scholarly journals Epstein-Barr Virus Protein EB2 Contains an N-Terminal Transferable Nuclear Export Signal That Promotes Nucleocytoplasmic Export by Directly Binding TAP/NXF1

2009 ◽  
Vol 83 (24) ◽  
pp. 12759-12768 ◽  
Author(s):  
Franceline Juillard ◽  
Edwige Hiriart ◽  
Nicolas Sergeant ◽  
Valérie Vingtdeux-Didier ◽  
Hervé Drobecq ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Epstein Barr Virus ◽  
Rna Binding ◽  
Nuclear Export Signal ◽  
Virus Protein ◽  
Barr Virus ◽  
Early Protein ◽  
Intronless Genes ◽  
Epstein Barr ◽  
Export Signal

ABSTRACT The Epstein-Barr virus early protein EB2 (also called BMLF1, Mta, or SM), which allows the nuclear export of a subset of early and late viral mRNAs derived from intronless genes, is essential for the production of infectious virions. An important feature of mRNA export factors is their capacity to shuttle continuously between the nucleus and the cytoplasm. In a previous study, we identified a novel CRM1-independent transferable nuclear export signal (NES) at the N terminus of EB2, between amino acids 61 and 146. Here we show that this NES contains several small arginine-rich domains that cooperate to allow efficient interaction with TAP/NXF1. Recruitment of TAP/NXF1 correlates with this NES-mediated efficient nuclear export when it is fused to a heterologous protein. Moreover, the NES can export mRNAs bearing MS2 RNA-binding sites from the nucleus when tethered to the RNA via the MS2 phage coat protein RNA-binding domain.

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

2000 ◽  
Vol 74 (13) ◽  
pp. 6068-6076 ◽  
Author(s):  
Géraldine Farjot ◽  
Monique Buisson ◽  
Madeleine Duc Dodon ◽  
Louis Gazzolo ◽  
Alain Sergeant ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Epstein Barr Virus ◽  
Consensus Sequence ◽  
Nuclear Export Signal ◽  
Site Directed Mutagenesis ◽  
Virus Protein ◽  
Nuclear Pore ◽  
Barr Virus ◽  
Epstein Barr ◽  
Export Signal

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.

scholarly journals Protein Kinase CK2 Phosphorylation of EB2 Regulates Its Function in the Production of Epstein-Barr Virus Infectious Viral Particles

2007 ◽  
Vol 81 (21) ◽  
pp. 11850-11860 ◽  
Author(s):  
Cahora Medina-Palazon ◽  
Henri Gruffat ◽  
Fabrice Mure ◽  
Odile Filhol ◽  
Valérie Vingtdeux-Didier ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Nuclear Export ◽  
Epstein Barr Virus ◽  
Nuclear Export Signal ◽  
Regulatory Subunit ◽  
Viral Mrnas ◽  
Barr Virus ◽  
Early Protein ◽  
Epstein Barr

ABSTRACT The Epstein-Barr Virus (EBV) early protein EB2 (also called BMLF1, Mta, or SM) promotes the nuclear export of a subset of early and late viral mRNAs and is essential for the production of infectious virions. We show here that in vitro, protein kinase CK2α and -β subunits bind both individually and, more efficiently, as a complex to the EB2 N terminus and that the CK2β regulatory subunit also interacts with the EB2 C terminus. Immunoprecipitated EB2 has CK2 activity that phosphorylates several sites within the 80 N-terminal amino acids of EB2, including Ser-55, -56, and -57, which are localized next to the nuclear export signal. EB2S3E, the phosphorylation-mimicking mutant of EB2 at these three serines, but not the phosphorylation ablation mutant EB2S3A, efficiently rescued the production of infectious EBV particles by HEK293BMLF1-KO cells harboring an EB2-defective EBV genome. The defect of EB2S3A in transcomplementing 293BMLF1-KO cells was not due to impaired nucleocytoplasmic shuttling of the mutated protein but was associated with a decrease in the cytoplasmic accumulation of several late viral mRNAs. Thus, EB2-mediated production of infectious EBV virions is regulated by CK2 phosphorylation at one or more of the serine residues Ser-55, -56, and -57.

