The Interaction of the Cellular Export Adaptor Protein Aly/REF with ICP27 Contributes to the Efficiency of Herpes Simplex Virus 1 mRNA Export

Herpes simplex virus 1 (HSV-1) protein ICP27 enables viral mRNA export by accessing the cellular mRNA export receptor TAP/NXF, which guides mRNA through the nuclear pore complex. ICP27 binds viral mRNAs and interacts with TAP/NXF, providing a link to the cellular mRNA export pathway. ICP27 also interacts with the mRNA export adaptor protein Aly/REF, which binds cellular mRNAs and also interacts with TAP/NXF. Studies using small interfering RNA (siRNA) knockdown indicated that Aly/REF is not required for cellular mRNA export, and similar knockdown studies during HSV-1 infection led us to conclude that Aly/REF may be dispensable for viral RNA export. Recently, the structural basis of the interaction of ICP27 with Aly/REF was elucidated at atomic resolution, and it was shown that three ICP27 residues, W105, R107, and L108, interface with the RNA recognition motif (RRM) domain of Aly/REF. Here, to determine the role the interaction of ICP27 and Aly/REF plays during infection, these residues were mutated to alanine, and a recombinant virus, WRL-A, was constructed. Virus production was reduced about 10-fold during WRL-A infection, and export of ICP27 protein and most viral mRNAs was less efficient. We conclude that interaction of ICP27 with Aly/REF contributes to efficient viral mRNA export.     

Herpes simplex virus ICP27 protein provides viral mRNAs with access to the cellular mRNA export pathway

The role of herpes simplex virus ICP27 protein in mRNA export is investigated by microinjection into Xenopus laevis oocytes. ICP27 dramatically stimulates the export of intronless viral mRNAs, but has no effect on the export of cellular mRNAs, U snRNAs or tRNA. Use of inhibitors shows, in contrast to previous suggestions, that ICP27 neither shuttles nor exports viral mRNA via the CRM1 pathway. Instead, ICP27-mediated viral RNA export requires REF and TAP/NXF1, factors involved in cellular mRNA export. ICP27 binds directly to REF and complexes containing ICP27, REF and TAP are found in vitro and in virally infected cells. A mutant ICP27 that does not interact with REF is inactive in viral mRNA export. We propose that ICP27 associates with viral mRNAs and recruits TAP/NXF1 via its interaction with REF proteins, allowing the otherwise inefficiently exported viral mRNAs to access the TAP-mediated export pathway. This represents a novel mechanism for export of viral mRNAs.     

REF, an evolutionary conserved family of hnRNP-like proteins, interacts with TAP/Mex67p and participates in mRNA nuclear export

Vertebrate TAP and its yeast ortholog Mex67p are involved in the export of messenger RNAs from the nucleus. TAP has also been implicated in the export of simian type D viral RNAs bearing the constitutive transport element (CTE). Although TAP directly interacts with CTE-bearing RNAs, the mode of interaction of TAP/Mex67p with cellular mRNAs is different from that with the CTE RNA and is likely to be mediated by protein-protein interactions. Here we show that Mex67p directly interacts with Yra1p, an essential yeast hnRNP-like protein. This interaction is evolutionarily conserved as Yra1p also interacts with TAP. Conditional expression in yeast cells implicates Yra1 p in the export of cellular mRNAs. Database searches revealed that Yra1p belongs to an evolutionarily conserved family of hnRNP-like proteins having more than one member in Mus musculus, Xenopus laevis, Caenorhabditis elegans, and Schizosaccharomyces pombe and at least one member in several species including plants. The murine members of the family directly interact with TAP. Because members of this protein family are characterized by the presence of one RNP-motif RNA-binding domain and exhibit RNA-binding activity, we called these proteins REF-bps for RNA and export factor binding proteins. Thus, Yra1p and members of the REF family of hnRNP-like proteins may facilitate the interaction of TAP/Mex67p with cellular mRNAs.     

