Epstein-Barr Virus EB2 Protein Exports Unspliced RNA via a Crm-1-Independent Pathway

Abstract

Human herpesviruses encode posttranscriptional activators that are believed to up-regulate viral replication by facilitating early and late gene expression. We have reported previously that the Epstein-Barr virus protein EB2 (also called M or SM) promotes nuclear export of RNAs that are poor substrates for spliceosome assembly, an effect that closely resembles the human immunodeficiency virus type 1 Rev-dependent nuclear export of unspliced viral RNA. Here we present experimental data showing that EB2 efficiently promotes the nuclear export of unspliced RNA expressed from a Rev reporter construct. Site-directed mutagenesis as well as domain swapping experiments indicate 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 nuclear 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 heterokaryon assay is, unlike Rev shuttling, not affected by LMB. We also show that overexpression of an N-terminal deletion mutant of Nup214/can, a major nucleoporin of the nuclear pore complex involved in several aspects of nuclear transport, blocks both Rev-and EB2-dependent nuclear export of RNA. These results strongly suggest that EB2 nuclear export of unspliced RNA is mediated by a Crm-1-independent pathway. Epstein-Barr virus (EBV) is a human gamma-herpesviruswidely spread in the adult human population. This virus is associated with several malignancies such as Burkitt's lymphoma, nasopharyngeal carcinoma, Hodgkin's disease, gastric carcinoma, breast carcinoma, and B-and T-cell lymphomas and induces the permanent proliferation (immortalization) of quiescent B lymphocytes in vitro. In EBV-associated tumors in vivo as well as in EBV-infected B cells proliferating ex vivo, entry into a productive cycle is a rare event and the transcription of the EBV genome is usually restricted to a few genes defining a latent state of the viral cycle (for reviews, see references 25 and 36). Although the molecular events occurring during the switch from latency to the productive cycle are now partially understood for in vitro-immortalized B cells (42), the functions of many EBV gene products expressed during the lytic cycle have only partially been characterized, and very little is known about certain others. Among these, the early nuclear protein EB2 (7), which is also called M or SM (8), was originally described as a promiscuous transcription factor, as it activates transient expression of the chloramphenicol acetyltransferase (CAT) gene placed under the control of many different promoters (26). We reported recently that EB2 could activate cytoplasmic accumulation of unspliced RNAs, particularly when they are poor substrates for spliceosome assembly, which suggested an effect of EB2 on either splicing or RNA export or both (4). A recent report, using heterokaryon assays, has revealed that EB2, like its h...

The Evolutionarily Conserved Kaposi's Sarcoma-associated Herpesvirus ORF57 Protein Interacts with REF Protein and Acts as an RNA Export Factor

“…ICP27 contains a CRM1-independent NES required for efficient protein export (18) and EBV MTA contains a novel CRM1-independent NES (69,73). However, ICP27 appears to mediate the export of some HSV-1 RNAs via a CRM1-dependent pathway (78).…”

“…Used extensively to study HIV-1 Rev (57,58), this assay was used to demonstrate that TAP protein increased CAT protein activity thereby inducing the cytoplasmic accumulation of intron-containing RNAs (48,62). This assay was also used to demonstrate that EBV MTA protein promotes RNA export (73). Expression of ORF57 significantly increased CAT protein activity, bypassing nuclear retention of inefficiently spliced cat mRNA and promoting its export to the cytoplasm.…”

Identification of a Novel Nuclear Localization Signal Common to 69- and 82-kDa Human Choline Acetyltransferase

“…To generate this subcellular distribution pattern, 69-kDa ChAT must also contain one or more NESs or be shuttled out of the nucleus by binding to a carrier protein containing an NES. Although LMB is a specific inhibitor of the Crm1-nuclear export pathway, there are a number of other pathways that move proteins out of the nucleus (16,19,(33)(34)(35). Therefore, in addition to utilizing the Crm1 pathway, 69-kDa ChAT could also be exported from the nucleus by other pathway(s) that are not inhibited by LMB.…”

A Novel Nuclear Export Signal and a REF Interaction Domain Both Promote mRNA Export by the Epstein-Barr Virus EB2 Protein

“…This suggests that ICP27-mediated mRNA export and ICP27 nucleocytoplasmic shuttling follow different pathways and are probably mediated by different ICP27 domains. However, the ICP27 CRM-1-independent nuclear export signal (NES) has not as yet been characterized.The EBV EB2 protein also shares properties with mRNA export factors: (i) it exports both intronless and intron-containing RNAs (22-24); (ii) it shuttles between the cytoplasm and the nucleus (22,24,25); and (iii) it is thought to carry two contiguous CRM-1-dependent NES (26). However, the EB2 nucleocytoplasmic shuttling has also been described to be CRM-1-independent (24).…”