Identification of domains within the human cytomegalovirus major immediate-early 86-kilodalton protein and the retinoblastoma protein required for physical and functional interaction with each other.
The human cytomegalovirus major immediate-early 86-kDa protein (IE2 86) plays an important role in the trans activation and regulation of HCMV gene expression. Previously, we demonstrated that IE2 86 contains three regions (amino acids [aa] 86 to 135, 136 to 290, and 291 to 364) that can independently bind to in vitro-translated Rb when IE2 86 is produced as a glutathione S-transferase fusion protein (M. H. Sommer, A. L. Scully, and D. H. Spector, J. Virol. 68:6223-6231, 1994). In this report, we have elucidated the regions of Rb involved in binding to IE2 86 and have further analyzed the functional nature of the interaction between these two proteins. We find that two domains on Rb, the A/B pocket and the carboxy terminus, can each independently form a complex with IE2 86. In functional assays, we demonstrate that IE2 86 and another IE protein, IE1 72, can counter the enlarged flat cell phenotype, but not the G1/S block, which results from expression of wild-type Rb in the human osteosarcoma cell line Saos-2. Mutational analysis reveals that there are two domains on IE2 86 that can independently affect Rb function. One region (aa 241 to 369) includes the major Rb-binding domain, while the second maps to the amino-terminal region (aa 1 to 85) common to both IE2 86 and IE1 72. These data show that Rb and IE2 86 physically and functionally interact with each other via at least two separate domains and provide further support for the hypothesis that IE2 86 may exert its pleiotropic effects through the formation of multimeric protein complexes.
Fortunato
E A
EA
Department of Biology, University of California, San Diego, La Jolla 92093-0357, USA.
Sommer
M H
MH
Yoder
K
K
Spector
D H
DH
D001665:Binding Sites; D002451:Cell Compartmentation; D003587:Cytomegalovirus / Q000235:genetics* / Q000378:metabolism; D019281:Dimerization; D019084:Fluorescent Antibody Technique, Indirect; D015967:Gene Expression Regulation, Viral; D006801:Humans; D017874:Immediate-Early Proteins / Q000737:chemistry* / Q000378:metabolism; D008562:Membrane Glycoproteins; D011485:Protein Binding; D016160:Retinoblastoma Protein / Q000737:chemistry* / Q000378:metabolism; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D015534:Trans-Activators; D014162:Transfection; D014407:Tumor Cells, Cultured; D014759:Viral Envelope Proteins; D014764:Viral Proteins
C069186:IE2 protein, Cytomegalovirus; D017874:Immediate-Early Proteins; D008562:Membrane Glycoproteins; D016160:Retinoblastoma Protein; D015534:Trans-Activators; C077406:UL115 protein, Human herpesvirus 5; D014759:Viral Envelope Proteins; D014764:Viral Proteins; C071440:glycoprotein H, Cytomegalovirus; C499358:glycoprotein H, Human cytomegalovirus; C439068:glycoprotein O, cytomegalovirus