Title: The roles of phosphorylation and the carboxy-terminus in the function of the human cytomegalovirus processivity factor, UL44
Description: Human cytomegalovirus (HCMV) is an important pathogen that causes disease in the immunocompromised and in neonates. HCMV encodes a protein kinase, UL97, which is an important antiviral drug target. UL44, an essential viral DNA replication protein, has been identified as a candidate substrate of UL97. UL97 has been shown to be important for efficient viral DNA replication and it has been proposed that this is dependent on UL97-mediated phosphorylation of UL44. The known biochemical activities of UL44 reside in the N-terminal region, while the C-terminal one-third of the protein contains a nuclear localization signal and 5 glycine-strings. The function, if any, of the C-terminus of in viral replication has not been identified. We identified sites of phosphorylation on UL44 by UL97 and cdk1 from in vitro kinase assays and from infected cells. Many of these sites overlapped and most are located in the C-terminus. An inhibitor of UL97 reduced the phosphorylation of specific peptides of UL44 during infection. A panel of HCMV mutants with altered UL44 phosphorylation sites exhibited a moderate (less than 10-fold) decrease in viral replication and replicated their DNA within three-fold of wild-type virus at all time points. These data provide further evidence that UL97 phosphorylates UL44 in infected cells, but provide little support for the hypothesis that phosphorylation of UL44 affects viral DNA synthesis.

We also generated HCMV mutants that expressed only the first 290 residues of UL44. These mutants replicated very poorly, which indicates that the C-terminal region of UL44 is required for efficient viral replication. Importantly, mature viral DNA replication compartments were not observed in cells transfected with the mutant construct. These data suggest that the C-terminus may be important for efficient replication compartment formation. This dissertation highlights the significance of phosphorylation and the C-terminus of UL44 in viral replication. Our data suggest that the effect of UL97 on DNA replication may not be mediated by phosphorylation of UL44. Further elucidation of the function of the C-terminus of this essential DNA replication protein should provide insight into the mechanism of viral DNA replication, a main focus in the development of antiviral drugs.