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

甲型病毒分为不同的地理隔离组，即新大陆(NW)和旧大陆 (哦)甲型病毒。不同的甲型病毒物种表现出对特定宿主和蚊子的特定适应 在人类和其他哺乳动物中传播并引发各种疾病。这些适应是由 病毒结构基因的进化，导致了六个主要的甲型病毒血清复合体的形成。 非结构蛋白nsP1、nsP2和nsp4的进化受其在RNA中的酶活性的限制 复制，这已经被很好地描述了。然而，NSP3在甲病毒生物学中的功能，除了 知道这种蛋白质是复制复合体的重要组成部分，人们知之甚少。氨基- NSP3的末端片段在甲型病毒物种之间具有高度的保守性。这个 羧基末端结构域高度磷酸化，显示出很低水平的序列一致性 甲型病毒，因此被称为高变区或HVD。我们最近的核磁共振研究表明 证明了基孔肯雅病毒(CHIKV)的NSP3特异性HVD是紊乱的。它包含线性图案， 与一组不同的蛋白质因子相互作用，这些相互作用对于CHIKV的复制是不可或缺的。 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。另一种级别的冗余是通过一些 相互作用的蛋白质家族独立地介导病毒复制复合体的形成。我们的中心假设 是细胞内NSP3 HVD结合蛋白介导CHIKV复制复合体的组装。在建议的 研究中，我们打算研究CHIKV HVD与细胞SH3结构域的相互作用机制。 含有蛋白质，主要集中在CD2AP蛋白质上。这种相互作用在CHIKV中起着亲病毒的作用 在脊椎动物细胞中复制，对蚊子细胞中的病毒复制更为关键。通过使用各种 生化和基于核磁共振的方法，我们将精确定义BIN1，CD2AP和 SH3KBP1在CHIKV HVD中的定位，分析BIN1-、CD2AP-和SH3KBP1-HVD复合体在CHIKV HVD中的定位 并确定SH3结构域蛋白在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和减毒疫苗的开发 不能通过蚊子传播的候选人。

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.