MULTIPLE MECHANISMS ARE IMPLICATED IN THE REGULATION OF NF-KAPPA-B ACTIVITY DURING HUMAN CYTOMEGALOVIRUS-INFECTION

Infection-induced activation of the human cytomegalovirus major immediate early enhancer/promoter has been shown to be regulated primarily by transcription factor NF-kappaB cis elements. However, the mechanism(s) by which human cytomegalovirus induces NF-kappaB activity is unknown. A study was therefore undertaken to determine how this virus would affect normal NF-kappaB regulation. Viral infection of fibroblasts resulted in the specific stimulation of promoters containing major histocompatibility complex NF-kappaB cis elements fused upstream of the chloramphenicol acetyltransferase reporter gene. Electrophoretic mobility shift assays of nuclear extracts derived from mock- and virus-infected cells showed dramatic and sustained increases in DNA-binding proteins specific for these NF-kappaB sequences. Experiments using MAD-3 IkappaB, a specific inhibitor of NF-kappaB, and antibodies directed against rel family members demonstrated that the induced binding activities contained p50 and p65 proteins but not c-rel. Northern analysis indicated maximal levels of p50 mRNA by 4 h postinfection, whereas p65 and MAD-3 IkappaB mRNA accumulation peaked at 48-72 h postinfection, suggesting different regulatory mechanisms for p50 and p65/IkappaB genes. Electrophoretic mobility shift assays with deoxycholate-treated cytoplasmic extracts demonstrated a 3- to 4-fold decrease in the cytosolic stores of NF-kappaB binding activity by 4 h postinfection. Western blots probed with antibodies directed against MAD-3 IkappaB or pp40 (a protein isolated from chicken with sequence and biochemical properties similar to those of MAD-3 IkappaB) indicated that a cross-reactive peptide of 39 kDa was no longer detectable after 24 h postinfection. These results demonstrate that the activation and maintenance of nuclear NF-kappaB DNA binding and enhancer activities upon human cytomegalovirus infection occurs by multiple mechanisms.