SR splicing factors serve as adapter proteins for TAP-dependent mRNA export

The only mammalian RNA binding adapter proteins known to partner with TAP/NXF1, the primary receptor for general mRNA export, are members of the REF family. We demonstrate that at least three shuttling SR (serine/arginine-rich) proteins interact with the same domain of TAP/NXF1 that binds REFs. Included are 9G8 and SRp20, previously shown to promote the export of intronless RNAs. A peptide derived from the N terminus of 9G8 inhibits the binding of both REF and SR proteins to TAP/NXF1 in vitro, and this finding argues for competitive interactions. In Xenopus oocytes, the N terminus of 9G8 exhibits a dominant-negative effect on mRNA export from the nucleus, while addition of excess TAP/NXF1 overcomes this inhibition. Thus, multiple adapters including SR proteins most likely cooperate to recruit multiple copies of TAP/NXF1 for efficient mRNA export.     

Cyclin-dependent kinase 1/cyclin B1 phosphorylates varicella-zoster virus IE62 and is incorporated into virions

Varicella-zoster virus (VZV), an alphaherpesvirus restricted to humans, infects differentiated cells in vivo, including T lymphocytes, keratinocytes, and neurons, and spreads rapidly in confluent cultured dermal fibroblasts (HFFs). In VZV-infected HFFs, atypical expression of cyclins D3 and B1 occurs along with the induction of cyclin-dependent kinase (CDK) activity. A specific CDK1 inhibitor blocked VZV spread, indicating an important function for this cellular kinase in VZV replication. CDK activity assays of infected cells revealed a large viral phosphoprotein that was identified as being the major immediate-early transactivator, IE62. Since IE62 colocalized with CDK1/cyclin B1 by confocal microscopy, we investigated whether this cellular kinase complex interacts with IE62. Using recombinant fragments of IE62 spanning the entire amino acid sequence, we found that purified CDK1/cyclin B1 phosphorylated IE62 at residues T10, S245, and T680 in vitro. Immunoprecipitation of cyclin B1 from VZV-infected HFFs indicated that IE62 was included in the complex within infected cells. The full-length IE62 protein, obtained by immunoprecipitation from infected cells, was also phosphorylated by purified CDK1/cyclin B1. Based on IE62/CDK1/cyclin B1 colocalization near viral assembly regions, we hypothesized that these cellular proteins could be incorporated into VZV virions with IE62. Purified virions were analyzed by immunoblotting for the presence of CDK1 and cyclin B1, and active CDK1 and cyclin B1 were present in the VZV tegument with IE62 and were sensitive to detergent treatment. Thus, IE62 is a substrate for CDK1/cyclin B1, and virions could deliver the active cellular kinase to nondividing cells that normally do not express it.     

The C-terminal domain of TAP interacts with the nuclear pore complex and promotes export of specific CTE-bearing RNA substrates

Messenger RNAs are exported from the nucleus as large ribonucleoprotein complexes (mRNPs). To date, proteins implicated in this process include TAP/Mex67p and RAE1/Gle2p and are distinct from the nuclear transport receptors of the beta-related, Ran-binding protein family. Mex67p is essential for mRNA export in yeast. Its vertebrate homolog TAP has been implicated in the export of cellular mRNAs and of simian type D viral RNAs bearing the constitutive transport element (CTE). Here we show that TAP is predominantly localized in the nucleoplasm and at both the nucleoplasmic and cytoplasmic faces of the nuclear pore complex (NPC). TAP interacts with multiple components of the NPC including the nucleoporins CAN, Nup98, Nup153, p62, and with three major NPC subcomplexes. The nucleoporin-binding domain of TAP comprises residues 508-619. In HeLa cells, this domain is necessary and sufficient to target GFP-TAP fusions to the nuclear rim. Moreover, the isolated domain strongly competes multiple export pathways in vivo, probably by blocking binding sites on the NPC that are shared with other transport receptors. Microinjection experiments implicate this domain in the export of specific CTE-containing RNAs. Finally, we show that TAP interacts with transportin and with two proteins implicated in the export of cellular mRNAs: RAE1/hGle2 and E1B-AP5. The interaction of TAP with nucleoporins, its direct binding to the CTE RNA, and its association with two mRNP binding proteins suggest that TAP is an RNA export mediator that may bridge the interaction between specific RNP export substrates and the NPC.     

