Epstein-Barr virus EB2 protein exports unspliced RNA via a Crm-1-independent pathway

Human herpesviruses encode posttranscriptional activators that are believed to up-regulate viral replication by Facilitating early and late gene expre ssion. We have reported previously that the Epstein-Barr virus protein EB2 (also called M or SM) promotes nuclear export of RNAs that are poor substra tes for spliceosome assembly, an effect that closely resembles the human im munodeficiency virus type 1 Rev-dependent nuclear export of unspliced viral RNA. Here we present experimental data showing that EB2 efficiently promot es the nuclear export of unspliced RNA expressed from a Rev reporter constr uct. Site-directed mutagenesis as well as domain swapping experiments indic ate that a leucine-rich region found in the EB2 protein, which matches the consensus sequence for the leucine-rich nuclear export signal, is not a nuc lear export signal per se. Accordingly, leptomycin B (LMB), a specific Crm- 1 inhibitor, impairs Rev- but not EB2-dependent nuclear export of unspliced RNA. Moreover, EB2 nucleocytoplasmic shuttling visualized by a heterokaryo n assay is, unlike Rev shuttling, not affected by LMB. We also show that ov erexpression of an N-terminal deletion mutant of Nup214/can, a major nucleo porin of the nuclear pore complex involved in several aspects of nuclear tr ansport, blocks both Rev- and EB2-dependent nuclear export of RNA. These re sults strongly suggest that EB2 nuclear export of unspliced RNA is mediated by a Crm-1-independent pathway.