EBNA1 can link the enhancer element to the initiator element of the Epstein-Barr virus plasmid origin of DNA replication

Abstract

The plasmid origin of DNA replication of Epstein-Barr virus, oriP, is replicated once per cell division, employing cellular replication machinery and only one viral protein. To understand how replication from this origin is initiated and regulated, we purified this viral protein, EBNA1. EBNA1 was expressed in CV-lp cells by using an infectious simian virus 40 vector containing the EBNA1 gene. It was purified in two chromatographic steps to apparent homogeneity. The purified protein is capable of supporting transcription of the luciferase gene from a reporter plasmid carrying the FR enhancer element to which EBNA1 binds. EBNAl does not have oriP-dependent ATPase activity, indicating that it does not carry out an energy-dependent step in the initiation of DNA replication. However, EBNAl does mediate an association between the two elements of oriP. We measured this association by binding one of the elements, the enhancer element, to a solid matrix and measuring retention by this element of the other one, the initiator element, in the presence of EBNAl. This retention is specific for DNA fragments containing EBNAl-binding sites. EBNA1 thus can link the two elements of the origin, providing a locally high concentration of EBNA1 at the site of initiation of DNA replication. We propose that this association is important either (i) to affect DNA structure to allow a cellular helicase to initiate DNA strand separation or (ii) to bind replication proteins to bring them to the origin of replication.

The plasmid replicon of EBV consists of multiplecis-acting elements that facilitate DNA synthesis by the cell and a viral maintenance element

“…© Oxford University Press oriP plasmids also differ from other viral replicons in that the origin-binding protein of EBV, EBNA-1, does not have an ATPase or helicase activity (Frappier and O'Donnell, 1991;Middleton and Sugden, 1992). One mechanism by which EBNA-1 could contribute to oriP replication, in parallel with those viral replicons requiring viral helicases such as SV40 (Stahl et al, 1986), BPV-1 and HSV-1 (Bruckner et al, 1991), would be for EBNA-1 to associate with a cellular helicase and tether it to DS, the site near or at which DNA synthesis from oriP initiates (Gahn and Schildkraut, 1989).…”

Dominant-negative inhibitors of EBNA-1 of Epstein-Barr virus

“…EBNA-1 is a site-specific DNA-binding protein which binds to clusters of sites in EBV that together constitute the viral plasmid origin of replication, oriP. Purified EBNA-1 lacks detectable enzymatic functions such as a DNA-dependent ATPase or a helicase (13,29) which are intrinsic to other viral origin-binding, replication proteins such as E1 of bovine papillomavirus type 1 or T antigen of simian virus 40. It is therefore not clear what EBNA-1 does to support replication of oriP-containing DNAs.…”

“…EBNA-1 associates multiple DNAs in vitro, to which it binds specifically, apparently through protein-protein binding (12,16,27,29,43). When the sites bound are intramolecular, this function is termed DNA looping; when they are intermolecular or unspecified, this function is termed DNA linking.…”

Functional Analyses of the EBNA1 Origin DNA Binding Protein of Epstein-Barr Virus

“…First, EBNA1 activates DNA replication from oriP (19,51). Since EBNA1 lacks enzymatic activities, origin activation is thought to involve the recruitment of host replication factors to oriP and/or the destabilization of the origin DNA (13,32). Second, EBNA1 governs the segregation of EBV episomes and FRcontaining plasmids by mediating the attachment of the FR to host cell metaphase chromosomes (11,18,27,35).…”