The Glc7p nuclear phosphatase promotes mRNA export by facilitating association of Mex67p with mRNA

mRNA export is mediated by Mex67p:Mtr2p/NXF1:p15, a conserved heterodimeric export receptor that is thought to bind mRNAs through the RNA binding adaptor protein Yra1p/REF. Recently, mammalian SR (serine/arginine-rich) proteins were shown to act as alternative adaptors for NXF1-dependent mRNA export. Npl3p is an SR-like protein required for mRNA export in S. cerevisiae. Like mammalian SR proteins, Npl3p is serine-phosphorylated by a cytoplasmic kinase. Here we report that this phosphorylation of Npl3p is required for efficient mRNA export. We further show that the mRNA-associated fraction of Npl3p is unphosphorylated, implying a subsequent nuclear dephosphorylation event. We present evidence that the essential, nuclear phosphatase Glc7p promotes dephosphorylation of Npl3p in vivo and that nuclear dephosphorylation of Npl3p is required for mRNA export. Specifically, recruitment of Mex67p to mRNA is Glc7p dependent. We propose a model whereby a cycle of cytoplasmic phosphorylation and nuclear dephosphorylation of shuttling SR adaptor proteins regulates Mex67p:Mtr2p/NXF1:p15-dependent mRNA export.     

An element in the 3' untranslated region of human LINE-1 retrotransposon mRNA binds NXF1(TAP) and can function as a nuclear export element

Export of unspliced mRNA to the cytoplasm is required for the replication of all retroviruses. In simian type D retroviruses, the RNA export is mediated by the constitutive transport element (CTE) that binds the cellular nuclear export factor 1, NXF1(TAP). To search for potential cellular RNA substrates for NXF1, we have set up an in vitro selection procedure, using an RNA library expressed from total human genomic DNA. A sequence that was isolated most frequently as independent clones exhibits extensive homology to the 3' untranslated region of expressed LINE1 (L1) retrotransposons. This region, termed L1-NXF1 binding element (L1-NBE) bears no structural resemblance to the viral CTE, but binds NXF1 as strongly as CTE, based on gel mobility shift competition assays. A deletion analysis of the NXF1 protein reveals that CTE and L1-NBE have different, but overlapping, binding domains on NXF1. Placed in an intron, L1-NBE is capable of mediating nuclear export of lariat RNA species in Xenopus laevis oocytes and of an unspliced HIV-1 derived RNA in human 293 cells, suggesting that it may function as a nuclear export element for the intronless L1 mRNA.     

Overexpression of TAP/p15 heterodimers bypasses nuclear retention and stimulates nuclear mRNA export

Human TAP and its yeast orthologue Mex67p are members of the multigene family of NXF proteins. A conserved feature of NXFs is a leucine-rich repeat domain (LRR) followed by a region related to the nuclear transport factor 2 (the NTF2-like domain). The NTF2-like domain of metazoan NXFs heterodimerizes with a protein known as p15 or NXT. A C-terminal region related to ubiquitin-associated domains (the UBA-like domain) is present in most, but not all NXF proteins. Saccharomyces cerevisiae Mex67p and Caenorhabditis elegans NXF1 are essential for the export of messenger RNA from the nucleus. Human TAP mediates the export of simian type D retroviral RNAs bearing the constitutive transport element, but the precise role of TAP and p15 in mRNA nuclear export has not yet been established. Here we show that overexpression of TAP/p15 heterodimers bypasses nuclear retention and stimulates the export of mRNAs that are otherwise exported inefficiently. This stimulation of mRNA export is strongly reduced by removing the UBA-like domain of TAP and abolished by deleting the LRR domain or the NTF2-like domain. Similar results are obtained when TAP/p15 heterodimers are directly tethered to the RNA export cargo. Our data indicate that formation of TAP/p15 heterodimers is required for TAP-mediated export of mRNA and show that the LRR domain of TAP plays an essential role in this process.     