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

2002 ◽  
Vol 278 (1) ◽  
pp. 335-342 ◽  
Author(s):  
Edwige Hiriart ◽  
Géraldine Farjot ◽  
Henri Gruffat ◽  
Minh Vu Chuong Nguyen ◽  
Alain Sergeant ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Epstein Barr Virus ◽  
Mrna Export ◽  
Nuclear Export Signal ◽  
Interaction Domain ◽  
Barr Virus ◽  
Epstein Barr ◽  
Export Signal

scholarly journals The C-Terminal Region but Not the Arg-X-Pro Repeat of Epstein-Barr Virus Protein EB2 Is Required for Its Effect on RNA Splicing and Transport

1999 ◽  
Vol 73 (5) ◽  
pp. 4090-4100 ◽  
Author(s):  
Monique Buisson ◽  
Fabienne Hans ◽  
Inca Kusters ◽  
Nathalie Duran ◽  
Alain Sergeant
Keyword(s):  
Nuclear Export ◽  
Epstein Barr Virus ◽  
Rna Binding ◽  
Direct Proof ◽  
Virus Protein ◽  
Functional Domain ◽  
Splice Sites ◽  
Barr Virus ◽  
Cytoplasmic Accumulation ◽  
Epstein Barr

ABSTRACT The Epstein-Barr virus BMLF1 gene product EB2 has been shown to efficiently transform immortalized Rat1 and NIH 3T3 cells, to bind RNA, and to shuttle from the nucleus to the cytoplasm. In transient-expression assays EB2 seems to affect mRNA nuclear export of intronless RNAs and pre-mRNA 3′ processing, but no direct proof of EB2 being involved in RNA processing and transport has been provided, and no specific functional domain of EB2 has been mapped. Here we significantly extend these findings and directly demonstrate that (i) EB2 inhibits the cytoplasmic accumulation of mRNAs, but only if they are generated from precursors containing weak (cryptic) 5′ splice sites, (ii) EB2 has no effect on the cytoplasmic accumulation of mRNA generated from precursors containing constitutive splice sites, and (iii) EB2 has no effect on the 3′ processing of precursor RNAs containing canonical and noncanonical cleavage-polyadenylation signals. We also show that in the presence of EB2, intron-containing and intronless RNAs accumulate in the cytoplasm. EB2 contains an Arg-X-Pro tripeptide repeated eight times, similar to that described as an RNA-binding domain in the herpes simplex virus type 1 protein US11. As glutathione S-transferase fusion proteins, both EB2 and the Arg-X-Pro repeat bound RNA in vitro. However, by using EB2 deletion mutants, we demonstrated that the effect of EB2 on splicing and RNA transport requires the C-terminal half of the protein but not the Arg-X-Pro repeat.

scholarly journals General and Target-Specific RNA Binding Properties of Epstein-Barr Virus SM Posttranscriptional Regulatory Protein

2009 ◽  
Vol 83 (22) ◽  
pp. 11635-11644 ◽  
Author(s):  
Zhao Han ◽  
Dinesh Verma ◽  
Chelsey Hilscher ◽  
Dirk P. Dittmer ◽  
Sankar Swaminathan
Keyword(s):  
Gene Expression ◽  
Nuclear Export ◽  
Epstein Barr Virus ◽  
Rna Binding ◽  
Regulatory Protein ◽  
Lytic Cycle ◽  
Binding Properties ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) SM protein is an essential nuclear shuttling protein expressed by EBV early during the lytic phase of replication. SM acts to increase EBV lytic gene expression by binding EBV mRNAs and enhancing accumulation of the majority of EBV lytic cycle mRNAs. SM increases target mRNA stability and nuclear export, in addition to modulating RNA splicing. SM and its homologs in other herpesvirus have been hypothesized to function in part by binding viral RNAs and recruiting cellular export factors. Although activation of gene expression by SM is gene specific, it is unknown whether SM binds to mRNA in a specific manner or whether its RNA binding is target independent. SM-mRNA complexes were isolated from EBV-infected B-lymphocyte cell lines induced to permit lytic EBV replication, and a quantitative measurement of mRNAs corresponding to all known EBV open reading frames was performed by real-time quantitative reverse transcription-PCR. The results showed that although SM has broad RNA binding properties, there is a clear hierarchy of affinities among EBV mRNAs with respect to SM complex formation. In vitro binding assays with two of the most highly SM-associated transcripts suggested that SM binds preferentially to specific sequences or structures present in noncoding regions of some EBV mRNAs. Furthermore, the presence of these sequences conferred responsiveness to SM. These data are consistent with a mechanism of action similar to that of hnRNPs, which exert sequence-specific effects on gene expression despite having multiple degenerate consensus binding sites common to a large number of RNAs.

