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Human cytomegalovirus (CMV) is a widespread human herpesvirus responsible for a number of complications and diseases, particularly in immunocompromised individuals such as AIDS patients, organ transplant recipients, and patients undergoing treatment for cancer. Although there are antiviral compounds that have been approved for the treatment of CMV, there are a number of issues with these current drugs including limited bioavailability, significant toxicity, and high rates of resistance. Additionally, all of these drugs have the same target, the viral DNA polymerase. Thus, there is a need for new antivirals that have novel targets and lower toxicity. The herpesvirus alkaline nuclease, pUL98 in CMV, represents a potential novel target for the development of new antivirals. This protein is critical for efficient replication of the virus and has no closely related human homolog. The goal of this research was to purify and characterize active pUL98, which was then used to design a miniaturized, high throughput assay for screening potential inhibitors of the nuclease. A fusion construct consisting of a double histidine tag incorporated into the N-terminus of pUL98 facilitated purification of the protein by sequential passage over Ni-chelating and Mono Q anion exchange columns. Activity of the nuclease was confirmed using an assay measuring the ability to release soluble, radioactive nucleotides from uniformly labelled DNA. Several compounds have been identified via in silico modeling that are predicted to interact with and thus interfere with the active site of pUL98. Two of these compounds, Acid Blue 129 and Acid Blue 40, have been found to inhibit CMV replication. Using the newly designed assay, it was determined that Acid Blue 129 has a greater inhibitory effect on the nuclease activity of pUL98 (IC50= 9.3 μM) than does Acid Blue 40 (IC50= 149 μM). Both the new assay and in silico modeling to identify potential inhibitors of a protein were validated in the course of these experiments. Furthermore, inhibition of pUL98 may be the mechanism by which Acid Blue 129, and the larger class of anthraquinone derivatives, has anti-CMV activity. Further studies are needed to identify what genetic changes result in resistance to Acid Blue 129, thus elucidating the mechanism of inhibition.