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

甲病毒分为不同的、地理上孤立的组，新世界（NW）和旧世界（Old World） (OW)甲病毒不同的甲病毒物种显示出对特定宿主和蚊子的特异性适应 在人类和其他哺乳动物中传播并诱发多种疾病。这些适应是由 病毒结构基因的进化，导致六种主要甲病毒血清复合体的形成。 非结构蛋白nsP 1、nsP 2和nsP 4的进化受到RNA酶活性的限制 复制，这已经得到了很好的表征。然而，nsP 3在甲病毒生物学中的功能，除了 虽然知道这种蛋白质是复制复合物的重要组成部分，但人们对它的了解却很少。氨基- nsP 3的末端片段证明甲病毒物种之间的高度保守性。的 羧基末端结构域是高度磷酸化的，在两个序列之间表现出非常低水平的序列同一性。 甲病毒，因此被称为高变结构域或HVD。我们最近的核磁共振研究 证明了基孔肯雅病毒（CHIKV）的nsP 3特异性HVD是无序的。它包含线性图案， CHIKV与一组不同的蛋白质因子相互作用，并且这些相互作用对于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。另一个层次的冗余是通过一些 相互作用的蛋白质家族独立地介导病毒复制复合物的形成。我们的核心假设 细胞nsP 3 HVD结合蛋白介导CHIKV复制复合物组装。拟议 研究中，我们打算研究CHIKV HVD与细胞SH 3结构域相互作用的机制， 含有蛋白质，主要集中在CD 2AP蛋白。这种相互作用在CHIKV中起着前病毒作用 在脊椎动物细胞中复制，并且对于蚊子细胞中的病毒复制更关键。通过使用各种 生物化学和NMR为基础的方法，我们将精确地定义结合位点的BIN 1，CD 2AP和 SH 3 KBP 1在CHIKV HVD中的定位，分析BIN 1-、CD 2AP-和SH 3 KBP 1-HVD复合物在CHIKV HVD中的定位。 感染的细胞，并确定了含有SH 3结构域的蛋白在CHIKV复制的不同步骤中的作用。我们 未来的研究将导致开发新的治疗方法和减毒疫苗 蚊子不能传播的候选人。

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.