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.

甲病毒分为不同的、地理上孤立的群体：新世界 (NW) 和旧世界 （OW）甲病毒。不同的甲病毒物种表现出针对特定宿主和蚊子的适应性 媒介并在人类和其他哺乳动物中诱发多种疾病。这些调整是由 病毒结构基因的进化，导致六种主要甲病毒血清复合物的形成。 非结构蛋白 nsP1、nsP2 和 nsP4 的进化受到它们在 RNA 中的酶活性的限制 复制，这已经得到了很好的表征。然而，nsP3 在甲病毒生物学中的功能除了 人们对这种蛋白质是复制复合物的重要组成部分了解较少。氨基- nsP3 的末端片段显示了甲病毒属物种之间的高度保守性。这 羧基末端结构域高度磷酸化，显示出非常低水平的序列同一性 α病毒，因此被称为高变域或HVD。我们最近的核磁共振研究 证明基孔肯雅病毒（CHIKV）的 nsP3 特异性 HVD 发生紊乱。它包含线性图案 与一组独特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子由蛋白质家族代表，其中每个成员都能够 支持病毒RNA复制。另一个级别的冗余是通过某些人的能力来实现的 相互作用的蛋白质家族独立介导病毒复制复合物的形成。我们的中心假设 细胞 nsP3 HVD 结合蛋白介导 CHIKV 复制复合物的组装。在提议的 研究中，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，主要集中在CD2AP蛋白质。这种相互作用在 CHIKV 中发挥促病毒作用 脊椎动物细胞中的复制，对于蚊子细胞中的病毒复制更为重要。通过使用各种 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 复合物在 CHIKV HVD 中的定位 感染细胞并定义含有 SH3 结构域的蛋白质在 CHIKV 复制的不同步骤中的作用。我们的 未来的研究将导致开发新的治疗方法和减毒疫苗 不能通过蚊子传播的候选者。