HUMAN CYTOMEGALOVIRUS IMMEDIATE-EARLY GENE-2 PROTEIN INTERACTS WITH ITSELF AND WITH SEVERAL NOVEL CELLULAR PROTEINS

The human cytomegalovirus immediate-early gene product 2 (IE2) is able to transactivate homologous and heterologous promoters alone or augme nted by immediate-early gene product 1 (IE1). IE2 has also been shown to autoregulate the major immediate-early promoter by directly binding to a cis repression signal located between the TATA box and the cap s ite. However, IE2 has not been shown to act directly through a specifi c DNA sequence in transactivating various promoters. To understand whe ther IE2 can be indirectly involved in DNA sequence-specific transacti vation through interactions with other transcriptional factors, we per formed a study of the interactions of IE2 with cellular proteins. In o rder to study these interactions, IE cDNAs were subcloned into a bacte rial expression vector, pGEX2T, by polymerase chain reaction amplifica tion to produce fusion proteins which were full-length as well as prot eins which contained various functional domains. We were able to demon strate IE2's ability to interact directly or indirectly with several c ellular proteins ranging from >200 to 14 kDa through glutathione S-tra nsferase-fusion protein precipitation and far-Western analysis. These interactions have been mapped to domains within IE2 which are known to be necessary for either transactivation or both transactivation and a utoregulation. All of the IE2-associated proteins are nuclear proteins , and a subset are phosphorylated. In vitro-synthesized S-35-IE2 prote in and bacterially expressed IE2 fusion proteins were used to study IE 2-IE2 interaction by binding assay and far-Western analysis. IE2-IE2 i nteractions were mapped to a domain containing a putative helix-turn-h elix motif located near the C terminus of IE2, between amino acids 456 and 539. However, IE2 was unable to directly interact with either IE1 , an alternatively spliced variant of IE2 (55 kDa), or IE2 deletion mu tants that did not contain the multimerization domain.