Identification of Residues Controlling dUTPase Enzyme Activity from Epstein‐Barr Virus

Epstein Barr‐Virus (EBV) is primarily known for causing infectious mononucleosis, but also increases the incidence of lymphomas and autoimmune disorders. There is currently no vaccine or treatment once infected by EBV. The EBV genome encodes a dUTPase enzyme. This enzyme catalyzes the reaction of dUTP to dUMP which is a precursor of the thymidine nucleotide. The enzyme activity regulates the dUTP/dTTP ratio and decreases misincorporation of uracil into newly synthesized DNA. A dUTPase knockout is lethal in mice, E. coli., and S. cerevisiae . The Murine gammaherpesvirus lacking its viral dUTPase has reduced viral replication. It is hypothesized that inhibition of EBV dUTPase would result in uracil misincorporation, double stranded DNA breaks, and a reduction in replication of the viral genome. Characterization of residues required for enzyme activity in EBV dUTPase was performed using sequence alignments and structural data. Mutagenesis and comparative enzyme kinetics on these residues identified aspartate 76 as being critical for enzyme activity. Analysis of structural data identified a histidine residue hypothesized to regulate substrate specificity. A small molecule binding site was identified using computational mapping of two EBV dUTPase 3D structures. We hypothesis that this allosteric binding site contains a serine that post transcriptionally regulates dUTPase activity. This research will guide the development of inhibitors that bind to critical dUTPase residues offering a potential treatment for EBV. Support or Funding Information This work was supported by, UWL College of Science and Health, UWL Research and Creativity, and Toce Deans Distinguished Fellowship