Human cytomegalovirus immediate-early gene 2 protein interacts with itself and with several novel cellular proteins.
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.
Furnari
B A
BA
Department of Microbiology and Immunology, University of North Carolina, Chapel Hill 27599.
Poma
E
E
Kowalik
T F
TF
Huong
S M
SM
Huang
E S
ES
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D003587:Cytomegalovirus / Q000235:genetics / Q000378:metabolism*; D005814:Genes, Viral; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D017874:Immediate-Early Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D008168:Lung; D008562:Membrane Glycoproteins; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides; D010761:Phosphorus Radioisotopes; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion; D013462:Sulfur Radioisotopes; D016385:TATA Box; D015534:Trans-Activators; D015533:Transcriptional Activation; D014759:Viral Envelope Proteins; D014763:Viral Matrix Proteins / Q000378:metabolism*; D014764:Viral Proteins
C088645:IE1 protein, cytomegalovirus; C069186:IE2 protein, Cytomegalovirus; D017874:Immediate-Early Proteins; D008562:Membrane Glycoproteins; D009838:Oligodeoxyribonucleotides; D010761:Phosphorus Radioisotopes; D011993:Recombinant Fusion Proteins; D013462:Sulfur Radioisotopes; D015534:Trans-Activators; C077406:UL115 protein, Human herpesvirus 5; D014759:Viral Envelope Proteins; D014763:Viral Matrix Proteins; D014764:Viral Proteins; C071440:glycoprotein H, Cytomegalovirus; C499358:glycoprotein H, Human cytomegalovirus; C439068:glycoprotein O, cytomegalovirus; D008715:Methionine; D005982:Glutathione Transferase