AN EPSTEIN-BARR-VIRUS WITH A 58-KILOBASE-PAIR DELETION THAT INCLUDES BARF0 TRANSFORMS B-LYMPHOCYTES IN-VITRO

A family of Epstein-Barr virus (EBV)-encoded RNAs found in nasopharyng eal carcinoma cells is also present at low levels in some latently inf ected and growth-transformed B lymphocytes (P. R. Smith, Y. Gao, L. Ka rran, M. D. Jones, D. Snudden, and B. E. Griffin, J. Virol. 67:3217-32 25, 1993). A molecular genetic approach using EBV recombinants was und ertaken to evaluate the role of these transcripts in primary B-lymphoc yte growth transformation and latent infection. Since the se transcrip ts arise from a 22-kbp segment of the EBV genome and construction of l arge deletion mutants is an improbable result after transfection of in fected cells with an EBV DNA fragment with a large deletion mutation, a new approach was taken to make a recombinant with the DNA encoding a ll of the BARF0 RNAs deleted. The approach derives from a recently des cribed strategy for making recombinants from five overlapping EBV cosm id-cloned DNAs (B. Tomkinson, E. Robertson, R. Yalamanchili, R. Longne cker, and E. Kieff, J. Virol. 67:7298-7306, 1993). A large segment of EBV DNA was deleted from the transfected cosmid DNAs by omitting a cos mid which included all of the DNA encoding the BARF0 RNA and by ligati ng the distal halves of the two flanking cosmids so as to create one c osmid which had ends that overlapped,vith the other two unaltered cosm ids. EBV recombinants with 58 kbp including BARF0 deleted resulted fro m transfecting the three overlapping EBV DNA fragments into P3HR-1 cel ls and simultaneously inducing lytic replication of the endogenous, tr ansformation-defective, P3HR-1 EBV. The endogenous P3HR-1 EBV provided lytic infection and packaging functions. EBV recombinants with intact transforming functions were then selected by infecting primary B lymp hocytes and growing the resultant transformed cells in lymphoblastoid cell lines. The efficiency of incorporation of the deletion into trans forming EBV recombinants was close to that of a known indifferent mark er, the type 1 EBNA 3A gene, indicating the absence of significant sel ection against the deletion. Cells infected with the deleted recombina nt grew similarly to those infected with wild-type recombinants and ha d a similar level of permissiveness for lytic EBV infection. Thus, the BARF0 transcript is not critical to primary B-lymphocyte growth trans formation or to latent infection. This methodology is useful for const ructing EBV recombinants which are specifically mutated at other sites in the three cosmids and is a step toward deriving a minimal transfor ming EBV genome.