Human cytomegalovirus (CMV) is a herpes virus and prototype of the beta herpes virus subfamily. CMV causes pneumonitis, blindness, and death among transplant and AIDS patients, and mental retardation and hearing loss among newborns. There is a pressing need for development of new antiviral drugs to treat CMV infections. All herpes viruses encode an alkaline nuclease (AN) and ANs are among the most highly conserved herpes virus proteins. However, the roles that ANs play in virus replication are not well understood and differ between subfamilies. DNase activities of ANs from the alpha herpes virus subfamily are proposed to promote recombination or facilitate DNA packaging by removing branches or unusual structures from newly replicated viral DNA. In contrast, ANs from the gamma herpes virus subfamily have RNase activity and function to shut off host protein translation by degrading mRNAs. Whether beta herpes virus ANs serve similar roles or have functions unique to the beta herpes virus subfamily is not known. The current application is a well-established multi-investigator collaboration focused on defining the structure and biochemical activities of the CMV AN, UL98, understanding its mechanistic roles in CMV replication, and identifying small molecule inhibitors of UL98 to explore its potential as an antiviral target. Our initial studies used homology modeling to predict the UL98 active site and mutagenesis of E. coli-expressed UL98 to confirm the importance of active site residues for DNase activity. A UL98-null virus was constructed and found to be profoundly growth-attenuated, demonstrating that UL98 is critically important for CMV replication and suggesting that small molecule inhibitors of UL98 may have potent antiviral activity. In support of the latter atanyl blue PRL, an inhibitor of UL98 nuclease activity, has been shown to inhibit CMV replication. The current application has one aim: to determine crystal structures of wild type UL98 and two catalytically-deficient UL98 mutants, as well as UL98 complexed with DNA and with the inhibitor atanyl blue PRL. Comparison of the UL98 structure with existing structures of gamma herpes virus ANs will allow identification of structural features that are unique to each of these proteins. The UL98 structure may also suggest novel functional domains that will help guide the design of mutations to dissect UL98's biochemical activities and functional roles in replication. Importantly, the structure of UL98 complexed with the inhibitor atanyl blue PRL will provide valuable mechanistic and structural insights and will inform and enable structure-based identification of additional UL98 inhibitors. Such inhibitors will serve as pharmacological probes to complement and extend genetic and biochemical studies of UL98's functions and may provide important lead structures for antiviral development. These advances will enable expanded pursuit of UL98 as an antiviral target and may ultimately lead to novel antivirals for treating CMV infections.
Human cytomegalovirus is a significant human pathogen for which a need exists for safer and more effective therapeutics. By determining the crystal structure of UL98 (the viral alkaline nuclease), the proposed studies will form a basis for: (i) future studies aimed at understanding the biochemical activities of UL98 and the role it plays in viral replication; and (ii) exploitation of UL98 as a target for anti-viral drug discovery.
