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

Abstract

A striking characteristic of mRNA export factors is that they shuttle continuously between the cytoplasm and the nucleus. This shuttling is mediated by specific factors interacting with peptide motifs called nuclear export signals (NES) and nuclear localization signals. We have identified a novel CRM-1-independent transferable NES and two nuclear localization signals in the Epstein-Barr virus mRNA export factor EB2 (also called BMLF1, Mta, or SM) localized at the N terminus of the protein between amino acids 61 and 146. We have also found that a previously described double NES (amino acids 213-236) does not mediate the nuclear shuttling of EB2, but is an interaction domain with the cellular export factor REF in vitro. This newly characterized REF interaction domain is essential for EB2-mediated mRNA export. Accordingly, in vivo, EB2 is found in complexes containing REF as well as the cellular factor TAP. However, these interactions are RNase-sensitive, suggesting that the RNA is an essential component of these complexes.In cells infected by human herpesviruses, viral mRNAs and proteins are trafficked through the nuclear pore complex. Several cellular factors that mediate the nucleocytoplasmic transport of mRNAs have now been identified (1-6). Interestingly, some human herpesviruses carry, in their genome, genes whose products are also mRNA export factors, such as HSV-1 1 ICP27 (7) and the EBV BMLF1 early gene product originally called EB2 (8), but later called Mta (9) or SM (10). Such genes are conserved among all human herpesviruses, suggesting a conserved function for their products. At least for HSV-1 and EBV, the inactivation of ICP27 (11) or EB2 (12), respectively, abolishes the production of infectious viral particles, demonstrating that ICP27 and EB2 are essential factors for viral mRNA export and that their function cannot be trans-complemented by cellular factors. Moreover, EB2 appears to have an effect on cellular mRNAs because it has transforming properties when expressed both in established cell lines such as Rat1 and NIH3T3 and in primary rat fibroblasts (13).Most of the HSV-1 and EBV early and late mRNAs are transcribed from intronless genes. However, it is now clearly established that the nuclear export of mRNAs is dramatically increased when a splicing event occurs (14). In effect, splicing leads to the deposition on the mRNA of a multiprotein export complex (called EJC for exon-exon junction complex), including REF/Aly (Yra1 in yeast), Y14, RNPS1, SRm160, and Magoh, 20 -24 nucleotides upstream of the exon-exon junction (2-5). Such a complex is thought to export mRNAs by recruiting TAP/Mex67p (15) to cellular messenger RNPs (16 -18). For cellular mRNAs generated from intronless genes, they are likely to be exported to the cytoplasm by cellular factors through nonspecific interactions with mRNA-bound adapters like REF (19) or through sequence-specific interactions with SRp20 or 9G8 (20) or U2AF (21). It is therefore tempting to speculate that EB2, like its HSV-1 functional homolog ICP27 (7), is an...

Spironolactone blocks Epstein–Barr virus production by inhibiting EBV SM protein function

“…SM binds EBV RNA and affects RNA stability (15)(16)(17)(18)(19). Although it preferentially enhances accumulation of some EBV mRNAs, SM action likely depends on inherent characteristics of inefficiently expressed RNAs, such as stability or nuclear exportability (8,9).…”

The Varicella-Zoster virus IE4 protein: A conserved member of the herpesviral mRNA export factors family and a potential alternative target in antiherpetic therapies

“…In infected cells, ICP27 was shown to relocate Aly/REF from sites of cellular splicing to sites of viral transcription [16]. This interaction with Aly/REF was first shown to occur through the ICP27 region that contains the RNAbinding domain [15], and was later demonstrated to be RNasesensitive, confirming that RNA also stabilizes the complex [9]. Direct interaction with TAP/NXF1 itself requires both ICP27 Nterminus and C-terminus [33], and was shown to be essential for both efficient export of viral transcripts and HSV-1 replication [34].…”

“…The Cterminal leucine-rich region of EB2 was shown to be implicated in the interaction with Aly/REF in vitro. Aly/REF co-immunoprecipitates also with EB2 from transfected cells through interaction with the same region, but this interaction is RNase-sensitive suggesting that in vivo these proteins are part of a ribonucleoprotein complex stabilized by RNA [9]. This Aly/REF binding motif was shown to be required for both EB2-mediated mRNA export and production of infectious virions.…”

“…It was also shown to contain an arginine-rich region at the N-terminus which resembles a bipartite NLS [6]. Similarly, EB2 was shown to contain two KH-rich motifs, mapping to residues 127-130 and 143-145, which function as two independent NLS sequences [9]. A basic arginine-rich region, mapping to residues 130-143, was also described in the Nterminal part of IE4 and was proposed to be good candidate for NLS [7].…”

“…Whereas ICP27 and EB2 possess a leucine-rich NES located in their N-terminal part, the requirement of CRM-1 for their shuttling has been much discussed. Since initial studies suggested that a LMB-sensitive leucine-rich region could be involved in the nucleocytoplasmic shuttling of EB2 [5,11], later studies identified the unique sequence responsible for the EB2 shuttling that was shown to be LMB-insensitive [9,12]. Similarly, although the export of ICP27 was first reported to be sensitive to LMB [13,14], the leucinerich N-terminal sequence that is required for an efficient ICP27 export was further shown to function as a CRM-1-independent NES [15,16].…”