Molecular Genetics of HSV-1 Capsids

描述(申请人提供)：HSV-1衣壳由七种蛋白质组成，它们一起形成一个大型复杂的组装。这些蛋白质的密切结合在病毒基因组周围形成一个保护壳，需要多个蛋白质-蛋白质相互作用。这些相互作用推动了这种结构的组装。大型蛋白质复合体的组装在许多细胞系统中都很明显，包括转录、蛋白质翻译和蛋白质降解。因此，确定这些多蛋白质组合如何结合、相互作用并表征其结构特征是生物学中的一个基本问题。保护病毒基因组的蛋白质外壳已经被研究为蛋白质如何相互作用和自组装成更高阶结构的范例。这一建议解决了HSV-1衣壳蛋白之间的相互作用，以及介导这些相互作用的残基。这些多蛋白质复合体的结构特征将从分离的复合体和衣壳的背景中进行分析。抗病毒靶标的验证取决于从相互作用结构域及其结构特征的鉴定中获得的信息。 具体目的1.鉴定VP5 N-末端疏水“口袋”或表面上与支架蛋白(22a)C-末端结合的残基。使用模拟该结构域的多肽来抑制蛋白质-蛋白质结合和衣壳组装，评估这种相互作用的抗病毒潜力。确定VP5仍能与22a的C末端结合的最小N端蛋白片段，并表征该复合体的结构性质。 具体目的2.用遗传学方法确定HSV-1蛋白(VP24)编码的衣壳成熟过程中的一个重要结构功能。确定第二个功能的特征，并使用蛋白质结构域交换实验来确定VP24对此功能至关重要的区域。 具体目的3.鉴定VP23的残基，这些残基是在三链组装过程中与VP19C相互作用；在二聚体形成过程中进行自我相互作用；以及在衣壳组装和壳的重新配置过程中与VP5相互作用所必需的。 具体目的4.利用电子冷冻显微镜和三维图像重建技术确定VP26和VP19C的N-末端在衣壳中的位置。确定衣壳中最大的被膜蛋白VPI/2的位置。

DESCRIPTION (provided by applicant): The HSV-1 capsid is comprised of seven proteins that together form a large complex assembly. The intimate association of these proteins to create a protective shell around the virus genome requires multiple protein-protein interactions. These interactions drive the assembly of this structure. Assemblies of large protein complexes are evident in a number of cellular systems including transcription, protein translation, and protein degradation. Thus, defining how these multi-protein assemblies associate, interact and characterization of their structural features is a fundamental problem in biology. The protein coat that protects the virus genomes has been studied as a paradigm for how proteins interact and self-assemble into higher order structures. This proposal addresses the interactions between the HSV-1 capsid proteins, and the residues that mediate these interactions. The structural features of these multi-protein complexes will be analyzed from isolated complexes and in the context of the capsid shell. The validation of an antiviral target depends on the information gained from identification of interactive domains and their structural characteristics. Specific Aim 1. Identify the residues in the hydrophobic "pocket" or surface in the N-terminus of VP5 that bind to the C-terminus of the scaffold protein (22a). Evaluate the antiviral potential of this interaction using peptides that mimic this domain to inhibit protein-protein binding and capsid assembly. Determine the minimal N-terminal protein fragment ofVP5 that can still bind to the C-terminal tail of 22a and characterize the structural properties of this complex. Specific Aim 2. Use genetic methods to confirm an essential structural function encoded by the HSV-1 protease (VP24) during capsid maturation. Characterize this second function and use protein-domain swapping experiments to identify the region of VP24 critical for this function. Specific Aim 3. Identification of the residues of VP23 that are required for interaction with VP19C during triplex assembly; for self-interaction during dimer formation; and for interaction with VP5 during capsid assembly and re-configuration of the shell. Specific Aim 4. Identify using electron cryo-microscopy and 3D image reconstruction the location of the N-terminus of VP26 and VP19C in the capsid shell. Determine the location of the largest tegument protein, VPI/2, in the capsid shell.