Characterization of an R-binding site mediating the R-induced activation of the Epstein-Barr virus BMLF1 promoter.
In cells latently infected with Epstein-Barr virus, the switch from latency to productive infection is linked to the expression of two Epstein-Barr virus transcription factors called EB1 and R. R is an enhancer factor, and an R-responsive element (RRE) has been identified in the BMLF1 promoter. In this study, we have used bidirectional deletion mutagenesis to delineate the BMLF1 RRE (RRE-M) to a 44-bp sequence. We also show that R expressed from a recombinant vaccinia virus protects RRE-M against digestion by DNase I. Using mobility shift assays and dimethyl sulfate interferences, we have characterized the contact points between in vitro-translated R and the DNA. R binds in vitro to one site by simultaneously contacting two sequences within the site, which are separated by 8 bp: 5'-catGTCCCtctatcatGGCGCagac-3'. Site-directed mutagenesis of this sequence completely impaired the binding of R in vitro and rendered the BMLF1 promoter nonresponsive to R. The results suggest that the R-inducible BMLF1 enhancer is composed of a single R-binding site, called RRE-M.
Gruffat
H
H
Laboratoire de Virologie Moléculaire, Ecole Normale Supérieure de Lyon, UMR 49 CNRS-ENS, France.
Duran
N
N
Buisson
M
M
Wild
F
F
Buckland
R
R
Sergeant
A
A
D001665:Binding Sites; D004279:DNA, Viral; D003850:Deoxyribonuclease I / Q000378:metabolism; D015967:Gene Expression Regulation, Viral; D006367:HeLa Cells; D004854:Herpesvirus 4, Human / Q000235:genetics*; D006801:Humans; D017874:Immediate-Early Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D015534:Trans-Activators / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014616:Vaccinia virus / Q000235:genetics / Q000502:physiology; D014764:Viral Proteins; D014779:Virus Replication / Q000235:genetics
C068382:BRLF1 protein, Human herpesvirus 4; D004279:DNA, Viral; D017874:Immediate-Early Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D014764:Viral Proteins; D003850:Deoxyribonuclease I