Defining the mechanism of cytomegalovirus kinase-mediated regulation of viral gene expression and the therapeutic implications

Human cytomegalovirus (HCMV) is a ubiquitous betaherpesvirus that establishes lifelong infections. HCMV is an opportunistic pathogen that causes disease in immunocompromised patients and is a common agent of congenital infection. Current approved treatments options are efficacious but concerns remain about their high toxicity and the rapid development of antiviral resistance. Additionally, the available anti-HCMV agents do not target early events of infection. Therefore, identifying new treatment options that are both safe and effective is of great importance.;The HCMV kinase, pUL97 is a serine/threonine-specific kinase with multiple important roles during infection. pUL97 acts as a viral cyclin-dependent kinase (Cdk) to stimulate cell cycle progression and functions to promote viral DNA synthesis, nuclear egress, and virion maturation. In addition, pUL97 is delivered upon infection as part of the virion, though its role prior to viral gene expression had not been explored. The importance of pUL97 during infection makes it an ideal target for antiviral treatment. The pUL97-specific kinase inhibitor, maribavir (MBV) was developed and is currently used in clinics as a second line anti-HCMV agent. MBV has low toxicity, though mutations in the UL27 open reading frame result in MBV resistance. pUL27 was found to induce p21Cip1 expression and cell cycle arrest. We hypothesized that pUL97 functions to stimulate viral immediate early (IE) gene expression, that pUL97 inhibition by MBV could be used as a safe anti-HCMV agent in a cell culture model of congenital infection, and that MBV resistance was influenced by p21Cip1 and Cdk activity.;The main aims of this thesis were to characterize the molecular mechanism by which pUL97 promotes IE gene expression, assess the effectiveness of MBV in a congenital HCMV model, and identify how p21Cip1 and Cdk activity contribute to MBV resistance. We found that inhibition or deletion of pUL97 resulted in a transient decrease in IE gene expression. pUL97 was sufficient to transactivate IE expression and virion-derived pUL97 increases IE expression. Interestingly, inhibiting histone deacetylases (HDACs), which function to repress transcription, rescued IE expression under pUL97 inhibition. We observed that pUL97 activity increased histone acetylation and decreased HDAC1 association with the major IE promoter (MIEP). Further, pUL97 interacted with and increased phosphorylation of HDAC1.;Previous studies have shown that IE and late viral events contribute to defects in cell culture models of congenital HCMV infection. We therefore tested the effectiveness of MBV for inhibiting infection in the embryonic stem cell-derived neuroprogenitor cell (NPC) model of congenital infection. MBV was demonstrated to effectively limit IE gene expression, expression of other viral genes, viral DNA synthesis and viral yield. Because antiviral resistance poses a major obstruction to treating infections in clinical infections we explored the mechanism of UL27-associated MBV resistance. We found that pUL97 interacts with and promotes phosphorylation of pUL27, while also blocking pUL27- mediated induction of p21Cip1 expression. These studies also resulted in the finding that the full antiviral effect of MBV required p21Cip1 and that UL27-associated MBV resistance required Cdk activity.;Work in this thesis provides mechanistic insight into the role of the pUL97 in activation of IE gene expression, suggests that inhibiting pUL97 in NPCs effectively reduces viral replication, and provides a potential mechanism for antiviral resistance to viral kinase inhibition. II.