CONSTITUTIVE BINDING OF EBNA1 PROTEIN TO THE EPSTEIN-BARR-VIRUS REPLICATION ORIGIN, ORIP, WITH DISTORTION OF DNA-STRUCTURE DURING LATENT INFECTION

Replication of the circular, 170 kb genome of Epstein-Barr virus (EBV) during latent infection is performed by the cellular replication mach inery under cell-cycle control. A single viral protein, EBNA1, directs the cellular replication apparatus to initiate replication within the genetically defined replication origin, oriP, at a cluster of four EB NA1 binding sites, referred to here as the physical origin of bidirect ional replication, or OBR. A second cluster of EBNA1 binding sites wit hin oriP, the 30 bp repeats, serves an essential role as a replication enhancer and also provides a distinct episome maintenance function th at is unrelated to replication. We examined the functional elements of oriP for binding by EBNA1 and possibly other proteins in proliferatin g Raji cells by generating in vivo footprints using two reagents, dime thylsulfate (DMS) and KMnO4. We also employed deoxyribonuclease I (DNa se I) with permeabilized cells. The in vivo and permeabilized cell foo tprints at the EBNA1 binding sites, particularly those obtained using DMS, gave strong evidence that all of these sites are bound by EBNA1 i n asynchronously dividing cells. No consistent evidence was found to s uggest binding by other proteins at any other sites within the functio nal regions of oriP. Thymines at symmetrical positions of the OBR with in oriP were oxidized when cells were treated with permanganate, sugge stive of bends or other distortions of DNA structure at these position s; binding of EBNA1 in vitro to total DNA from Raji cells induced reac tivity to permanganate at identical positions. The simplest interpreta tion of the results, which were obtained using asynchronously dividing cells, is that EBNA1 binds to its sites at oriP and holds the OBR in a distorted conformation throughout most of the cell cycle, implying t hat replication is initiated by a cellular mechanism and is not limite d by an availability of EBNA1 for binding to oriP.