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

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.

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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

Journal of Virology ◽

10.1128/jvi.73.5.4090-4100.1999 ◽

1999 ◽

Vol 73 (5) ◽

pp. 4090-4100 ◽

Cited By ~ 36

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.

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Epstein-Barr Virus mRNA Export Factor EB2 Is Essential for Production of Infectious Virus

Journal of Virology ◽

10.1128/jvi.76.19.9635-9644.2002 ◽

2002 ◽

Vol 76 (19) ◽

pp. 9635-9644 ◽

Cited By ~ 64

Author(s):

Henri Gruffat ◽

Julien Batisse ◽

Dagmar Pich ◽

Bernhard Neuhierl ◽

Evelyne Manet ◽

...

Keyword(s):

Dna Replication ◽

Nuclear Export ◽

Epstein Barr Virus ◽

Alternative Pathway ◽

Virus Production ◽

Gene Encoding ◽

Barr Virus ◽

Cytoplasmic Accumulation ◽

Epstein Barr ◽

Simplex Virus

ABSTRACT The splicing machinery which positions a protein export complex near the exon-exon junction mediates nuclear export of mRNAs generated from intron-containing genes. Many Epstein-Barr virus (EBV) early and late genes are intronless, and an alternative pathway, independent of splicing, must export the corresponding mRNAs. Since the EBV EB2 protein induces the cytoplasmic accumulation of intronless mRNA, it is tempting to speculate that EB2 is a viral adapter involved in the export of intronless viral mRNA. If this is true, then the EB2 protein is essential for the production of EBV infectious virions. To test this hypothesis, we generated an EBV mutant in which the BMLF1 gene, encoding the EB2 protein, has been deleted (EBVBMLF1-KO). Our studies show that EB2 is necessary for the production of infectious EBV and that its function cannot be transcomplemented by a cellular factor. In the EBVBMLF1-KO 293 cells, oriLyt-dependent DNA replication was greatly enhanced by EB2. Accordingly, EB2 induced the cytoplasmic accumulation of a subset of EBV early mRNAs coding for essential proteins implicated in EBV DNA replication during the productive cycle. Two herpesvirus homologs of the EB2 protein, the herpes simplex virus type 1 protein ICP27 and, the human cytomegalovirus protein UL69, only partly rescued the phenotype of the EBVBMLF1-KO mutant, indicating that some EB2 functions in virus production cannot be transcomplemented by ICP27 and UL69.

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

Journal of Virology ◽

10.1128/jvi.01483-09 ◽

2009 ◽

Vol 83 (22) ◽

pp. 11635-11644 ◽

Cited By ~ 12

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.

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

Journal of Virology ◽

10.1128/jvi.01359-10 ◽

2010 ◽

Vol 84 (22) ◽

pp. 11781-11789 ◽

Cited By ~ 30

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.

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

Journal of Virology ◽

10.1128/jvi.01276-09 ◽

2009 ◽

Vol 83 (24) ◽

pp. 12759-12768 ◽

Cited By ~ 26

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.

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Association with the Cellular Export Receptor CRM 1 Mediates Function and Intracellular Localization of Epstein-Barr Virus SM Protein, a Regulator of Gene Expression

Journal of Virology ◽

10.1128/jvi.73.8.6872-6881.1999 ◽

1999 ◽

Vol 73 (8) ◽

pp. 6872-6881 ◽

Cited By ~ 51

Author(s):

Sarah M. Boyle ◽

Vivian Ruvolo ◽

Ashish K. Gupta ◽

Sankar Swaminathan

Keyword(s):

Nuclear Export ◽

Epstein Barr Virus ◽

Rna Binding ◽

Lytic Cycle ◽

Nuclear Pore ◽

Leptomycin B ◽

Barr Virus ◽

Cytoplasmic Translocation ◽

Epstein Barr

ABSTRACT Splicing and posttranscriptional processing of eukaryotic gene transcripts are linked to their nuclear export and cytoplasmic expression. Unspliced pre-mRNAs and intronless transcripts are thus inherently poorly expressed. Nevertheless, human and animal viruses encode essential genes as single open reading frames or in the intervening sequences of other genes. Many retroviruses have evolved mechanisms to facilitate nuclear export of their unspliced mRNAs. For example, the human immunodeficiency virus RNA-binding protein Rev associates with the soluble cellular export receptor CRM 1 (exportin 1), which mediates nucleocytoplasmic translocation of Rev-HIV RNA complexes through the nuclear pore. The transforming human herpesvirus Epstein-Barr virus (EBV) expresses a nuclear protein, SM, early in its lytic cycle; SM binds RNA and posttranscriptionally activates expression of certain intronless lytic EBV genes. Here we show that both the trans-activation function and cytoplasmic translocation of SM are dependent on association with CRM 1 in vivo. SM is also shown to be associated in vivo with other components of the CRM 1 export pathway, including the small GTPase Ran and the nucleoporin CAN/Nup214. SM is shown to be present in the cytoplasm, nucleoplasm, and nuclear envelope of transfected cells. Mutation of a leucine-rich region (LRR) of SM inhibited CRM 1-mediated cytoplasmic translocation and SM activity, as did leptomycin B, an inhibitor of CRM 1 complex formation. Surprisingly, however, leptomycin B treatment and mutation of the LRR both led to SM becoming more tightly attached to intranuclear structures. These findings suggest a model in which SM is not merely a soluble carrier protein for RNA but rather is bound directly to intranuclear proteins, possibly including the nuclear pore complex.

