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J Virol. 1994 March; 68(3): 1449-1458

An Epstein-Barr virus with a 58-kilobase-pair deletion that includes BARF0 transforms B lymphocytes in vitro.

E S Robertson, B Tomkinson and E Kieff

Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115.

ABSTRACT

A family of Epstein-Barr virus (EBV)-encoded RNAs found in nasopharyngeal carcinoma cells is also present at low levels in some latently infected and growth-transformed B lymphocytes (P. R. Smith, Y. Gao, L. Karran, M. D. Jones, D. Snudden, and B. E. Griffin, J. Virol. 67:3217-3225, 1993). A molecular genetic approach using EBV recombinants was undertaken to evaluate the role of these transcripts in primary B-lymphocyte growth transformation and latent infection. Since the se transcripts arise from a 22-kbp segment of the EBV genome and construction of large deletion mutants is an improbable result after transfection of infected cells with an EBV DNA fragment with a large deletion mutation, a new approach was taken to make a recombinant with the DNA encoding all of the BARF0 RNAs deleted. The approach derives from a recently described strategy for making recombinants from five overlapping EBV cosmid-cloned DNAs (B. Tomkinson, E. Robertson, R. Yalamanchili, R. Longnecker, 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 cosmid which included all of the DNA encoding the BARF0 RNA and by ligating the distal halves of the two flanking cosmids so as to create one cosmid which had ends that overlapped with the other two unaltered cosmids. EBV recombinants with 58 kbp including BARF0 deleted resulted from transfecting the three overlapping EBV DNA fragments into P3HR-1 cells and simultaneously inducing lytic replication of the endogenous, transformation-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 lymphocytes and growing the resultant transformed cells in lymphoblastoid cell lines. The efficiency of incorporation of the deletion into transforming EBV recombinants was close to that of a known indifferent marker, the type 1 EBNA 3A gene, indicating the absence of significant selection against the deletion. Cells infected with the deleted recombinant grew similarly to those infected with wild-type recombinants and had a similar level of permissiveness for lytic EBV infection. Thus, the BARF0 transcript is not critical to primary B-lymphocyte growth transformation or to latent infection. This methodology is useful for constructing EBV recombinants which are specifically mutated at other sites in the three cosmids and is a step toward deriving a minimal transforming EBV genome.

J Virol. 1994 March; 68(3): 1449-1458




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