Identification and mapping of dimerization and DNA-binding domains in the C terminus of the IE2 regulatory protein of human cytomegalovirus

Abstract

The 80-kDa IE2 nuclear phosphoprotein encoded by the human cytomegalovirus (HCMV) major immediateearly (MIE) gene behaves both as a nonspecific transactivator of heterologous reporter genes and as a specific repressor of its own promoter-enhancer region. To begin to examine the biochemical properties of the IE2 protein, we prepared panels of N-terminal and C-terminal truncation mutants by in vitro translation procedures. In cross-linking experiments, the C-terminal half of IE2 (which is sufficient for down-regulation) formed dimers but N-terminal segments did not do so. Cotranslated Oct2/IE2 fusion proteins containing the same IE2 C-terminal region from codons 266 to 579 also formed mixed-subunit DNA-bound oligomeric complexes in gel mobility shift assays. Furthermore, an IE2 domain bounded by codons 388 to 542 proved to immunoprecipitate as heterodimers with cotranslated subunits containing known epitopes for specific antibodies. Deletion up to codon 428 or truncation back to codon 504 prevented this interaction. In direct gel shift DNA-binding assays, a bacterial GST/IE2(346-579) fusion protein bound to a 30-mer oligonucleotide probe encompassing the major immediate-early gene negative cis-regulatory target DNA sequence but failed to bind to a single-base-pair insertion mutant probe (ACRS). This specific DNA-binding activity was abolished by further deletion up to codon 388 on the N-terminal side or by truncation at codon 542 on the C-terminal side. Therefore, the minimal DNA-binding domain requires additional amino acid motifs on both sides of the dimerization domain. This segment of IE2 is functionally important for both transactivation and down-regulation and contains several highly conserved amino acid motifs that are shared amongst the equivalent HCMV, simian CMV, mouse CMV, rat CMV, and human herpesvirus 6 proteins from other betaherpesviruses. 145 to 151 (NLS-1) and 321 to 328 (NLS-2) and several consensus casein kinase II substrate motifs such as 203-6201

PIAS1 enhances SUMO‐1 modification and the transactivation activity of the major immediate–early IE2 protein of human cytomegalovirus

“…2B,C). Although we could not test whether the full length IE2 also binds to PIAS1 in this assay due to the low expression level of the GST‐IE2(full length) fusion protein in E. coli , our results suggest that IE2 may bind to PIAS1 through the C‐terminal domain of IE2, which is well conserved among equivalent proteins of betaherpesviruses [32].…”

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

“…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).…”