CDNA cloning and transient expression of the Epstein-Barr virus-determined nuclear antigen EBNA3B in human cells and identification of novel transcripts from its coding region

Abstract

Recombinant plasmids containing sequences from the BamHI-E rightward reading frames 2a and 2b (BERF2a and 2b) of the Epstein-Barr virus (EBV) genome were isolated from a library of cDNA clones which had been previously made from the EBV B95-8 lymphoblastoid cell line (M. Bodescot, O. Brison, and M. Perricaudet, Nucleic Acids Res. 14:7103-7114, 1986). The characterization of these clones in combination with RNase mapping experiments led to the identification of one leftward and several rightward transcripts traversing the EBV-determined nuclear antigen EBNA3B coding region. One cDNA (T7) contains a continuous open reading frame generated by the splicing together of BERF2a and BERF2b. The T7 clone was used to reconstruct a complete fused BERF2a/2b open reading frame in an adenovirus-based expression vector. Western immunoblotting and immunofluorescence experiments using human 293 cells showed that the recombinant plasmid is capable of expressing a protein with a size, immunological characteristics, and a subcellular localization indistinguishable from those of native B95-8 EBNA3B.