Coupling of Mitotic Chromosome Tethering and Replication Competence in Epstein-Barr Virus-Based Plasmids

Abstract

The Epstein-Barr virus (EBV) replicates once per cell cycle and segregates with high efficiency yet does not encode the enzymes needed for DNA replication or the proteins required to contact mitotic spindles. The virus-encoded EBNA-1 (EBV nuclear antigen 1) and latent replication origin (oriP) are 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 tethering are linked through the EBNA-1 protein. We show that deleting EBNA-1 or oriP disrupts mitotic chromosome tethering but removing the dyad symmetry element of oriPdoes not. Microscopic and biochemical approaches demonstrated that an EBNA-1 mutant lacking residues 16 to 372 bound to oriPplasmids but did not support their mitotic chromosome association and that the mutant lost the ability of wild-type EBNA-1 to associate with interphase chromatin. Importantly, the transient-replication abilities of various mutant forms of EBV plasmids, including the mutant form with the EBNA-1 internal deletion, 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 replication machinery and enable the plasmids to adhere to host chromosomes to increase their segregation efficiency.

ACKNOWLEDGMENTS

We thank A. Belmont for generously providing the lac operator/lac repressor-GFP system. We also thank F. Hanaoka for pYN3215-bsr, J. L. Kolman for a pEPB construct, T. Koga for pMBL19, N. Somia for pCLMFG-MCS and 293gp/bsr cells, and J. Middeldorp and G. Miller for anti EBNA-1 serum. We thank H. Miyoshi for helpful suggestions about retroviral vectors and Wahl laboratory members for critically reading the manuscript.

This work was supported by a grant from the California Cancer Research Program (99-00573V-10074) and the Pioneer Fund Fellowship (T.K.).