The Role of ATP Hydrolysis in HSV-1 Terminase Activity

ATP 水解在 HSV-1 终止酶活性中的作用

• 批准号: 6694560
• 负责人: Carol Lynn Duffy
• 金额: $ 4.81万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-01 至 2005-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6694560
• 关键词: Escherichia coli; adenosinetriphosphatase; artificial chromosomes; bioenergetics; cell line; chemical kinetics; endodeoxyribonuclease; enzyme activity; enzyme structure; functional /structural genomics; herpes simplex virus 1; hydrolysis; postdoctoral investigator; protein protein interaction; protein purification; protein structure function; recombinant virus; virus DNA; virus assembly; virus protein

DESCRIPTION (provided by applicant): The goal of this project is a thorough analysis of the ATPase activity associated with the herpes simplex virus type 1 packaging enzyme, or terminase. The process of DNA packaging is both conserved among members of the Herpesviridae and distinct from cellular processes. Thus studies on the HSV-1 terminase will provide knowledge important for the generation of anti-viral agents for herpesviruses associated with a number of life-threatening diseases. The hydrolysis of ATP is required for herpesvirus DNA packaging. The UL15 and UL28 proteins have been proposed to comprise the HSV-1 terminase and, therefore, to possess the many activities required for DNA packaging including ATPase activity. The specific aims of this proposal utilize biochemical and genetic approaches to identify and analyze the ATPase center of the HSV-1 terminase. Purified UL15 and UL28 proteins will be tested for ATPase activity and the kinetics of ATP hydrolysis will be determined. ATP-interacting residues will be identified through the sequencing of protease-derived peptides from 8-N3-[alpha-32p]ATP-photolabeled protein. Proteins containing amino acid changes in ATP-interacting residues will be generated, purified, and studied to confirm the importance of those residues to ATP hydrolysis. Finally, HSV-1 mutants carrying the above mutations will be generated and examined in vivo to identify specific DNA packaging activities that require the newly discovered ATPase center. These studies will greatly increase the understanding of the process of DNA packaging by all herpesviruses and will extend our knowledge of ATP-driven molecular motors.

描述(由申请人提供)：该项目的目标是彻底分析与单纯疱疹病毒1型包装酶或终止酶相关的ATPase活性。DNA包装过程在疱疹病毒科成员中既是保守的，又不同于细胞过程。因此，对HSV-1终止酶的研究将为开发与多种危及生命的疾病相关的疱疹病毒的抗病毒药物提供重要的知识。疱疹病毒DNA包装需要ATP的水解酶。UL15和UL28蛋白被认为是HSV-1终止酶的组成部分，因此具有DNA包装所需的许多活性，包括ATPase活性。这项建议的具体目的是利用生化和遗传学方法来鉴定和分析HSV-1终止酶的ATPase中心。将对纯化的UL15和UL28蛋白进行ATPase活性测试，并确定ATP水解酶的动力学。ATP相互作用残基将通过对8-N3-[α-32P]ATP光标记蛋白中的蛋白酶衍生多肽的测序来鉴定。将产生、纯化和研究含有ATP相互作用残基中氨基酸变化的蛋白质，以确认这些残基对ATP水解的重要性。最后，携带上述突变的HSV-1突变体将被产生并在体内进行检测，以确定需要新发现的ATPase中心的特定DNA包装活动。这些研究将大大增加对所有疱疹病毒包装DNA过程的理解，并将扩大我们对ATP驱动的分子马达的知识。