EPSTEIN-BARR-VIRUS RECOMBINANTS FROM OVERLAPPING COSMID FRAGMENTS

Five overlapping type 1 Epstein-Barr virus (EBV) DNA fragments constit uting a complete replication- and transformation-competent genome were cloned into cosmids and transfected together into P3HR-1 cells, along with a plasmid encoding the Z immediate-early activator of EBV replic ation. P3HR-1 cells harbor a type 2 EBV which is unable to transform p rimary B lymphocytes because of a deletion of DNA encoding EBNA LP and EBNA 2, but the P3HR-1 EBV can provide replication functions in trans and can recombine with the transfected cosmids. EBV recombinants whic h have the type 1 EBNA LP and 2 genes from the transfected EcoRI-A cos mid DNA were selectively and clonally recovered by exploiting the uniq ue ability of the recombinants to transform primary B lymphocytes into lymphoblastoid cell lines. PCR and immunoblot analyses for seven dist inguishing markers of the type 1 transfected DNAs identified cell line s infected with EBV recombinants which had incorporated EBV DNA fragme nts beyond the transformation marker-rescuing EcoRI-A fragment. Approx imately 10% of the transforming virus recombinants had markers mapping at 7, 46 to 52, 93 to 100, 108 to 110, 122, and 152 kbp from the 172- kbp transfected genome. These recombinants probably result from recomb ination among the transfected cosmid-cloned EBV DNA fragments. The one recombinant virus examined in detail by Southern blot analysis has al l the polymorphisms characteristic of the transfected type 1 cosmid DN A and none characteristic of the type 2 P3HR-1 EBV DNA. This recombina nt was wild type in primary B-lymphocyte infection, growth transformat ion, and lytic replication. Overall, the type 1 EBNA 3A gene was incor porated into 26% of the transformation marker-rescued recombinants, a frequency which was considerably higher than that observed in previous experiments with two-cosmid EBV DNA cotransfections into P3HR-1 cells (B. Tomkinson and E. Kieff, J. Virol. 66:780-789,1992). Of the recomb inants which had incorporated the marker-rescuing cosmid DNA fragment and the fragment encoding the type 1 EBNA 3A gene, most had incorporat ed markers from at least two other transfected cosmid DNA fragments, i ndicating a propensity for multiple homologous recombinations. The fre quency of incorporation of the nonselected transfected type 1 EBNA 3C gene, which is near the end of two of the transfected cosmids, was 26% overall, versus 3% in previous experiments using transfections with t wo EBV DNA cosmids. In contrast, the frequency of incorporation of a 1 2-kb EBV DNA deletion which was near the end of two of the transfected cosmids was only 13%. Other than through incorporation into recombina nts arising from among the five cosmids, this marker was rarely incorp orated into recombinants which had any marker from the P3HR-1 genome. Thus, the five-cosmid transfection strategy is particularly useful for incorporation of a nonselected marker mapping near the end of a trans fected cosmid or near the site of a large deletion.