The splicing machinery which positions a protein export complex near the exon-exon junction mediates nuclear export of mRNAs generated from intron-containing genes. Many Epstein-Barr virus (EBV) early and late genes are intronless, and an alternative pathway, independent of splicing, must export the corresponding mRNAs. Since the EBV EB2 protein induces the cytoplasmic accumulation of intronless mRNA, it is tempting to speculate that EB2 is a viral adapter involved in the export of intronless viral mRNA. If this is true, then the EB2 protein is essential for the production of EBV infectious virions. To test this hypothesis, we generated an EBV mutant in which the BMLF1 gene, encoding the EB2 protein, has been deleted (EBV BMLF1-KO ). Our studies show that EB2 is necessary for the production of infectious EBV and that its function cannot be transcomplemented by a cellular factor. In the EBV BMLF1-KO 293 cells, oriLyt-dependent DNA replication was greatly enhanced by EB2. Accordingly, EB2 induced the cytoplasmic accumulation of a subset of EBV early mRNAs coding for essential proteins implicated in EBV DNA replication during the productive cycle. Two herpesvirus homologs of the EB2 protein, the herpes simplex virus type 1 protein ICP27 and, the human cytomegalovirus protein UL69, only partly rescued the phenotype of the EBV BMLF1-KO mutant, indicating that some EB2 functions in virus production cannot be transcomplemented by ICP27 and UL69.

The Epstein‐Barr virus (EBV) genome codes for several transcriptional trans‐activators. One of them, the BZLF1 open reading frame (ORF)‐encoded product EB1, is able to induce the productive cycle in infected B cells. From the cloning and characterization of full‐length cDNAs, we found that EB1 could be made from three overlapping messenger RNAs expressed under the control of two different promoters that we call P1 and P2. The first mRNA, 1 kb long, is made from the P1 promoter and codes for EB1 alone. The two other mRNAs, respectively 3 and 4 kb long and made by facultative splicing, are bicistronic mRNAs. They code not only for the trans‐activator EB1 but also for a second EBV transcriptional trans‐activator R, encoded by the BRLF1 ORF. In effect, authentic EB1 and R proteins are expressed from the 3 and 4 kb long cDNAs as demonstrated by identification of the proteins with specific antisera. In addition, EB1 and R expressed from the 3 and 4 kb cDNAs activate transcription from their specific targets in the EBV early promoter DR.