scholarly journals Epstein-Barr Virus SM Protein Utilizes Cellular Splicing Factor SRp20 To Mediate Alternative Splicing

2010 ◽  
Vol 84 (22) ◽  
pp. 11781-11789 ◽  
Author(s):  
Dinesh Verma ◽  
Swarna Bais ◽  
Melusine Gaillard ◽  
Sankar Swaminathan
Keyword(s):  
Alternative Splicing ◽  
Splice Site ◽  
Nuclear Export ◽  
Epstein Barr Virus ◽  
Rna Binding ◽  
Lytic Cycle ◽  
Splicing Factor ◽  
Barr Virus ◽  
Epstein Barr ◽  
Sm Protein

ABSTRACT Epstein-Barr virus (EBV) SM protein is an essential nuclear protein produced during the lytic cycle of EBV replication. SM is an RNA-binding protein with multiple mechanisms of action. SM enhances the expression of EBV genes by stabilizing mRNA and facilitating nuclear export. SM also influences splicing of both EBV and cellular pre-mRNAs. SM modulates splice site selection of the host cell STAT1 pre-mRNA, directing utilization of a novel 5′ splice site that is used only in the presence of SM. SM activates splicing in the manner of SR proteins but does not contain the canonical RS domains typical of cellular splicing factors. Affinity purification and mass spectrometry of SM complexes from SM-transfected cells led to the identification of the cellular SR splicing factor SRp20 as an SM-interacting protein. The regions of SM and SRp20 required for interaction were mapped by in vitro and in vivo assays. The SRp20 interaction was shown to be important for the effects of SM on alternative splicing by the use of STAT1 splicing assays. Overexpression of SRp20 enhanced SM-mediated alternative splicing and knockdown of SRp20 inhibited the SM effect on splicing. These data suggest a model whereby SM, a viral protein, recruits and co-opts the function of cellular SRp20 in alternative splicing.

scholarly journals Epstein-Barr Virus Protein Kinase BGLF4 Is a Virion Tegument Protein That Dissociates from Virions in a Phosphorylation-Dependent Process and Phosphorylates the Viral Immediate-Early Protein BZLF1

2006 ◽  
Vol 80 (11) ◽  
pp. 5125-5134 ◽  
Author(s):  
Risa Asai ◽  
Ai Kato ◽  
Kentaro Kato ◽  
Mikiko Kanamori-Koyama ◽  
Ken Sugimoto ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Epstein Barr Virus ◽  
Tegument Protein ◽  
Virus Protein ◽  
Barr Virus ◽  
Early Protein ◽  
Dependent Process ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) BGLF4 is a viral protein kinase that is expressed in the lytic phase of infection and is packaged in virions. We report here that BGLF4 is a tegument protein that dissociates from the virion in a phosphorylation-dependent process. We also present evidence that BGLF4 interacts with and phosphorylates BZLF1, a key viral regulator of lytic infection. These conclusions are based on the following observations. (i) In in vitro tegument release assays, a significant fraction of BGLF4 was released from virions in the presence of physiological NaCl concentrations. (ii) Addition of physiological concentrations of ATP and MgCl2 to virions enhanced BGLF4 release, but phosphatase treatment of virions significantly reduced BGLF4 release. (iii) A recombinant protein containing a domain of BZLF1 was specifically phosphorylated by purified recombinant BGLF4 in vitro, and BGLF4 altered BZLF1 posttranslational modification in vivo. (iv) BZLF1 was specifically coimmunoprecipitated with BGLF4 in 12-O-tetradecanoylphorbol-13-acetate-treated B95-8 cells and in COS-1 cells transiently expressing both of these viral proteins. (v) BGLF4 and BZLF1 were colocalized in intranuclear globular structures, resembling the viral replication compartment, in Akata cells treated with anti-human immunoglobulin G. Our results suggest that BGLF4 functions not only in lytically infected cells by phosphorylating viral and cellular targets but also immediately after viral penetration like other herpesvirus tegument proteins.

