UC San Diego

This dissertation further explores some of the many facets by which human cytomegalovirus (HCMV) subverts and utilizes components of the ubiquitin-proteasome system in facilitating a productive infection. HCMV infection causes severe cell cycle deregulation and arrest, mediated in part by the deactivation of the anaphase-promoting complex (APC), one of the main E3 ubiquitin ligases involved in cell cycle regulation. By characterizing the effects on the APC subunits and its co-activator Cdh1 during the infection, I have further delineated the mechanism(s) by which APC Cdh1 is disabled during the infection. Cdh1 becomes abnormally phosphorylated early in the infection in a Cdk-independent manner, which may inhibit its ability to bind and activate the APC. UL97 is identified as the viral protein kinase involved in mediating Cdh1 phosphorylation. Analysis of the APC core subunits reveals that the complex dissociates during the infection with the TPR subunits relocalizing to the cytoplasm while APC1 remains nuclear, which is caused by the proteasome- mediated degradation of APC5 and APC4. Studies utilizing a UL97-deletion virus indicate that Cdh1 phosphorylation and APC dissociation occur independently despite similar kinetics. The possible redundancy of these mechanisms underlies the importance of deactivating the APC during the infection. The targeting of an intermediate component in the ubiquitin-proteasome pathway is necessary as inhibition of the proteasome is also found to be detrimental to viral replication. Proteasome inhibition assays show that proteasome activity is required at all stages of the infection. Moreover, proteasome activity increases as the infection progresses, and proteasome subunits relocalize in and around viral replication centers. Characterization of the peri-replication center region shows it to be proteolytically and transcriptionally active. Taken together, these results suggest that proteasomes (or specific subunits) may play a direct role in facilitating viral DNA replication and transcription