Characterization of the cytomegalovirus alkaline nuclease active site

• Main Author: Kuchta, Alison L
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-2008
• Subjects: Microbiology
• ISBN: 054978036X; 9780549780366

Human Cytomegalovirus (CMV) produces an alkaline nuclease, UL98, that is highly conserved among herpesviruses and has both endonuclease and exonuclease activities in vitro. This protein is thought to be important for viral replication and therefore represents a potential target for antiviral development; however, little is known about its structure or role in viral replication. Herpesvirus alkaline nucleases are classified within the PD-(D/E)XK superfamily of deoxyribonucleases based on their enzymatic activities, sequence conservation, and predicted structural similarities. Structure computational algorithms were used to generate a model of pUL98 based on the known structure of lambda exonuclease. The model predicted that residues D254, E278, and K280 of pUL98 represent the critical D, E, and K active site residues that define the PD-(D/E)XK superfamily, while R164 and S252 correspond to residues proposed in lambda exonuclease as binding the 5’-phosphate end of the DNA substrate. N-terminally 6x-His tagged UL98 was expressed in E. coli, affinity column purified, and confirmed to have exonuclease activities. Amino acid substitutions D254A, E278A, K280A, and S252A each essentially inactivated exonuclease activity, while R164A decreased exonuclease activity by >90%, confirming the structural model's predictions that these residues comprise the active sie of UL98. Endonuclease activity was examined as well and was intact in R164A, slightly attenuated in K280A and S252A, and significantly decreased in D254A and E278A. These results help elucidate roles of each residue in the active site. An amber null mutation of UL98 was constructed in the virus and demonstrated that UL98 is very important for viral replication as titers were reduced from 106 to < 102 pfu/ml. A second viral mutation, UL98-R164A, was constructed and demonstrated that a > 90% decrease in exonuclease activity does not impair viral replication. These results should facilitate the design of future experiments that will further elucidate the role of ANs in herpes viral replication and aid in discovery of inhibitors that target UL98.