Molecular Genetics of HSV DNA Polymerase Gene

描述(由申请人提供)：这项研究的长期目标是详细了解疱疹病毒DNA聚合酶和针对它们的药物。这些酶包括一个催化亚基(POL)和一个辅助亚基，刺激长链DNA合成，都是原型？DNA聚合酶，也是抗病毒药物的优秀靶点。后一种特性尤其与健康有关，因为治疗疱疹病毒感染需要新药。在本应用中，针对这些蛋白质及其相互作用的辅助亚基、催化亚基和药物的未回答问题被解决。具体目的1是研究单纯疱疹病毒(HSV)UL42和人巨细胞病毒(HCMV)UL44等辅助亚基与DNA相互作用的不同方式，使它们紧密结合，但又沿DNA线性扩散，从而允许DNA合成。单分子方法将被用来分析这些蛋白质如何在DNA上移动，特别是野生型UL42或紧密结合的突变体是否必然螺旋移动，或者例如，可以沿着螺旋的一侧移动。移动这些亚基所需的力将与停止或减缓催化亚基所需的力进行比较。X射线结晶学将被用来了解蛋白质-DNA相互作用的分子细节。具体目的2是从酶功能、与碱基切除修复(BER)酶尿嘧啶DNA糖基酶(UNG)的结合、病毒复制和抗病毒耐药机制等方面，研究拇指、手掌和手指结构域的催化亚基的结构域的作用。在HSV Pol的晶体结构中观察到两个结构域，即Pre-NH2和NH2，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，将设计突变酶并对其进行相关的生化活性检测。突变病毒将被设计并检测病毒在细胞和小鼠模型中的复制。用酶学方法研究突变株HCMV POLS在3‘-5’外切酶结构域上抵抗更昔洛韦(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UL44相互作用以及抑制HCMV复制的新化合物。UL44和另一种复制蛋白UL84之间的相互作用对病毒复制的重要性，以及它是否可以被用作药物靶点，将结合生化、分子遗传学和细胞生物学方法进行研究，包括努力开发一种新的技术来绘制蛋白质-蛋白质相互作用图。如果UL44-UL84相互作用看起来有希望成为药物靶点，将随机筛选抑制这种相互作用的化合物。 公共卫生相关性：疱疹病毒在一般人群中引起广泛疾病，在免疫力受损的人中引起严重疾病。对抗这些病毒的新药有相当大的需求。拟议的研究不仅应该提供有助于药物发现和了解病毒如何对现有药物产生抗药性的信息，而且直接旨在发现新的抗疱疹病毒药物。

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 ?-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. PUBLIC HEALTH RELEVANCE: Herpesviruses cause widespread disease in the population at large and severe disease in people with impaired immunity. There is considerable need for new drugs to combat these viruses. The research proposed should not only provide information that could aid in drug discovery and understanding how viruses become resistant to current drugs, but aims directly to discover new anti-herpesvirus drugs.