A set of lambda phages containing overlapping fragments of Epstein-Barr virus (EBV) defective DNA has been cloned from P3HR-1-superinfected Raji cells. Mapping data obtained using these cloned DNA fragments confirmed the structure of P3HR-1 defective DNA previously deduced directly from virion DNA (M.-S. Cho, G. W. Bornkamm, and H. zur Hausen, 1984, J. Virol., in press). The ability of the cloned defective DNA fragments to induce EBV antigens in transfected baby hamster kidney (BHK) cells was tested using indirect immunofluorescence assays. Up to 5% of those cells receiving a defective DNA fragment BamHI-W′C′ transiently expressed a de novo nuclear antigen which was identified as being a component of the EAD complex by its reactivity with characterized EBV-positive human sera. A 20-kb clone of P3HR-1 defective DNA (EcoRI-C1) was found to induce the synthesis of a component of the VCA complex. One percent of cells transfected with this clone showed cytoplasmic fluorescence when tested with either VCA+ human sera or EBV anti-VCA monoclonal antibody. Subcloning of the EcoRI-C1 fragment localized the VCA gene to a 4.1-kb segment which maps within the BamHI-A fragment of the standard genome. This segment contains a single large open reading frame of 2.6 kb (B. Barrell, A. Bankier, R. Baer, P. Biggin, P. Deininger, P. Farrell, T. Gibson, G. Hatfull, G. Hudson, S. Stachweli, and C. Sequin, 1984, Nature (London), in press). None of the defective DNA clones were capable of inducing EBV-specific nuclear antigens (EBNAs) which is consistent with the absence of the known EBNA coding regions from the defective genome.
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