Molecular Genetics of HSV-1 Capsids

描述（由申请人提供）：HSV-1衣壳由7种蛋白质组成，它们共同形成一个大型复杂组装体。这些蛋白质紧密结合在一起，在病毒基因组周围形成一个保护壳，这需要多种蛋白质-蛋白质相互作用。这些相互作用驱动了这种结构的组装。大蛋白质复合物的组装在许多细胞系统中是明显的，包括转录、蛋白质翻译和蛋白质降解。因此，定义这些多蛋白质组装体如何缔合、相互作用以及它们的结构特征的表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被研究为蛋白质如何相互作用和自组装成更高级结构的范例。该建议解决了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。这些多蛋白质复合物的结构特征将从分离的复合物和衣壳壳的背景下进行分析。抗病毒靶点的验证依赖于从鉴定相互作用结构域及其结构特征中获得的信息。 具体目标1。鉴定VP 5 N末端疏水“口袋”或表面中与支架蛋白C末端结合的残基（22 a）。使用模拟该结构域的肽来抑制蛋白质-蛋白质结合和衣壳组装，评价这种相互作用的抗病毒潜力。确定仍能与22 a的C-末端尾部结合的VP 5的最小N-末端蛋白片段，并表征该复合物的结构特性。 具体目标2。使用遗传学方法确认衣壳成熟过程中HSV-1蛋白酶（VP 24）编码的基本结构功能。描述第二个功能，并使用蛋白质结构域交换实验来鉴定VP 24的该功能的关键区域。 具体目标3。鉴定在三链体组装期间与VP 19 C相互作用;在二聚体形成期间自身相互作用;以及在衣壳组装和壳的重新配置期间与VP 5相互作用所需的VP 23残基。 具体目标4。使用电子冷冻显微镜和3D图像重建鉴定VP 26和VP 19 C的N-末端在衣壳壳中的位置。确定衣壳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.