Activation of the NF-κB pathway in human cytomegalovirus-infected cells is necessary for efficient transactivation of the major immediate-early promoter

We previously demonstrated that human cytomegalovirus (HCW) infection induced the activation of the cellular transcription factor NF-kappaB. Here, we investigate the mechanism for the HCW-induced NF-kappaB activation and the role that the induced NF-kappaB plays in transactivation of the major immediate-early promoter (MIEP) and production of immediate-early (IE) proteins. Using a dominant-negative inhibitor of NF-kappaB, the IkappaB-superrepressor, we demonstrated that active NF-kappaB is critical for transactivation of the HCMV MIEP. Investigation of the mechanisms of NF-kappaB activation following HCMV infection showed a rapid and sustained decrease in the inhibitors of NF-kappaB, IkappaBalpha and IkappaBbeta. Because the IkappaB kinases (IKKs) regulate the degradation of the IkappaBs, virus-mediated changes in the IKKs were examined next. Using dominant-negative forms of the IKKs, we showed significant decreases in transactivation of the MIEP in the presence of these mutants. In addition, protein levels of members of the IKK complex and IKK kinase activity were upregulated throughout the time course of infection. Lastly, the role NF-kappaB plays in HCMV IE mRNA and protein production during infection was examined. Using aspirin and MG-132, we demonstrated that production of IE protein and mRNA was significantly decreased and delayed in infected cells treated with these drugs. Together, the results of these studies suggest that virus-mediated NF-kappaB activation, through the dysregulation of the IKK complex, plays a primary role in the initiation of the HCMV gene cascade in fibroblasts and may provide new targets for therapeutic intervention.