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

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The 80-kDa immediate-early regulatory protein IE2 of human cytomegalovirus (HCMV) functions as an essential positive transactivator of downstream viral promoters, but it also specifically down-regulates transcription from the major immediate-early promoter through a 14-bp DNA target motif known as the cis-repression signal (CRS) located at the transcription start site. The IE2 protein purified from bacteria as a fusion product of either staphylococcal Protein A/IE2(290-579) or glutathione-S-transferase (GST)/IE2(346-579) bound specifically to a [32P]-labeled CRS oligonucleotide probe in an in vitro electrophoretic mobility shift assay (EMSA). In contrast, no direct interaction with the CRS probes could be detected with IE2 wild-type protein in extracts from infected or transfected mammalian cells or when synthesized by in vitro translation. However, in vitro phosphorylation of GST/IE2(346-579) by incubation with either the catalytic subunit of protein kinase A (PKA) or a HeLa cell nuclear extract strongly inhibited its DNA-binding activity. This process required ATP hydrolysis and could be reversed by subsequent incubation with bacterial alkaline phosphatase. Importantly, dephosphorylation of the constitutively expressed native IE2 protein present in a nuclear extract from the U373(A45) cell line unmasked a specific CRS DNA-binding activity that could be supershifted with anti-IE2 monoclonal antibody (mAb). A series of high-molecular-weight hetero-oligomeric DNA-bound structures of intermediate mobility were formed in EMSA assays when a mixture of staphylococcal Protein A/IE2 and GST/IE2 was coincubated with the CRS probe. Coincubation with a DNA-binding negative but dimerization-competent GST/IE2 deletion mutant competitively inhibited DNA-binding by staphylococcal Protein A/IE2, whereas coincubation with a GST/IE2 deletion mutant that lacked the ability to both dimerize and bind to DNA failed to influence the mobility of the DNA-bound staphylococcal Protein A/IE2 protein. Therefore, IE2 appears to bind to DNA as a higher-order oligomer in which the presence of subunits with mutant DNA-binding domains interferes with the overall DNA-binding function. A series of point mutations introduced into each of nine conserved motifs throughout the DNA-binding and dimerization domain, all of which abolish the ability of the transfected intact IE2 protein to autoregulate the MIE promoter, also all lacked the ability to bind to CRS sequences as GST/IE2(346-379) fusion proteins. Detailed analysis of point mutations in the 14-bp CRS target DNA binding motif revealed that IE2 binds in a relatively sequence-independent manner to 10-bp-long A/T-rich DNA elements bounded on each side by CG dinucleotides. Moreover, the A/T-rich minor groove binding agent distamycin, but not the G/C-rich minor groove binding agent chromomycin-A3, actively competed with IE2 for binding to the CRS motif in a dose-dependent fashion. In conclusion, IE2 binds preferentially as multimerized dimers to A/T-rich sequences in the minor groove that are flanked...