Role of cooperative interactions of HSV multifunctional protein ICP27

描述（由申请人提供）：单纯疱疹病毒1 （HSV-1）引起的疾病范围从疼痛性皮肤病变到角膜炎和脑炎。HSV-1编码一种名为ICP27的基本蛋白质，该蛋白质在病毒感染期间参与多种功能。ICP27具有与病毒mRNA和多种宿主细胞蛋白相互作用的有趣能力，劫持它们以促进病毒的产生。因此，ICP27可以作为抗病毒干预的靶点。抗病毒药物的未来发展需要了解这些重要的多组分复合物如何协同组装以及如何调节组装。我们将研究协同性在ICP27介导的蛋白-蛋白和蛋白- rna相互作用中的作用，并表征其复合物的结合界面，以确定ICP27在病毒感染过程中如何在不同复合物中组装，从而揭示其功能的分子机制。这些结构信息将用于探索相互作用界面残基在感染过程中在ICP27功能中的作用。ICP27由许多结构化和非结构化结构域组成，它们参与大量不同的蛋白质-蛋白质和蛋白质- rna相互作用。我们假设这些相互作用可能是多位点的，因此是合作的，由主序列中遥远的区域介导。我们假设瞬态相互作用也是可能的，特别是当涉及未折叠区域时。我们假设至少一些ICP27的相互作用是由磷酸化和/或精氨酸甲基化调节的。我们将从两个方面验证这些假设：1)研究ICP27的直接结合界面

DESCRIPTION (provided by applicant): Herpes simplex virus 1 (HSV-1) causes diseases that range from painful skin lesions to keratitis and encephalitis. HSV-1 encodes an essential protein called ICP27 that is involved in a diversity of functions during viral infection. ICP27 has the intriguing ability to interact with viral mRNA and a multiplicity of host cell proteins, hijacking hem to benefit virus production. Thus, ICP27 could conceivably serve as a target for antiviral intervention. The future development of antivirals requires an understanding of how the cooperative assembly of these essential multicomponent complexes occurs and how the assembly is regulated. We will study the role of cooperativity in protein-protein and protein-RNA interactions mediated by ICP27 and characterize the binding interfaces of its complexes to determine how ICP27 is assembled in different complexes during viral infection, with the goal of revealing the molecular mechanism of its function. The structural information will be used to explore the role of interaction interface residues in ICP27 function during infection. ICP27 consists of a number of structured as well as intrinsically-unstructured domains, which participate in a large number of diverse protein-protein and protein-RNA interactions. We hypothesize that these interactions may be multi-site and therefore cooperative, mediated by regions that may be distant in the primary sequence. We postulate that transient interactions also are possible, especially when unfolded regions are involved. We hypothesize that at least some of ICP27's interactions are regulated by phosphorylation and/or arginine methylation. We will test these hypotheses in two specific aims: 1) To study the direct binding interfaces of ICP27 with cellular proteins and viral mRNA by generating shorter protein constructs in which structural stability is not perturbed, and expressing them in amounts sufficient for structural biology studie including traditional and novel Isotopically-Discriminated (IDIS) NMR spectroscopy as well as biophysical approaches. Cooperativity will be analyzed in interactions between short functional fragments of intrinsically unstructured N-terminal ICP27 and partners that interact within this region, including viral RNA and host cell proteins. In vitro post-translational modifications of ICP27 will be performed to explore how ICP27-multi-protein complex assembly is affected by phosphorylation and arginine methylation. We will also endeavor to perform structural studies of folded domains from the C-terminal part of ICP27 and to characterize interactions with its cellular protein partners. Because ICP27 undergoes a head-to-tail intramolecular interaction in vivo, a chimera containing appropriate fragments of the N- and C-termini could be created to study how these regions interact. 2) To explore the role of interaction interface residues in ICP27 function during viral infection, recombinant viruses will be constructed bearing point mutations at interaction interfaces and will be characterized for in vivo interactions and ICP27 functional activities and effects on viral infection.