Replication and maintenance of the 170-kb circular chromosome of Epstein-Barr virus (EBV) during latent infection are generally believed to depend upon a single viral gene product, the nuclear protein EBNA-1. EBNA-1 binds to two clusters of sites at oriP, an 1,800-bp sequence on the EBV genome which can support replication and maintenance of artificial plasmids introduced into cell lines that contain EBNA-1. To investigate the importance of EBNA-1 to latent infection by EBV, we introduced a frameshift mutation into the EBNA-1 gene of EBV by recombination along with a flanking selectable marker. EBV genomes carrying the frameshift mutation could be isolated readily after superinfecting EBV-positive cell lines, but not if recombinant virus was used to infect EBV-negative B-cell lines or to immortalize peripheral blood B cells. EBV mutants lacking almost all of internal repeat 3, which encode a repetitive glycine and alanine domain of EBNA-1, were generated in the same way and found to immortalize B cells normally. An EBNA-1-deficient mutant of EBV was isolated and found to be incapable of establishing a latent infection of the cell line BL30 at a detectable frequency, indicating that the mutant was less than 1% as efficient as an isogenic, EBNA-1-positive strain in this assay. The data indicate that EBNA-1 is required for efficient and stable latent infection by EBV under the conditions tested. Evidence from other studies now indicates that autonomous maintenance of the EBV chromosome during latent infection does not depend on the replication initiation function of oriP. It is therefore likely that the viral chromosome maintenance (segregation) function of oriP and EBNA-1 is what is required.

The Epstein-Barr virus (EBV) genome contains an open reading frame, BHRF1, that encodes a presumptive membrane protein with sequence similarity to the proto-oncogene bcI2, which is linked to human B-cell follicular lymphoma. Potential roles for BHRF1 in EBV's ability to growth transform human B cells and to replicate in B cells in culture were investigated by generating EBV mutants that lack most of the open reading frame. This was accomplished by recombination of plasmids carrying mutations in BHRF1 with the transformation-defective EBV strain P3HR1. Because BHRF1 resides close to the deletion in P3HR1 that renders this strain transformation defective, B-cell transformation could be used to select for recombination events in the region. B-cell clones were established by recombinants which lacked most of the BHRF1 open reading frame, although most of these initial B-cell transformants also carried nonrecombinant (BHRF1+) P3HR1 genomes, at levels ranging from a fraction of a copy to four copies per cell. Secondary B-cell transformants that lacked BHRF1+ EBV at detectable levels were found to release transforming, BHRF1deficient EBV at levels that were within the normal range for EBV-immortalized B-cell clones. These studies demonstrate that BHRF1 is nonessential for growth transformation of B cells and for virus replication and release from these cells in culture.

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