scholarly journals Nuclear-Cytoplasmic Shuttling Is Not Required for the Epstein-Barr Virus EBNA-LP Transcriptional Coactivation Function

2009 ◽  
Vol 83 (14) ◽  
pp. 7109-7116 ◽  
Author(s):  
Paul D. Ling ◽  
Jie Tan ◽  
RongSheng Peng
Keyword(s):  
Nuclear Export ◽  
Epstein Barr Virus ◽  
Nuclear Export Signal ◽  
Nuclear Body ◽  
Simple Diffusion ◽  
Nuclear Localization Signals ◽  
Barr Virus ◽  
Histone Deacetylase 4 ◽  
Epstein Barr ◽  
Cellular Compartments

ABSTRACT Epstein-Barr virus (EBV) EBNA-LP is a transcriptional coactivator of EBNA2 that works though interaction with the promyelocytic leukemia nuclear-body-associated protein Sp100A. EBNA-LP localizes predominantly in the nucleus through the action of nuclear localization signals in the repeated regions of the protein. EBNA-LP has also been detected in the cytoplasm, and a previous study suggested that some of the EBNA-LP coactivation function is mediated by relocalizing histone deacetylase 4 (HDAC4) from the nucleus to the cytoplasm. Although EBNA-LP can be found in the cytoplasm, it has no obvious nuclear export signal, and there is no direct evidence for active shuttling between these cellular compartments. Whether active shuttling between the nucleus and cytoplasm is required for coactivation remains to be clarified. To address these issues, we tested a variety of EBNA-LP isoforms and mutants for nuclear-cytoplasmic shuttling activity in an interspecies heterokaryon assay and for the ability to associate with HDAC4. EBNA-LP isoforms smaller than 42 kDa shuttle efficiently in the heterokaryon assay via a crm-1-independent mechanism. In addition, no specific EBNA-LP domain that mediates nuclear export could be identified. In contrast, an EBNA-LP 62-kDa isoform does not demonstrate detectable shuttling in the heterokaryon assay yet still coactivates EBNA2 similarly to the smaller EBNA-LP isoforms. All of the EBNA-LP mutants tested, including the coactivation-deficient ΔCR3 mutant and the nonshuttling 62-kDa isoform, were capable of associating with HDAC4. Taken together, our results suggest that simple diffusion may account for the nuclear export observed with smaller isoforms of EBNA-LP, that nuclear-cytoplasmic shuttling is not required for efficient EBNA-LP coactivation function, and that competence for HDAC4 association is not sufficient to mediate nuclear-cytoplasmic shuttling or EBNA-LP coactivation in the absence of a functional interaction with Sp100A.

scholarly journals Mta Has Properties of an RNA Export Protein and Increases Cytoplasmic Accumulation of Epstein-Barr Virus Replication Gene mRNA

1998 ◽  
Vol 72 (12) ◽  
pp. 9526-9534 ◽  
Author(s):  
O. John Semmes ◽  
Lin Chen ◽  
Robert T. Sarisky ◽  
Zhigang Gao ◽  
Ling Zhong ◽  
...  
Keyword(s):  
Rna Processing ◽  
Nuclear Export ◽  
Epstein Barr Virus ◽  
Rna Binding ◽  
Mrna Levels ◽  
Transfected Cells ◽  
Barr Virus ◽  
Replication Gene ◽  
Cytoplasmic Accumulation ◽  
Epstein Barr