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Curcumin Inhibits Proliferation of Epstein–Barr Virus-Associated Human Nasopharyngeal Carcinoma Cells by Inhibiting EBV Nuclear Antigen 1 Expression

BioMed Research International ◽

10.1155/2019/8592921 ◽

2019 ◽

Vol 2019 ◽

pp. 1-10

Author(s):

Limei Liu ◽

Jiaomin Yang ◽

Wuguang Ji ◽

Chao Wang

Keyword(s):

Nasopharyngeal Carcinoma ◽

Epstein Barr Virus ◽

Lytic Replication ◽

Nuclear Antigen ◽

Mrna Levels ◽

Curcumin Treatment ◽

Antitumor Effects ◽

Barr Virus ◽

Cycle Arrest ◽

Epstein Barr

This investigation aims to study the effect of curcumin on the proliferation, cycle arrest, and apoptosis of Epstein–Barr virus- (EBV-) positive nasopharyngeal carcinoma (NPC) cells. EBV+ NPC cells were subjected to curcumin treatment. The cell viability was evaluated with the CCK-8. Cell cycle and apoptosis were analyzed by flow cytometry analysis. Expression (protein and mRNA) levels were detected with western blotting and quantitative real-time PCR, respectively. Curcumin efficiently reduced the viability of EBV+ NPC cells. Curcumin induced the cycle arrest of the HONE1 and HK1-EBV cells positive for EBV. Moreover, curcumin treatment promoted the NPC cell apoptosis, via the mitochondria- and death receptor-mediated pathways. Furthermore, curcumin decreased the expression of EBNA1 in the HONE1 and HK1-EBV cells and inhibited the transcriptional level of EBNA1 in the HeLa cells. Curcumin induced EBNA1 degradation via the proteasome-ubiquitin pathway. In addition, curcumin inhibited the proliferation of HONE1 and HK1-EBV cells positive for EBV, probably by decreasing the expression level of EBNA1. In both the HONE1 and HK1-EBV cells, curcumin inhibited the EBV latent and lytic replication. Curcumin could reduce the EBNA1 expression and exert antitumor effects against NPC in vitro.

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

Journal of Virology ◽

10.1128/jvi.01421-07 ◽

2007 ◽

Vol 81 (21) ◽

pp. 11850-11860 ◽

Cited By ~ 21

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.

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Mitosis-Specific Hyperphosphorylation of Epstein-Barr Virus Nuclear Antigen 2 Suppresses Its Function

Journal of Virology ◽

10.1128/jvi.78.7.3542-3552.2004 ◽

2004 ◽

Vol 78 (7) ◽

pp. 3542-3552 ◽

Cited By ~ 15

Author(s):

Wei Yue ◽

Matthew G. Davenport ◽

Julia Shackelford ◽

Joseph S. Pagano

Keyword(s):

Epstein Barr Virus ◽

Regulation Of Gene Expression ◽

Cyclin B1 ◽

Nuclear Antigen ◽

Mrna Levels ◽

Type Iii ◽

Barr Virus ◽

M Phase ◽

Epstein Barr

ABSTRACT The Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA-2) is a key gene expressed in EBV type III latent infection that can transactivate numerous promoters, including those for all the other type III viral latency genes as well as cellular genes responsible for cell proliferation. EBNA-2 is essential for EBV-mediated immortalization of primary B lymphocytes. We now report that EBNA-2, a phosphoprotein, is hyperphosphorylated specifically in mitosis. Evidence that the cyclin-dependent kinase p34cdc2 may be involved in this hyperphosphorylation includes (i) coimmunoprecipitation of EBNA-2 and p34cdc2, suggesting physical association; (ii) temporal correlation between hyperphosphorylation of EBNA-2 and an increase in p34cdc2 kinase activity; and (iii) ability of purified p34cdc2/cyclin B1 kinase to phosphorylate EBNA-2 in vitro. Hyperphosphorylation of EBNA-2 appears to suppress its ability to transactivate the latent membrane protein 1 (LMP-1) promoter by about 50%. The association between EBNA-2 and PU.1 is also decreased by about 50% in M-phase-arrested cells, as shown by coimmunoprecipitation from cell lysates, suggesting that hyperphosphorylation of EBNA-2 impairs its affinity for PU.1. Finally, endogenous LMP-1 mRNA levels in M phase are around 55% of those in asynchronously growing cells. These results suggest that regulation of gene expression during type III latency may be regulated in a cell-cycle-related manner.

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