Coupling of mitotic chromosome tethering and replication competence in Epstein-Barr virus-based plasmids

The Epstein-Barr virus (EBV) replicates once per cell cycle and segregates with high efficiency yet does not encode the enzymes needed for DNA replica tion or the proteins required to contact mitotic spindles. The virus-encode d EBNA-1 (EBV nuclear antigen 1) and latent replication origin (oriP) nt e required for both replication and segregation. We developed a sensitive and specific fluorescent labeling strategy to analyze the interactions of both EBNA-1 with viral episomes and viral episomes with host chromosomes. This enabled investigation of the hypothesis that replication and chromosome tet hering are linked through the EBNA-1 protein. We show that deleting EBNA-1 or oriP disrupts mitotic chromosome tethering but removing the dyad symmetr y element of oriP does not. Microscopic and biochemical approaches demonstr ated that an EBNA-1 mutant lacking residues 16 to 372 bound to oriP plasmid s but did not support their mitotic chromosome association and that the mut ant lost the ability of wild-type EBNA-1 to associate with interphase chrom atin, Importantly, the transient-replication abilities of various mutant fo rms of EBV plasmids, including the mutant form with the EBNA-1 internal del etion, correlated directly with their chromosome-tethering abilities. These data lead us to propose that EBNA-1 recruits oriP-containing plasmids into chromatin subdomains in interphase nuclei to both engage the host replicat ion machinery anti enable the plasmids to adhere to host chromosomes to inc rease their segregation efficiency.