The Role of ATP Hydrolysis in HSV-1 Terminase Activity

Cornell University Ithaca, Ithaca NY

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Abstract

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.