Mechanistic understanding of SH3 domain-containing protein function in alphavirus replication

甲病毒复制中含 SH3 结构域的蛋白质功能的机制理解

• 批准号: 9804946
• 负责人: ILYA V. FROLOV
• 金额: $ 18.8万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-06-12 至 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9804946
• 关键词: Acute; Africa; Alphavirus; Alphavirus Infections; Arthralgia; Arthritis; Asia; Attenuated Vaccines; Binding; Binding Proteins; Binding Sites; Biochemical; Biology; C-terminal; CD2-associated protein; Cell membrane; Cells; Chikungunya virus; Chronic; Complex; Culicidae; Data; Development; Disease; Dissection; Europe; Evolution; Exanthema; Exhibits; Family member; Fever; Future; Gene Structure; Genes; Geography; Histones; Human; Individual; Integration Host Factors; Investigation; Knowledge; Mammals; Mediating; Nonstructural Protein; Outcome; Pathogenicity; Phenotype; Plants; Play; Protein Family; Proteins; Public Health; RNA Viruses; RNA replication; Research; Research Proposals; Role; Rubella; SH3 Domains; Set protein; Structural Genes; Structure; Symptoms; Therapeutic; Tissues; Vaccines; Viral; Virus; Virus Assembly; Virus Replication; base; member; mutant; novel therapeutics; novel vaccines; pandemic disease; protein function; structural viral genes; therapeutic development; therapeutic vaccine; vaccine candidate; vector; vector mosquito; vector-induced; viral RNA; virus host interaction

Alphaviruses are divided into distinct, geographically isolated groups, the New World (NW) and Old World (OW) alphaviruses. Different alphavirus species display adaptations specific to particular hosts and mosquito vectors and induce diverse diseases in humans and other mammals. These adaptations are driven by the evolution of viral structural genes, which has resulted in the formation of six major alphavirus serocomplexes. Evolution of the nonstructural proteins nsP1, nsP2 and nsP4 is restricted by their enzymatic activities in RNA replication, which have been well characterized. However, functions of nsP3 in alphavirus biology, besides knowing that this protein is an essential component of replication complexes, are less understood. The amino- terminal fragment of nsP3 demonstrates a high level of conservation between alphavirus species. The carboxy-terminal domain is highly phosphorylated, exhibits a very low level of sequence identity between alphaviruses, and is thusly referred to as the hypervariable domain or HVD. Our recent NMR studies have demonstrated that nsP3-specific HVD of chikungunya virus (CHIKV) is disordered. It contains linear motifs that interact with a distinct set of protein factors, and these interactions are indispensable for CHIKV replication. Most of the interacting host factors are represented by protein families, where each member is capable of supporting viral RNA replication. Another level of redundancy is achieved by the abilities of some of the interacting protein families to independently mediate viral replication complex formation. Our central hypothesis is that cellular nsP3 HVD-binding proteins mediate assembly of CHIKV replication complexes. In the proposed research, we intend to investigate the mechanism of CHIKV HVD interactions with cellular SH3 domain- containing proteins, with the main focus on the CD2AP protein. This interaction plays a pro-viral role in CHIKV replication in vertebrate cells and is more critical for viral replication in mosquito cells. By using a variety of biochemical and NMR-based approaches, we will precisely define binding sites for BIN1, CD2AP and SH3KBP1 in CHIKV HVD, analyze localization of BIN1-, CD2AP- and SH3KBP1-HVD complexes in the infected cells and define the role of SH3 domain containing proteins in different steps of CHIKV replication. Our future studies will result in development of new means of therapeutic treatment and attenuated vaccine candidates that cannot be transmitted by mosquitoes.

α病毒分为独特的，地理孤立的群体，新世界（NW）和旧世界 （OW）α病毒。不同的α病毒物种显示针对特定宿主和蚊子的适应性 向量并诱导人类和其他哺乳动物的多种疾病。这些改编是由 病毒结构基因的进化，导致形成六个主要的α病毒血清复合体。 非结构蛋白NSP1，NSP2和NSP4的进化受RNA中的酶促活性的限制 复制，已被很好地表征。但是，NSP3在α病毒生物学中的功能，此外 知道这种蛋白是复制复合物的重要组成部分，知之甚少。氨基 - NSP3的末端碎片显示α病毒物种之间的守恒水平很高。这 羧基末端结构域是高度磷酸化的，表现出非常低的序列身份水平 α病毒，因此被称为高变量域或HVD。我们最近的NMR研究已有 证明Chikungunya病毒（CHIKV）的NSP3特异性HVD是无序的。它包含线性主题 与一组不同的蛋白质因子相互作用，这些相互作用对于CHIKV复制是必不可少的。 大多数相互作用的宿主因素由蛋白质家族表示，每个成员都有能力 支持病毒RNA复制。通过某些能力来实现另一个冗余的水平 相互作用的蛋白质家族独立介导病毒复制复合复合物的形成。我们的中心假设 是细胞NSP3 HVD结合蛋白介导CHIKV复制复合物的组装。在提议中 研究，我们打算研究CHIKV HVD与细胞SH3结构域相互作用的机制 含有蛋白质，主要关注CD2AP蛋白。这种相互作用在Chikv中起着促病毒的作用 在脊椎动物细胞中复制，对于蚊子细胞中的病毒复制更为重要。通过使用多种 基于生化和NMR的方法，我们将精确定义BIN1，CD2AP和 chikv HVD中的SH3KBP1，分析BIN1-，CD2AP-和SH3KBP1-HVD复合物的定位 感染细胞并定义含有蛋白质的SH3结构域在CHIKV复制的不同步骤中的作用。我们的 未来的研究将导致新的治疗方法发展并减弱疫苗 蚊子不能传播的候选人。