Interaction of Epstein-Barr virus nuclear antigen 1 with the viral latent origin of replication.
The Epstein-Barr virus latent origin of replication (oriP) requires only one viral protein, the Epstein-Barr virus nuclear antigen 1 (EBNA-1), for activity. oriP consists of two spatially separated, essential sequence elements, regions I and II, both of which contain multiple EBNA-1-binding sites. Region II contains, or is close to, the site at which DNA synthesis initiates. The role of region I, a transcriptional enhancer in cells that express EBNA-1, in replication is not understood. To determine how the binding of EBNA-1 to sites in region II leads to the initiation of DNA synthesis and to investigate the role of region I, EBNA-1 has been overproduced in insect cells by using a baculovirus vector and purified to homogeneity, and the interaction of EBNA-1 with oriP has been examined. Footprinting experiments demonstrated that EBNA-1 binds to oriP in a sequence-specific manner and bends or untwists the DNA at two symmetry-related sites in region II. Distortion of region I by EBNA-1 was not detected, suggesting that differences in the spacing of binding sites in regions I and II and resulting protein-protein interactions underlie differences in their biological properties. KMnO4 footprinting experiments did not reveal significant single-stranded structures in region II, suggesting that cellular proteins may recognize the EBNA--region II complex and unwind the DNA duplex. Region I did not quantitatively or qualitatively alter the interaction of EBNA-1 with region II. The contribution of an A + T-rich sequence in region II to replication was investigated by a mutational analysis. The results indicated that the overall A + T-rich nature of this sequence is not essential for replication of oriP-bearing plasmids. Nuclease protection experiments performed with these mutagenized plasmids provided additional evidence for protein-protein interactions in region II.
Hearing
J
J
Department of Medicine, State University of New York, Stony Brook 11794.
Mülhaupt
Y
Y
Harper
S
S
D000818:Animals; D000956:Antigens, Viral / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D004261:DNA Replication; D004279:DNA, Viral / Q000235:genetics* / Q000302:isolation & purification; D003850:Deoxyribonuclease I; D004591:Electrophoresis, Polyacrylamide Gel; D019309:Epstein-Barr Virus Nuclear Antigens; D004854:Herpesvirus 4, Human / Q000235:genetics* / Q000276:immunology; D006801:Humans; D007313:Insecta; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D016366:Open Reading Frames; D015183:Restriction Mapping; D014162:Transfection
D000956:Antigens, Viral; D004279:DNA, Viral; D019309:Epstein-Barr Virus Nuclear Antigens; D009838:Oligodeoxyribonucleotides; D003850:Deoxyribonuclease I