Cloning and regulation of the promoter of pseudorabies virus (TNL strain) glycoprotein E gene.
The nucleotide sequence upstream to the glycoprotein E (gE) gene of pseudorabies virus (PrV, TNL strain) was cloned from the genomic virus DNA by polymerase chain reaction (PCR) and its DNA sequences were determined. The DNA segment, which was supposed to contain the gE promoter, was subcloned into a chloramphenicol acetyltransferase (CAT) reporter gene and the resulting plasmid was named pgEp-B-CAT. To examine the promoter function of this upstream sequence of gE gene, we transfected pgEp-B-CAT DNA into L-M cells and the promoter activity was analyzed by CAT assay. Results showed that our DNA fragment could exhibit promoter activity. Furthermore, we transfected L-M cells with pgEp-B-CAT for 48 h, then superinfected cells with pseudorabies virus, and performed CAT assay. It was found that PrV superinfection could slightly enhance the activity of gE promoter, suggesting that factors produced during viral infection could stimulate the promoter. To explore the possible mechanism of regulation at transcriptional level, the pgEp-B-CAT plasmid were cotransfected with eukaryotic vectors expressing viral regulatory proteins IE or EP0, and results indicated that the gE promoter was activated by IE protein whereas it was inhibited by EP0 protein. Moreover, the effect of exogenous IE or EP0 on the protein level of gE in PrV-infected cells was examined; conclusion similar to that of CAT assay were obtained.
Chang
Yuan-Yen
YY
Department of Veterinary Medicine, College of Veterinary Medicine, National Chung-Hsing University, Taichung, Taiwan.
Wong
Min-Liang
ML
Lin
Hui-Wen
HW
Chang
Tien-Jye
TJ
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular; D015967:Gene Expression Regulation, Viral; D005814:Genes, Viral / Q000502:physiology; D011558:Herpesvirus 1, Suid / Q000235:genetics*; D017874:Immediate-Early Proteins / Q000235:genetics; D007739:L Cells; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic; D014759:Viral Envelope Proteins / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics
D017874:Immediate-Early Proteins; D014759:Viral Envelope Proteins; D014764:Viral Proteins; C089580:glycoprotein E, Suid herpesvirus 1; C087086:glycoprotein E2, classical swine fever virus; C047583:pseudorabies virus immediate early proteins; C070914:early protein O, Pseudorabies virus