Human cytomegalovirus immediate-early gene 2 protein interacts with itself and with several novel cellular proteins

Abstract

The human cytomegalovirus immediate-early gene product 2 (IE2) is able to transactivate homologous and heterologous promoters alone or augmented 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 site. However, IE2 has not been shown to act directly through a specific DNA sequence in transactivating various promoters. To understand whether IE2 can be indirectly involved in DNA sequence-specific transactivation through interactions with other transcriptional factors, we performed a study of the interactions of IE2 with cellular proteins. In order to study these interactions, IE cDNAs were subcloned into a bacterial expression vector, pGEX2T, by polymerase chain reaction amplification to produce fusion proteins which were full-length as well as proteins which contained various functional domains. We were able to demonstrate IE2's ability to interact directly or indirectly with several cellular proteins ranging from > 200 to 14 kDa through glutathione S-transferase-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 autoregulation. All of the IE2-associated proteins are nuclear proteins, and a subset are phosphorylated. In vitro-synthesized 35S-IE2 protein and bacterially expressed IE2 fusion proteins were used to study IE2-IE2 interaction by binding assay and far-Western analysis. IE2-IE2 interactions were mapped to a domain containing a putative helix-turn-helix 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 mutants that did not contain the multimerization domain.

Regulation of human cytomegalovirus US3 gene transcription by a cis-repressive sequence

“…The mIE gene complex is the most extensively characterized of the immediate-early transcription units. Regulation of mIE gene expression is controlled by a complex regulatory region that includes the promoter, the enhancer (which contains a number of imperfect repeat elements), a modulator region, nuclear factor 1 binding sites, and additional sequences found 3Ј of the TATA box (1,4,13,18,24,29). Transcription from the mIE promoter results in the expression of the IE1 and IE2 genes through multiple spliced RNAs (37,38).…”

“…Interaction of IE2 with the crs element appears to be directly responsible for down regulation of the mIE promoter (22,25). Down regulation by IE2 requires only the COOH terminus of the protein; this protein region is also involved in transactivation and dimer formation (9,13). Like other transcriptional activators, IE2 forms contacts with general transcriptional factors, including the TATA-binding protein of TFIID and TFIIB (5,14,20).…”

The Use of Recombinant Baculoviruses for Sustained Expression of Human Cytomegalovirus Immediate Early Proteins in Fibroblasts

“…Coexpression of IE1 72 augments the regulatory properties of IE2 86, a major transactivator of HCMV and heterologous promoters. IE2 86 can interact with itself and the viral 75 kDa early protein (UL84), as well as with the components of the basal transcription complex (TBP and TFIIB), several TBP-associated factors, and specific transcription factors and products of the tumor suppressor genes p53 and Rb (Caswell et al, 1993;Chiou et al, 1993;Choi et al, 1995;Furnari et al, 1993;Hagemeier et al, 1992;Jupp et al, 1993;Lang et al, 1995;Lukac et al, 1994Lukac et al, , 1997Schwartz et al, 1996;Scully et al, 1995;Sommer et al, 1994;Spector and Tevethia, 1994;Speir et al, 1994;Yoo et al, 1996). IE2 86 can also bind to DNA in a site-specific but sequence-tolerant manner (Arlt et al, 1994;Cherrington et al, 1991;Huang and Stinski, 1995;Liu et al, 1991;Pizzorno and Hayward, 1990;Rodems et al, 1998;Schwartz et al, 1994;Scully et al, 1995).…”

The human cytomegalovirus UL37 immediate-early regulatory protein is an integral membrane N-glycoprotein which traffics through the endoplasmic reticulum and Golgi apparatus

“…Of the IE products required for HCMV DNA synthesis, HCMV major IE proteins IE1 and IE2 are both nuclear phosphoproteins which are known to bind DNA and to interact with cellular transcription factors (2,8,16,17,21,28,29,32,37). In contrast, three of the remaining HCMV IE loci, UL37, US3, and UL119-117, predictably encode type I membranebound N-glycoproteins (7,26,30,53).…”