Molecular Genetics of HSV DNA Polymerase Gene

描述（由申请人提供）：本研究的长期目标是详细了解疱疹病毒DNA聚合酶和靶向药物。这些酶包括一个催化亚基（Pol）和一个刺激长链DNA合成的辅助亚基，它们都是原型α-样DNA聚合酶，也是抗病毒药物的极好靶点。后一种特性尤其与健康有关，因为需要新的药物来治疗疱疹病毒感染。在这个应用程序中，未回答的问题，关于辅助亚基，催化亚基，靶向这些蛋白质和它们的相互作用的药物得到解决。具体目标1是研究辅助亚单位，如单纯疱疹病毒（HSV）UL 42和人巨细胞病毒（HCMV）UL 44与DNA相互作用的不寻常和不同的方式，使它们紧密结合，但沿着DNA线性扩散，以允许进行性DNA合成。单分子方法将用于分析这些蛋白质如何在DNA上移动，特别是野生型UL 42或紧密结合突变体是否必然螺旋移动或可以例如沿着螺旋的沿着移动。将移动这些亚基所需的力与停止或减慢催化亚基所需的力进行比较。X射线晶体学将用于了解蛋白质-DNA相互作用的分子细节。具体目标2是调查的催化亚基的N-末端的拇指，手掌和手指结构域的酶功能，结合到碱基切除修复（BER）酶尿嘧啶DNA糖基化酶（UNG），病毒复制和抗病毒药物耐药性的机制的结构域的作用。在HSV Pol的晶体结构中已经观察到两个结构域，pre-NH 2和NH 2，但它们在酶功能和病毒复制中的作用尚不清楚。为了解决这些问题，突变酶将被工程化并测定相关的生物化学活性。将对突变病毒进行工程改造，并在细胞和小鼠模型中测定病毒复制。利用酶学分析研究在其3 '-5'核酸外切酶结构域中具有取代的突变HCMV Pol抵抗更昔洛韦（GCV）作用的机制。具体目标3是使用基于结构的方法发现抑制HCMV Pol和UL 44的相互作用以及HCMV复制的新化合物。UL 44和另一种复制蛋白UL 84之间的相互作用对病毒复制的重要性，以及它是否可以作为药物靶点，将使用生物化学，分子遗传学和细胞生物学方法的组合进行研究，包括努力开发一种新的技术来绘制蛋白质-蛋白质相互作用。如果UL 44-UL 84相互作用看起来有希望作为药物靶标，则将进行抑制这种相互作用的化合物的随机筛选。

DESCRIPTION (provided by applicant): The long-term objective of this research is a detailed understanding of herpesvirus DNA polymerases and drugs that target them. These enzymes, which include a catalytic subunit (Pol) and an accessory subunit that stimulates long-chain DNA synthesis, are both prototype α-like DNA polymerases and excellent targets for antiviral drugs. This latter property is especially health-related, as new drugs are needed for treatment of herpesvirus infections. In this application, unanswered questions regarding accessory subunits, catalytic subunits, and drugs that target these proteins and their interaction are addressed. Specific aim 1 is to investigate the unusual and different manners by which the accessory subunits, such as herpes simplex virus (HSV) UL42 and human cytomegalovirus (HCMV) UL44, interact with DNA so that they bind tightly, yet diffuse linearly along the DNA to permit processive DNA synthesis. Single-molecule approaches will be used to analyze how these proteins move on DNA, particularly whether wild type UL42 or a tight-binding mutant necessarily moves helically or can, for example, move along one side of the helix. The force required to move these subunits will be compared with the force required to stop or slow the catalytic subunits. X-ray crystallography will be used to understand the molecular details of the protein-DNA interaction. Specific aim 2 is to investigate the roles of structural domains of the catalytic subunits that are N-terminal to the thumb, palm, and fingers domains in terms of enzymatic functions, binding to the base excision repair (BER) enzyme uracil DNA glycosylase (UNG), viral replication, and mechanisms of antiviral drug resistance. Two structural domains, pre-NH2 and NH2, have been observed in the crystal structure of HSV Pol, but their roles in enzyme function and viral replication are unknown. To address these questions, mutant enzymes will be engineered and assayed for relevant biochemical activities. Mutant viruses will be engineered and assayed for viral replication both in cells and in a mouse model. The mechanisms by which mutant HCMV Pols with substitutions in their 3'-5' exonuclease domain resist ganciclovir (GCV) action will be investigated using enzymological analyses. Specific aim 3 is to discover new compounds that inhibit the interaction of HCMV Pol and UL44, and HCMV replication, using a structure-based approach. The importance of the interaction between UL44 and another replication protein, UL84, for viral replication, and whether it can be exploited as a drug target, will be investigated using a combination of biochemical, molecular genetic, and cell biological approaches, including efforts to develop a new technique to map protein-protein interactions. Should the UL44-UL84 interaction look promising as a drug target, random screening for compounds that inhibit this interaction will be undertaken.