Identifying cellular kinases that regulate HSV-1 ICP0 activities and viral replication.

Herpes simplex virus 1 (HSV-1) infects a significant number of people worldwide and is the number one cause of infectious blindness in western industrialized countries. HSV-1 causes a lifelong latent infection that can be reactivated by various stressors. HSV-1 E3 ubiquitin ligase phosphoprotein, infected cell protein 0 (ICP0), is a potent global transcriptional activator of genes and is required for productive viral replication and reactivation from latency. While the phosphorylation state of ICP0 is important for regulating many activities of ICP0, there is only a minimal amount known about which specific cellular kinases are important for modulating these activities. Studies with HSV-1 have been done with Roscovitine (Rosco), which inhibits CDK1, CDK2, CDK3, CDK5, CDK7, and CDK9 have shown that Rosco impairs HSV-1 replication, reactivation, the transactivation activity of ICP0, and it alters the posttranslational modification state of ICP0. It is not known which of these specific kinases are required for regulating these activities. This thesis summarizes my studies to identify specific cellular kinases that are required for the transactivation activity of ICP0 and regulate its other key functions to promote efficient viral replication and reactivation from latency. To begin to elucidate which specific cellular kinases are important for the transactivation activity of ICP0, we developed a high throughput cell-based assay using HSV-1 GFP reporter viruses with small interfering (siRNA) in a 96-well plate format. siRNA library used in screens was against ~700 known and putative cellular kinases, from which I identified several (~21) potential regulators of ICP0’s ability to stimulate viral gene expression. From the list of kinase candidates, we focused our studies on cyclin-dependent kinases (CDKs) 1, 2, 4 and 6, which are associated with the cell cycle and did not appear to have an inhibitory effect on ICP0 protein expression in siRNA-transfected cells. We decided to use specific CDK inhibitors to examine the role activities of specific CDKs play in modulating ICP0 function. Our data shows that inhibiting CDK1 impaired HSV-1 replication in an ICP0-dependent manner, while the replication of wild-type (WT) HSV-1 and an ICP0-null mutant was diminished when the CDK2 inhibitor was tested. We then examined the effect of these CDK inhibitors on viral protein expression in WT HSV-1 infected cells. While the CDK1 inhibitor reduced levels of UL42, an early protein; the CDK2 inhibitor had a much greater effect by decreasing or inhibiting UL42, ICP8, US11, and VP5 expression. None of the CDK inhibitors appeared to have an effect on ICP0 localization or ND10 disruption. Finally, using a quiescent infection model, we demonstrated that inhibitors of CDKs 1 and 2 repressed ICP0-induced reactivation of viral gene expression. These results strongly suggest that CDK1 regulates an aspect of ICP0’s transactivation activity, which in turn enhances HSV-1 lytic infection and reactivation. While CDK2 also appears to regulate (at least in part) ICP0’s ability to stimulate gene expression during lytic replication and reactivation, it also aids HSV-1 productive infection cell culture in an ICP0-independent fashion