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The minimal replicator of Epstein-Barr virus oriP.
J Virol. 2000 May; 74(10):4512-22.JV

Abstract

oriP is a 1.7-kb region of the Epstein-Barr virus (EBV) chromosome that supports the replication and stable maintenance of plasmids in human cells. oriP contains two essential components, called the DS and the FR, both of which contain multiple binding sites for the EBV-encoded protein, EBNA-1. The DS appears to function as the replicator of oriP, while the FR acts in conjunction with EBNA-1 to prevent the loss of plasmids from proliferating cells. Because of EBNA-1's role in stabilizing plasmids through the FR, it has not been entirely clear to what extent EBNA-1 might be required for replication from oriP per se, and a recent study has questioned whether EBNA-1 has any direct role in replication. In the present study we found that plasmids carrying oriP required EBNA-1 to replicate efficiently even when assayed only 2 days after plasmids were introduced into the cell lines 143B and 293. Significantly, using 293 cells it was demonstrated that the plasmid-retention function of EBNA-1 and the FR did not contribute significantly to the accumulation of replicated plasmids, and the DS supported efficient EBNA-1-dependent replication in the absence of the FR. The DS contains two pairs of closely spaced EBNA-1 binding sites, and a previous study had shown that both sites within either pair are required for activity. However, it was unclear from previous work what additional sequences within the DS might be required. We found that each "half" of the DS, including a pair of closely spaced EBNA-1 binding sites, had significant replicator activity when the other half had been deleted. The only significant DNA sequences that the two halves of the DS share in common, other than EBNA-1 binding sites, is a 9-bp sequence that is present twice in the "left half" and once in the "right half." These nonamer repeats, while not essential for activity, contributed significantly to the activity of each half of the DS. Two thymines occur at unique positions within EBNA-1 binding sites 1 and 4 at the DS and become sensitive to oxidation by permanganate when EBNA-1 binds, but mutation of each to the consensus base, adenine, actually improved the activity of each half of the DS slightly. In conclusion, the DS of oriP is an EBNA-1-dependent replicator, and its minimal active core appears to be simply two properly spaced EBNA-1 binding sites.

Authors+Show Affiliations

Department of Genetics, Roswell Park Cancer Institute, Buffalo, New York 14263, USA. Yates@sc3101.med.buffalo.eduNo affiliation info availableNo affiliation info available

Pub Type(s)

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

Language

eng

PubMed ID

10775587

Citation

Yates, J L., et al. "The Minimal Replicator of Epstein-Barr Virus OriP." Journal of Virology, vol. 74, no. 10, 2000, pp. 4512-22.
Yates JL, Camiolo SM, Bashaw JM. The minimal replicator of Epstein-Barr virus oriP. J Virol. 2000;74(10):4512-22.
Yates, J. L., Camiolo, S. M., & Bashaw, J. M. (2000). The minimal replicator of Epstein-Barr virus oriP. Journal of Virology, 74(10), 4512-22.
Yates JL, Camiolo SM, Bashaw JM. The Minimal Replicator of Epstein-Barr Virus OriP. J Virol. 2000;74(10):4512-22. PubMed PMID: 10775587.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - The minimal replicator of Epstein-Barr virus oriP. AU - Yates,J L, AU - Camiolo,S M, AU - Bashaw,J M, PY - 2000/4/25/pubmed PY - 2000/5/16/medline PY - 2000/4/25/entrez SP - 4512 EP - 22 JF - Journal of virology JO - J Virol VL - 74 IS - 10 N2 - oriP is a 1.7-kb region of the Epstein-Barr virus (EBV) chromosome that supports the replication and stable maintenance of plasmids in human cells. oriP contains two essential components, called the DS and the FR, both of which contain multiple binding sites for the EBV-encoded protein, EBNA-1. The DS appears to function as the replicator of oriP, while the FR acts in conjunction with EBNA-1 to prevent the loss of plasmids from proliferating cells. Because of EBNA-1's role in stabilizing plasmids through the FR, it has not been entirely clear to what extent EBNA-1 might be required for replication from oriP per se, and a recent study has questioned whether EBNA-1 has any direct role in replication. In the present study we found that plasmids carrying oriP required EBNA-1 to replicate efficiently even when assayed only 2 days after plasmids were introduced into the cell lines 143B and 293. Significantly, using 293 cells it was demonstrated that the plasmid-retention function of EBNA-1 and the FR did not contribute significantly to the accumulation of replicated plasmids, and the DS supported efficient EBNA-1-dependent replication in the absence of the FR. The DS contains two pairs of closely spaced EBNA-1 binding sites, and a previous study had shown that both sites within either pair are required for activity. However, it was unclear from previous work what additional sequences within the DS might be required. We found that each "half" of the DS, including a pair of closely spaced EBNA-1 binding sites, had significant replicator activity when the other half had been deleted. The only significant DNA sequences that the two halves of the DS share in common, other than EBNA-1 binding sites, is a 9-bp sequence that is present twice in the "left half" and once in the "right half." These nonamer repeats, while not essential for activity, contributed significantly to the activity of each half of the DS. Two thymines occur at unique positions within EBNA-1 binding sites 1 and 4 at the DS and become sensitive to oxidation by permanganate when EBNA-1 binds, but mutation of each to the consensus base, adenine, actually improved the activity of each half of the DS slightly. In conclusion, the DS of oriP is an EBNA-1-dependent replicator, and its minimal active core appears to be simply two properly spaced EBNA-1 binding sites. SN - 0022-538X UR - https://www.unboundmedicine.com/medline/citation/10775587/The_minimal_replicator_of_Epstein_Barr_virus_oriP_ DB - PRIME DP - Unbound Medicine ER -