Constitutive activation of Epstein-Barr virus (EBV) nuclear antigen 1 gene transcription by IRF1 and IRF2 during restricted EBV latency.
The Epstein-Barr virus (EBV) EBNA1 gene promoter active in the type I program of restricted viral latency was recently identified and shown to reside in the viral BamHI Q fragment. This promoter, Qp, is active in a wide variety of cell lines and has an architecture reminiscent of eukaryotic housekeeping gene promoters (B. C. Schaefer, J. L. Strominger, and S. H. Speck, Proc. Natl. Acad. Sci. USA 92:10565-10569, 1995; B. C. Schaefer, J. L. Strominger, and S. H. Speck, Mol. Cell. Biol. 17:364-377, 1997). Here we demonstrate by deletion analysis that the important cis-acting elements regulating Qp are clustered in a relatively small region (ca. 80 bp) surrounding the site of transcription initiation. Immediately upstream of the site of initiation is a region which is protected from DNase I digestion by crude nuclear extracts. Electrophoretic mobility shift analyses (EMSA) employing probes spanning this region demonstrated the presence of two major protein complexes. Deletion analysis of Qp demonstrated that at least one of these complexes plays an important role in Qp activity. Evidence that interferon response factor 2 (IRF2) is a major constituent of the most prominent EMSA complex and that IRF1 may be a minor component of this complex is presented. Transfections into IRF1-/-, IRF2-/-, and IRF1,2-/- fibroblasts demonstrated that absence of both IRF1 and IRF2 reduced Qp activity to approximately the same extent as mutation of the IRF-binding site in Qp, strongly implicating IRF2, and perhaps IRF1, in the regulation of Qp activity. Notably, transcription from Qp was not inducible by either alpha or gamma interferon in EBV-negative B cells but rather was shown to be constitutively activated by IRF1 and IRF2. This observation suggests that IRF1 and IRF2 have a previously unrecognized role as constitutive activators of specific genes. Additionally, data presented indicate that a protein complex containing the nonhistone architectural protein HMG-I(Y) binds to the region identified as the major transcription initiation site for Qp. This observation raises the possibility that HMG-I(Y)-induced DNA bending plays a role in the initiation of transcription from Qp.
Schaefer
B C
BC
Division of Tumor Virology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115, USA.
Paulson
E
E
Strominger
J L
JL
Speck
S H
SH
D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D002457:Cell Extracts; D002460:Cell Line; D002461:Cell Line, Transformed; D004279:DNA, Viral / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D019309:Epstein-Barr Virus Nuclear Antigens / Q000235:genetics*; D015967:Gene Expression Regulation, Viral / Q000187:drug effects; D025741:HMGA1a Protein; D004854:Herpesvirus 4, Human / Q000235:genetics* / Q000502:physiology; D006609:High Mobility Group Proteins / Q000378:metabolism; D006801:Humans; D050856:Interferon Regulatory Factor-1; D050836:Interferon Regulatory Factor-2; D016898:Interferon-alpha / Q000494:pharmacology; D007371:Interferon-gamma / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D015534:Trans-Activators / Q000378:metabolism*; D014157:Transcription Factors; D014158:Transcription, Genetic / Q000235:genetics; D017735:Virus Latency / Q000235:genetics*
D002457:Cell Extracts; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D019309:Epstein-Barr Virus Nuclear Antigens; D006609:High Mobility Group Proteins; C494257:IRF1 protein, human; C494229:IRF2 protein, human; D050856:Interferon Regulatory Factor-1; D050836:Interferon Regulatory Factor-2; D016898:Interferon-alpha; C494258:Irf1 protein, mouse; C494230:Irf2 protein, mouse; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D025741:HMGA1a Protein; D007371:Interferon-gamma