Second-site homologous recombination in Epstein-Barr virus: insertion of type 1 EBNA 3 genes in place of type 2 has no effect on in vitro infection

This study was undertaken to develop a general strategy for the introduction of mutations into specific sites in the Epstein-Barr virus (EBV) genome. Previous approaches were limited by the need for physical linkage of the transfected EBV DNA fragment to a positive selection marker. In our experiments, a positive selection marker was introduced into one site in the EBV genome and a distant, nonlinked, marker was introduced into another site. Each marker was on a large EBV DNA fragment and was inserted into the genome by transfection into cells carrying a resident EBV genome. The resident EBV genome was simultaneously induced to replicate by using a cotransfected expression plasmid for the EBV immediate-early transactivator, Z (J. Countryman, H. Jenson, R. Seibl, H. Wolf, and G. Miller, J. Virol. 61:3672-3679, 1987; G. Miller, M. Rabson, and L. Heston, J. Virol. 50:174-182, 1984). Eleven percent of the resultant EBV genomes which incorporated the positive selection marker also incorporated the nonlinked marker. Both markers uniformly targeted the homologous EBV genome site. In this way novel EBV recombinants were constructed in which the EBV type 1 EBNA 3A, EBV type 1 EBNA 3A and 3B, or EBV type 1 EBNA 3A, 3B, and 3C genes were introduced into a largely type 2 EBV genome, replacing the corresponding type 2 gene(s). No difference was observed in primary B-lymphocyte growth transformation, in latent EBV gene expression, or in spontaneous lytic EBV gene expression. These new recombinants should be useful for ongoing analyses of the type specificity of the immune response.