavirus replication proteins

观察小核糖核酸病毒复制蛋白的构象重排

基本信息

批准号：
10461745
负责人：
David Douglas Boehr
金额：
$ 37.98万
依托单位：
PENNSYLVANIA STATE UNIVERSITY, THE
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-07-15 至 2025-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10461745
关键词：
1-Phosphatidylinositol 4-Kinase 3-Dimensional Active Sites Affinity Amino Acid Substitution Antiviral Agents Behavior Binding Binding Proteins Biogenesis Biological Assay Biological Models Biological Process Biophysics Capsid Proteins Cells Complement Coronavirus Crystallization Development Drug Targeting Elements Enterovirus Enterovirus 68 Environment Enzymes Event Family Picornaviridae Funding Genome Grant Heart Human Human poliovirus Immune response Lead Life Cycle Stages Lipid Binding Lipids Membrane Membrane Proteins Molecular Conformation Motion Mutagenesis NMR Spectroscopy Nucleotides Organelles Peptide Hydrolases Peptides Phosphatidylinositols Play Polymerase Polyproteins Process Production Protein Conformation Proteins Proteolysis Proteolytic Processing Proteome RNA RNA Binding RNA Viruses RNA replication RNA-Directed RNA Polymerase Replication-Associated Process Rhinovirus Ribonucleotides Roentgen Rays Role Sampling Specificity Structure Surface System Thermodynamics Translation Process Viral Viral Proteins Virus Virus Diseases Virus Inhibitors Virus Replication Work antiviral drug development attenuation base emerging pathogen flexibility inhibitor insight nanosecond novel pathogenic virus phosphodiester protein function vaccine development viral RNA

项目摘要

Project Summary Some of the most important new and (re)emerging pathogens are positive-strand RNA viruses, including coronavirus and picornaviruses Enterovirus D68, Enterovirus A71 and even poliovirus. These viruses can directly use their RNA genome to guide the synthesis of a large polyprotein, which must then be proteolyzed into its component parts, including the capsid proteins and enzymes important for genome replication and encapsidation. Virus RNA genomes are rather small, and so these viruses have evolved strategies to essentially expand their functional proteomes. For example, the picornavirus 3C protein is a multi-functional protein that has protease activity, binds RNA control sequences important for coordinating replication and translation processes, and binds phosphoinositide lipids found in virus “replication organelles”, which act to protect the virus from host cell defenses. All of these activities are encoded within its small 20 kDa structure. Another strategy to expand functional protein content is for proteolytic precursors to have different functions than their fully processed counterparts. For example, 3C is also found as part of the 3CD protein, but the 3CD protein has different protease specificity, and different RNA and lipid binding affinities. The 3CD protein also has a 3D domain; the 3D protein is the RNA-dependent RNA polymerase but 3CD does not possess polymerase activity. By itself, 3CD also upregulates phosphoinositide lipid production and induces membrane proliferation, events important for replication organelle biogenesis. How the different and emergent functions of 3CD arise is poorly understood; X-ray crystal structures indicate that 3CD is merely a composite of the 3C and 3D proteins joined together by a small flexible linker. We propose that structural dynamics, that is, the ability to sample multiple structural conformations, is the missing ingredient in understanding virus protein function. We propose that 3C fluctuates among many conformations, providing 3C the ability to access and coordinate its many functions, and we propose that 3CD fluctuates into different conformations, providing it with alternative functions. These dynamic excursions can be further modified by interactions with RNA, lipids and protein binding partners to coordinate virus protein function. We will evaluate these protein structural dynamics through solution-state nuclear magnetic resonance spectroscopy, which provide atomic-level detail of protein motions from the picosecond to second timescales, and complement these studies with mutagenesis studies, functional assays and cell-based approaches to better understand the roles of protein structural dynamics in the virus life cycle. The completed work will provide new opportunities for rational anti-viral strategies, for example, by finding molecules that bind to alternative protein conformations and/or disrupt functionally-important motions, as already validated for 3D.

项目总结