ABSTRACT The Epstein-Barr virus (EBV) Zta and Mta regulatory proteins were previously found to be required for efficient replication of oriLyt in cotransfection-replication assays, but the contribution of Mta to the replication process was unknown. We now demonstrate that Mta regulates replication gene expression. Using the polymerase processivity factor BMRF1 as an example, we found that in transfected cells, total BMRF1 mRNA levels were unaffected by Mta but that the amounts of cytoplasmic BMRF1 RNA and protein were greatly increased in the presence of Mta. Mta also increased cytoplasmic accumulation of the BALF2, BALF5, BSLF1, and BBLF4 replication gene mRNAs but did not affect cytoplasmic levels of BBLF2/3 mRNA. Thus, five of the six core replication genes require Mta for efficient accumulation of cytoplasmic RNA. The contribution of Mta to posttranscriptional RNA processing was examined. Examination of Mta localization in transfected cells by indirect immunofluorescence revealed that Mta colocalized with the splicing factor SC35. We also found that Mta has RNA binding activity. GlutathioneS-transferase–Mta bound to BMRF1 and BMLF1 transcripts but not to a control cellular gene RNA. Mta contains a consensus leucine-rich nuclear export signal. Such signal sequences are characteristic of proteins that undergo nuclear export. Examination of Mta localization in a heterokaryon assay provided evidence that Mta shuttles between the nucleus and the cytoplasm. Our experiments indicate that Mta functions in RNA processing and transport and mediates cytoplasmic accumulation of a number of EBV early mRNAs.

scholarly journals Epstein-Barr Virus Protein EB2 Stimulates Translation Initiation of mRNAs through Direct Interactions with both Poly(A)-Binding Protein and Eukaryotic Initiation Factor 4G

2017 ◽  
Vol 92 (3) ◽  
Author(s):  
Fabrice Mure ◽  
Baptiste Panthu ◽  
Isabelle Zanella-Cléon ◽  
Frédéric Delolme ◽  
Evelyne Manet ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Epstein Barr Virus ◽  
Rna Binding ◽  
Binding Protein ◽  
Initiation Factor ◽  
Translation Efficiency ◽  
Eukaryotic Initiation Factor ◽  
Barr Virus ◽  
Intronless Genes ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) expresses several mRNAs produced from intronless genes that could potentially be unfavorably translated compared to cellular spliced mRNAs. To overcome this situation, the virus encodes an RNA-binding protein (RBP) called EB2, which was previously found to both facilitate the export of nuclear mRNAs and increase their translational yield. Here, we show that EB2 binds both nuclear and cytoplasmic cap-binding complexes (CBC and eukaryotic initiation factor 4F [eIF4F], respectively) as well as the poly(A)-binding protein (PABP) to enhance translation initiation of a given messenger ribonucleoparticle (mRNP). Interestingly, such an effect can be obtained only if EB2 is initially bound to the native mRNPs in the nucleus. We also demonstrate that the EB2-eIF4F-PABP association renders translation of these mRNPs less sensitive to translation initiation inhibitors. Taken together, our data suggest that EB2 binds and stabilizes cap-binding complexes in order to increase mRNP translation and furthermore demonstrate the importance of the mRNP assembly process in the nucleus to promote protein synthesis in the cytoplasm. IMPORTANCE Most herpesvirus early and late genes are devoid of introns. However, it is now well documented that mRNA splicing facilitates recruitment on the mRNAs of cellular factors involved in nuclear mRNA export and translation efficiency. To overcome the absence of splicing of herpesvirus mRNAs, a viral protein, EB2 in the case of Epstein-Barr virus, is produced to facilitate the cytoplasmic accumulation of viral mRNAs. Although we previously showed that EB2 also specifically enhances translation of its target mRNAs, the mechanism was unknown. Here, we show that EB2 first is recruited to the mRNA cap structure in the nucleus and then interacts with the proteins eIF4G and PABP to enhance the initiation step of translation.

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