Interaction of bovine herpesvirus 4 (BHV-4) immediate early 2 gene product with BHV-4 thymidine kinase promoter-regulatory region.
To understand the mechanism of regulation of bovine herpesvirus 4 (BHV-4) early gene expression by immediate-early (IE) gene products, we studied activation of expression of the BHV-4 early gene encoding thymidine kinase (TK). BHV-4 encodes two IE RNAs. IE RNA 2 encodes a protein with predicted amino acid sequence similarity to the Epstein-Barr virus (EBV) R transactivator. The BHV-4 TK promoter-regulatory region was specifically transactivated approximately 9-fold by the IE2 gene product in transient expression cotransfection assays. Deletion analysis localized the IE2 responsive element(s) within a 72 bp fragment 5' to the start of transcription. Multiple copies of this 72 bp fragment conferred IE2-responsiveness on an enhancerless simian virus 40 (SV40) promoter and mediated IE2 transactivation in either orientation. Gel retardation assays demonstrated a sequence-specific complex between the IE2 gene product and the 72 bp DNA fragment, and indicated that the IE2 gene product binds DNA as a dimer. Sequences contained in a 25 bp subfragment were sufficient for IE2-DNA complex formation. No nucleotide sequence similarity was noted between the TK IE2-binding fragment and the consensus binding site for the EBV R transactivator. A single amino acid change in the putative DNA-binding domain of the IE2 gene product abolished both complex formation in vitro and transactivation activity in vivo.
Zhang
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Department of Pathobiology, Auburn University, Alabama 36849-5519, USA.
van Santen
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VL
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002460:Cell Line; D004279:DNA, Viral / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004742:Enhancer Elements, Genetic / Q000235:genetics; D018142:Gammaherpesvirinae / Q000201:enzymology / Q000235:genetics*; D015967:Gene Expression Regulation, Viral / Q000235:genetics*; D017874:Immediate-Early Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics; D013539:Simian virus 40 / Q000235:genetics; D013937:Thymidine Kinase / Q000096:biosynthesis / Q000235:genetics; D015534:Trans-Activators / Q000378:metabolism*; D014158:Transcription, Genetic / Q000235:genetics; D015533:Transcriptional Activation / Q000235:genetics
D004279:DNA, Viral; D004268:DNA-Binding Proteins; C079185:IE2 protein, bovine herpesvirus type 4; D017874:Immediate-Early Proteins; D012367:RNA, Viral; D015534:Trans-Activators; D013937:Thymidine Kinase