Open AccessInitiation of DNA Replication within oriP Is Dispensable for Stable Replication of the Latent Epstein-Barr Virus Chromosome after Infection of Established Cell Lines
Author(s) -
Paolo Norio,
Carl L. Schildkraut,
John L. Yates
Publication year - 2000
Publication title -
journal of virology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.617
H-Index - 292
eISSN - 1070-6321
pISSN - 0022-538X
DOI - 10.1128/jvi.74.18.8563-8574.2000
Subject(s) - biology , dna replication , pre replication complex , origin recognition complex , origin of replication , licensing factor , control of chromosome duplication , viral replication , plasmid , genetics , replication factor c , ter protein , virus , dna replication factor cdt1 , chromosome , virology , gene , eukaryotic dna replication , microbiology and biotechnology
The 165-kb circularized chromosome of Epstein-Barr virus (EBV) is replicated in latently infected cells once per cell cycle by host proteins during S phase. Replication initiates at multiple sites on latent EBV chromosomes, including within a 1.8-kb region calledoriP , which can provide both replication and stabilization for recombinant plasmids in the presence of the EBV-encoded protein, EBNA-1. Replication initiates at or near the dyad symmetry component (DS) oforiP , which depends on multiple EBNA-1 binding sites for activity. To test the importance of the replication function oforiP , the DS was deleted from the viral genome. EBV mutants lacking the DS and carrying a selectable gene could establish latent infections in BL30 cells, in which circular, mutant viral chromosomes were stably maintained. Analysis of replication fork movement using two-dimensional gel electrophoresis showed that the deletion of the DS reduced the initiation events to an undetectable level within theoriP region so that this segment was replicated exclusively by forks entering the region from either direction. A significant slowing or stalling of replication forks that occurs normally at the approximate position of the DS was also eliminated by deletion of the DS. The results confirm the DS as both a replication origin and a place where replication forks pause. Since the replication function oforiP is dispensable at least in certain cell lines, the essential role of EBNA-1 for infection of these cell lines is likely to be that of stabilizing the EBV chromosome by associating with the 30-bp repeats oforiP . The results also imply that in established cell lines, the EBV chromosome can be efficiently replicated entirely from origins that are activated by cellular factors. Presumably, initiation of replication at the DS, mediated by EBNA-1, is important for the natural life cycle of EBV, perhaps in establishing latent infections of normal B cells.