Genetic evidence that EBNA-1 is needed for efficient, stable latent infection by Epstein-Barr virus

The life cycle of Epstein-Barr virus (EBV) appears to depend upon a mechanism to replicate and maintain the viral genome in an autonomous state in expanding B-cell populations during the initial phase of latency (20–22). Maintenance of the viral chromosome in proliferating cells required that it be duplicated each cell cycle and passed on to daughter nuclei during mitosis. During latent infection, the circularized EBV genome replicates during the S phase of the cell cycle (14), apparently under a cellular control mechanism that limits each viral chromosome to one round of replication per S phase (1, 47). EBV chromosomes associate with the condensed cellular chromosomes during mitosis, ensuring that all copies of the EBV chromosome are enveloped within daughter nuclei as they form at the close of mitosis (15). An 1,800-bp region of the EBV genome, oriP, was identified based on its ability to confer long-term maintenance of recombinant plasmids under selection in human cell lines (44). oriP requires only a single EBV-encoded protein, EBNA-1, for this activity (31, 48). oriP provides both replication and segregation functions to plasmids that carry it through two distinct and essential functional elements (4, 35), both of which bind EBNA-1 at multiple sites (34). A cluster of four EBNA-1 binding sites, usually called the DS for a dyad symmetry element contained within it, supports plasmid replication (42) and is the point from which replication forks diverge in both directions, at least within a few hundred base pairs, the resolution achieved by a two-dimensional gel analysis of replication intermediates (9). The other essential element of oriP is a repetitive array of EBNA-1 binding sites, a family of 30-bp repeats called the FR, which is located nearly 1 kb away from the DS. The FR supports very little plasmid replication by itself, but in the presence of EBNA-1, it allows plasmids to be retained with their genes active for prolonged periods of time after being introduced into cells (23, 35). It was shown that oriP could confer mitotic stability to a 600-kb, circularized, cloned segment of a human chromosome in cells containing EBNA-1 and cause the artificial plasmid to associate with condensed human chromosomes during mitosis (36). EBNA-1 itself associates with human metaphase chromosomes (12) and also has the capacity to link together DNA molecules to which it is bound, by self-interaction (8, 32, 38). The mechanism behind the plasmid retention function of the FR and EBNA-1 is therefore likely to involve EBNA-1-mediated tethering of plasmids to human chromosomes during mitosis to prevent their loss to the cytoplasm. It has long been assumed that oriP and EBNA-1 are together responsible for supporting the replication and maintenance of the circular EBV chromosome during latent infection of mitotically active cells, but a direct test of this has been lacking. However, in recent years, evidence has accumulated to suggest that the replication initiation function of oriP is dispensable to EBV. Two-dimensional gel analysis of replication intermediates has indicated that on the EBV chromosome, oriP is replicated passively from distant origins most of the time, with initiation occurring within oriP only a fraction of the time (29). An isolate of the cell line X50-7 was found to carry a variant EBV genome which had sustained a deletion that removed all of oriP except for the FR (43); when tested on plasmids, the FR does not support significant replication in the absence of the DS of oriP (4, 31, 35, 42, 44). Recent genetic studies in this laboratory have confirmed that the DS of oriP can be deleted from EBV without seriously compromising its ability to maintain its chromosome autonomously during latent infection (our unpublished data). Could the viral chromosomal maintenance function, or segregation function, of EBNA-1 and oriP be redundant, too? EBNA-1 and oriP have been conserved during the evolution of the close relatives of EBV that infect Old World primates (6, 30, 46). However, the FR of oriP is also a potent EBNA-1-dependent enhancer of transcription, and EBNA-1 and the FR appear to be important for proper transcription from two EBV promoters that give rise to expression of genes required to immortalize B cells (10, 33, 39). Among the more distantly related gamma herpesviruses that are known to support autonomous replication of their viral genomes during latent infection, none appears to have a homolog of EBNA-1 or of oriP (2, 5, 24, 40). It therefore seemed possible that the plasmid maintenance property of EBNA-1 and oriP could be functionally redundant, like the replication initiation function. To test the importance of EBNA-1 for latent infection by EBV, we introduced a frameshift mutation into the EBNA-1 gene of EBV by homologous recombination. Viruses carrying this mutation could not immortalize B cells nor stably infect an EBV-negative B-cell line. This suggests that the chromosome maintenance function of EBNA-1 and oriP is required for efficient and stable latent infection.