title: The functionally active IE2 immediate-early regulatory protein of human cytomegalovirus is an 80-kilodalton polypeptide that contains two distinct activator domains and a duplicated nuclear localization signal.
abstract: The IE2 region of the human cytomegalovirus (CMV) strain Towne major immediate-early (MIE) gene encodes a transcriptional transactivator that stimulates expression from a variety of heterologous target promoters but specifically down-regulates its own promoter. By immunofluorescence and Western immunoblot analysis with monospecific peptide antisera, we found that human CMV MIE exon 5 encodes four overlapping polypeptides, two present at immediate-early times (80 and 55 kDa) and two others detected only at late times after infection (55 and 40 kDa). However, only the 80-kDa version (579 amino acids), which is derived from the small upstream exons 2 and 3 fused to the intact exon 5 region, was functionally active in both transactivation and autoregulation as assessed by cotransfection experiments. These results confirm the corrected assignment of the coding capacity of the exon 5 region based on amino acid homology with the equivalent IE2 protein from simian CMV (Colburn). In transient DNA transfection assays, IE2 expression plasmids also produced a predominant full-length 80-kDa protein, which was localized in a distinctive reticular pattern in the nucleus. Two short basic nuclear localization signals in IE2 were identified by deletion analysis and by conversion of a test cytoplasmic herpes simplex virus protein into a form that localized in the nucleus after insertion of either of these two human CMV motifs. Functional assays with MIE region plasmids containing deletions or truncations in exon 5 revealed that both transactivation and autoregulation required several distinct domains within the COOH half of the IE2 protein, whereas a region between codons 99 and 194 could be discarded. Three segments at the NH2 end of the protein between codons 1 to 85, 86 to 98, and 195 to 290 were also essential for transactivation but played no role in autoregulation. Finally, in domain swap experiments, GAL4-fusion proteins containing either an NH2-terminal 51-amino-acid domain from exon 3 (codons 25 to 85) or the COOH-terminal 33-amino-acid domain from exon 5 (codons 544 to 579) identified two distinct activator domains from IE2, both of which have acidic characteristics.
authors.affiliation: Department of Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine, Baltimore, Maryland 21205.
authors.forename: M C
authors.initials: MC
authors.lastname: Pizzorno
authors.forename: M A
authors.initials: MA
authors.lastname: Mullen
authors.forename: Y N
authors.initials: YN
authors.lastname: Chang
authors.forename: G S
authors.initials: GS
authors.lastname: Hayward
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology; D000956:Antigens, Viral / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D015153:Blotting, Western; D002451:Cell Compartmentation; D003587:Cytomegalovirus / Q000235:genetics*; D004252:DNA Mutational Analysis; D005455:Fluorescent Antibody Technique; D015967:Gene Expression Regulation, Viral; D005814:Genes, Viral; D017874:Immediate-Early Proteins; D066298:In Vitro Techniques; D008562:Membrane Glycoproteins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D013329:Structure-Activity Relationship; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014709:Vero Cells; D014759:Viral Envelope Proteins; D014764:Viral Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D015678:Viral Structural Proteins / Q000235:genetics
chemical_list: D000914:Antibodies, Viral; D000956:Antigens, Viral; C069186:IE2 protein, Cytomegalovirus; D017874:Immediate-Early Proteins; D008562:Membrane Glycoproteins; D009687:Nuclear Proteins; D015534:Trans-Activators; C077406:UL115 protein, Human herpesvirus 5; D014759:Viral Envelope Proteins; D014764:Viral Proteins; D015678:Viral Structural Proteins; C071440:glycoprotein H, Cytomegalovirus; C499358:glycoprotein H, Human cytomegalovirus; C439068:glycoprotein O, cytomegalovirus