Characterization of human cytomegalovirus immediate-early protein phosphorylation and its role in transcriptional activation

The human cytomegalovirus (HCMV) major immediate-early proteins (IEPs), IEP86 (IE2579aa) and IEP72 (IE1491aa), are promiscuous transcriptional activators of many viral and cellular promoters. Their function is thought to be crucial for the process of temporal viral gene activation as well as for inducing the expression of cellular factors that support viral replication. We have investigated the phosphorylation of the HCMV major IEPs, and the effects of phosphorylation on their ability to function as transcriptional activators. We found that cellular kinases are largely responsible for mediating IEP phosphorylation during HCMV infection. Serum-independent as well as serum-inducible kinases phosphorylated multiple domains of IEP86 and IEP72 in vitro. Alanine substitution mutagenesis was performed on specific serines or threonines found in consensus mitogen-activated protein kinase (MAPK) motifs of IEP86 and IEP72. These mutations in IEP86 and IEP72 clearly eliminated specific phosphorylation in vivo. Analysis of these mutations in vitro showed that T27 and T233/S234 were the major sites in IEP86 for serum-inducible as well as purified ERK2-mediated phosphorylation. IEP86 T555 was phosphorylated by another MAPK family member, JNK1. IEP86 S144 was strongly phosphorylated by p34cdc2/cyclin B kinase. IEP72 S406 was phosphorylated by ERK2 in vitro. In transient transfection analyses, IEP86 isoforms containing alanines at T233/S234, or a combination of alanines at T 27/S144/T233/S234, displayed mildly increased transcriptional activation of a synthetic promoter, suggesting that phosphorylation at these sites in wild type IEP86 may result in reduction of its transcriptional activation ability. However, further analysis showed that the same mutations of IEP86 in the context of other MIE proteins displayed reduced transcriptional activation ability. This indicated that phosphorylation at these sites may increase IEP86\u27s ability to synergize with other MIE proteins in activating transcription. In vitro studies showed that specific kinases reduced the affinity of protein-protein interactions between IEP86 and cellular transcription factors, potentially providing a mechanism for the effects of phosphorylation on the ability of IEP86 to activate transcription