Structural Studies on Noroviruses

诺如病毒的结构研究

• 批准号: 7774781
• 负责人: Bidadi Venkataram Prasad
• 金额: $ 29.17万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-15 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7774781
• 关键词: Accounting; Acute; Animals; Antiviral Agents; Bacterial Gastroenteritis; Blood Group Antigens; Caliciviridae; Calicivirus; Capsid; Capsid Proteins; Complex; Cysteine; Epidemic; Eukaryotic Initiation Factors; Exhibits; Experimental Designs; Family; Family Picornaviridae; Fingers; Funding; Genome; Genotype; Goals; Grant; Human; Link; Molecular; Molecular Target; N-terminal; Norovirus; Norwalk virus; Pattern; Pattern Recognition; Peptide Hydrolases; Peptide Initiation Factors; Polymerase; Polyproteins; Proteins; Proteolytic Processing; RNA-Directed RNA Polymerase; Ribosomes; Roentgen Rays; Role; Sequence Analysis; Serine Protease; Set protein; Site; Staging; Structural Protein; Structure; Substrate Specificity; System; Therapeutic; Time; Translation Initiation; Viral; Viral Genome; Virus; Virus Replication; Walkers; base; design; drug discovery; helicase; inhibitor/antagonist; insight; novel; nucleoside triphosphatase; pathogen; prevent; programs; protein function; receptor; small molecule; viral RNA

This project focuses on continuing our X-ray crystallographic studies on Norovirus capsids and some ofthe non-structural proteins encoded by the norovirus genome that are critical for norovirus replication. Based on the substantial progress made in the previous funding period, the new specific aims in this proposal are designed to further our understanding of the structural basis of various virus related functions that could be helpful in the design of experimental strategies in Project 2, and identify novel targets for developing effective antiviral drugs for noroviruses in conjunction with proposed studies in Project 1. In Specific Aim 1, our focus will be to further investigate the structural basis of genotype-specific recognition of HBGA by noroviruses. In Specific Aim 2, our goal is to delineate the structural determinants of substrate specificity in the NV protease, making use of a novel system that we discovered during the current grant period. In addition, we will perform structural analysis on small molecule inhibitors identified in Project 1 in complex with the protease. These structures will facilitate the design of inhibitors with increased potency. In Specific Aim 3, we will determine the structure of a norovirus VPg. Several studies on both animal andtlhuman caliciviruses have strongly implicated this protein as a "cap substitute" for translation initiation of the viral RNA and in a primer function during genome replication. We have expressed and purified VPg ,''VPg-Pro, VPg-Pro-Pol and obtained crystals of VPg-Pro demonstrating the feasibility of obtaining the structure of this important protein and setting the stage for structural studies to understand the molecular basis of its interaction with ribosomal initiation factors and viral polymerase, and the mechanism of uridylylation. In Specific aim 4, we will determine the structure ofthe p41 of Norwalk virus. p41 protein is an NTPase/helicase analogous to picornavirus 2C protein. Based on sequence analysis, p41 exhibits motifs such as A, B, B', C and Arg finger found in SF3-2 helicases. Although, the function of this protein is unclear, one hypothesis is that it provides a mechanism to prevent viral ssRNA from annealing during the strand synthesis by the viral polymerase. We have been able to express and purify this protein to homogeneity.

该项目的重点是继续我们对诺如病毒capsids和某些X射线晶体学研究 非结构性蛋白质是由诺如病毒基因组编码的，这对于诺如病毒复制至关重要。基于 在上一个资金期间取得的实质进展，该提案中的新特定目标是 旨在进一步了解我们可能是各种病毒相关功能的结构基础 有助于项目2中实验策略的设计，并确定开发有效的新目标 诺病毒的抗病毒药物与项目1中的拟议研究结合。在特定目标1中，我们的重点 将进一步研究诺病毒对HBGA基因型特异性识别的结构基础。在 具体目标2，我们的目标是描述NV蛋白酶底物特异性的结构决定因素， 利用我们在当前赠款期间发现的新型系统。此外，我们将表演 针对蛋白酶复杂的项目1中鉴定的小分子抑制剂的结构分析。这些 结构将促进抑制剂的设计，并增加效力。在特定目标3中，我们将确定 诺如病毒VPG的结构。关于动物和tlhuman蜡civires的几项研究都有很强的 将这种蛋白与病毒RNA的翻译启动和引物功能相暗示。 在基因组复制期间。我们已经表达并纯化的VPG，''VPG-Pro，VPG-Pro-Pol，并获得了 VPG-Pro的晶体表明，获得该重要蛋白质的结构的可行性和 为结构研究奠定了阶段，以了解其与核糖体相互作用的分子基础 起始因子和病毒聚合酶以及尿液磷酸的机理。在特定目标4中，我们将 确定Norwalk病毒的p41的结构。 p41蛋白是类似于NTPase/Helicase Picornavirus 2C蛋白。基于序列分析，P41表现出诸如A，B，B'，C和Arg Finger之类的基序 在SF3-2解旋酶中发现。虽然，该蛋白质的功能尚不清楚，但一个假设是它提供了 一种预防病毒聚合酶链合成过程中病毒ssRNA退火的机制。我们 能够将这种蛋白质表达和纯化为同质性。