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Interaction of Epstein-Barr virus nuclear antigen 1 with the viral latent origin of replication.
J Virol. 1992 Feb; 66(2):694-705.JV

Abstract

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.

Authors+Show Affiliations

Department of Medicine, State University of New York, Stony Brook 11794.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

1309908

Citation

Hearing, J, et al. "Interaction of Epstein-Barr Virus Nuclear Antigen 1 With the Viral Latent Origin of Replication." Journal of Virology, vol. 66, no. 2, 1992, pp. 694-705.
Hearing J, Mülhaupt Y, Harper S. Interaction of Epstein-Barr virus nuclear antigen 1 with the viral latent origin of replication. J Virol. 1992;66(2):694-705.
Hearing, J., Mülhaupt, Y., & Harper, S. (1992). Interaction of Epstein-Barr virus nuclear antigen 1 with the viral latent origin of replication. Journal of Virology, 66(2), 694-705.
Hearing J, Mülhaupt Y, Harper S. Interaction of Epstein-Barr Virus Nuclear Antigen 1 With the Viral Latent Origin of Replication. J Virol. 1992;66(2):694-705. PubMed PMID: 1309908.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Interaction of Epstein-Barr virus nuclear antigen 1 with the viral latent origin of replication. AU - Hearing,J, AU - Mülhaupt,Y, AU - Harper,S, PY - 1992/2/1/pubmed PY - 1992/2/1/medline PY - 1992/2/1/entrez SP - 694 EP - 705 JF - Journal of virology JO - J Virol VL - 66 IS - 2 N2 - 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. SN - 0022-538X UR - https://www.unboundmedicine.com/medline/citation/1309908/Interaction_of_Epstein_Barr_virus_nuclear_antigen_1_with_the_viral_latent_origin_of_replication_ L2 - http://jvi.asm.org/cgi/pmidlookup?view=long&pmid=1309908 DB - PRIME DP - Unbound Medicine ER -