TAP (NXF1) belongs to a multigene family of putative RNA export factors with a conserved modular architecture

Vertebrate TAP (also called NXF1) and its yeast orthologue, Mex67p, have been implicated in the export of mRNAs from the nucleus. The TAP protein includes a noncanonical RNP-type RNA binding domain, four leucine-rich repeats, an NTF2-like domain that allows heterodimerization with p15 (also called NXT1), and a ubiquitin-associated domain that mediates the interaction with nucleoporins. Here we show that TAP belongs to an evolutionarily conserved family of proteins that has more than one member in higher eukaryotes. Not only the overall domain organization but also residues important for p15 and nucleoporin interaction are conserved in most family members. We characterize two of four human TAP homologues and show that one of them, NXF2, binds RNA, localizes to the nuclear envelope, and exhibits RNA export activity. NXF3, which does not bind RNA or localize to the nuclear rim, has no RNA export activity. Database searches revealed that although only one p15 (nxt) gene is present in the Drosophila melanogaster and Caenorhabditis elegans genomes, there is at least one additional p15 homologue (p15-2 [also called NXT2]) encoded by the human genome. Both human p15 homologues bind TAP, NXF2, and NXF3. Together, our results indicate that the TAP-p15 mRNA export pathway has diversified in higher eukaryotes compared to yeast, perhaps reflecting a greater substrate complexity.     

Kaposi's sarcoma-associated herpesvirus ORF57 interacts with cellular RNA export cofactors RBM15 and OTT3 to promote expression of viral ORF59

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes ORF57, which promotes the accumulation of specific KSHV mRNA targets, including ORF59 mRNA. We report that the cellular export NXF1 cofactors RBM15 and OTT3 participate in ORF57-enhanced expression of KSHV ORF59. We also found that ectopic expression of RBM15 or OTT3 augments ORF59 production in the absence of ORF57. While RBM15 promotes the accumulation of ORF59 RNA predominantly in the nucleus compared to the levels in the cytoplasm, we found that ORF57 shifted the nucleocytoplasmic balance by increasing ORF59 RNA accumulation in the cytoplasm more than in the nucleus. By promoting the accumulation of cytoplasmic ORF59 RNA, ORF57 offsets the nuclear RNA accumulation mediated by RBM15 by preventing nuclear ORF59 RNA from hyperpolyadenylation. ORF57 interacts directly with the RBM15 C-terminal portion containing the SPOC domain to reduce RBM15 binding to ORF59 RNA. Although ORF57 homologs Epstein-Barr virus (EBV) EB2, herpes simplex virus (HSV) ICP27, varicella-zoster virus (VZV) IE4/ORF4, and cytomegalovirus (CMV) UL69 also interact with RBM15 and OTT3, EBV EB2, which also promotes ORF59 expression, does not function like KSHV ORF57 to efficiently prevent RBM15-mediated nuclear accumulation of ORF59 RNA and RBM15's association with polyadenylated RNAs. Collectively, our data provide novel insight elucidating a molecular mechanism by which ORF57 promotes the expression of viral intronless genes.     

An interaction between KSHV ORF57 and UIF provides mRNA-adaptor redundancy in herpesvirus intronless mRNA export

The hTREX complex mediates cellular bulk mRNA nuclear export by recruiting the nuclear export factor, TAP, via a direct interaction with the export adaptor, Aly. Intriguingly however, depletion of Aly only leads to a modest reduction in cellular mRNA nuclear export, suggesting the existence of additional mRNA nuclear export adaptor proteins. In order to efficiently export Kaposi's sarcoma-associated herpesvirus (KSHV) intronless mRNAs from the nucleus, the KSHV ORF57 protein recruits hTREX onto viral intronless mRNAs allowing access to the TAP-mediated export pathway. Similarly however, depletion of Aly only leads to a modest reduction in the nuclear export of KSHV intronless mRNAs. Herein, we identify a novel interaction between ORF57 and the cellular protein, UIF. We provide the first evidence that the ORF57-UIF interaction enables the recruitment of hTREX and TAP to KSHV intronless mRNAs in Aly-depleted cells. Strikingly, depletion of both Aly and UIF inhibits the formation of an ORF57-mediated nuclear export competent ribonucleoprotein particle and consequently prevents ORF57-mediated mRNA nuclear export and KSHV protein production. Importantly, these findings highlight that redundancy exists in the eukaryotic system for certain hTREX components involved in the mRNA nuclear export of intronless KSHV mRNAs.