Replication licensing of the EBV oriP minichromosome

Abstract

The latent EBV genome may persist in the integrated form as well as the circular episomal form. However, most of the latent viral DNA molecules are known to exist in the circular episomal form, which binds to host chromosomes during mitosis. The DS element of oriP in the circular episomal DNA functions as a replication origin. As it replicates once in a single S phase, it is possible that oriP is regulated by the cellular replication licensing mechanism including the MCM family of replication licensing factors. Transient replication analysis using the oriP plasmid and HeLa/EB1 cells revealed that the DS element requires early G1 phase for the next round of replication, the same cell-cycle window in which the replication licensing of cellular chromatin occurs. After this phase, the sedimentation velocity of the oriP minichromosome increases. MCM2 associates with the oriP minichromosome at late G1 but not at G2/M, and this association requires the DS element in the plasmid. The interaction of EBNA1 and the MCM proteins on the DS element was also suggested. These results suggested that the cellular licensing mechanism controls the replication from oriP. This also suggested a similarity in the replication machinery of the cellular chromatin and the latent EBV genome. In addition to DS-dependent replication, the EBV genome replicates in a manner independent of the DS element in several cultured cell lines. The DS-dependent replication is likely to be suppressed in these cell lines by the expression of other viral proteins. In contrast, EBV-positive Burkitt's lymphoma and circulating EBV-infected B cells express only EBNA1 or both EBNA1 and LMP2. DS-dependent replication may play a major role in these EBNA1-only cells, and the licensing regulation of oriP is important for maintenance of the EBV genome during this latent period of the viral life cycle. EBNA1 is required for efficient nuclear retention and partitioning of oriP-carrying plasmid by its binding to the FR element, thus providing stable persistence of the latent EBV genome during cell division. The copy number of latent EBV DNA molecules in B-cell lines remains fairly constant during multiple passage in culture. However, very little is known about the mechanism by which the viral DNA molecules are equally segregated into daughter cells. To understand the mechanisms responsible for stable nuclear retention and partitioning of the latent viral genome, it is essential to analyze the episomal and integrated viral DNAs at a single-cell level by FISH and other techniques.