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 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 Identification and Characterization of the Physiological Gene Targets of the Essential Lytic Replicative Epstein-Barr Virus SM Protein

2015 ◽  
Vol 90 (3) ◽  
pp. 1206-1221 ◽  
Author(s):  
Jacob Thompson ◽  
Dinesh Verma ◽  
DaJiang Li ◽  
Tim Mosbruger ◽  
Sankar Swaminathan
Keyword(s):  
Dna Replication ◽  
Mechanism Of Action ◽  
Epstein Barr Virus ◽  
Rna Binding ◽  
Lytic Cycle ◽  
Barr Virus ◽  
Ebv Dna ◽  
Epstein Barr ◽  
Sm Protein ◽  
Target Rna

ABSTRACTEpstein-Barr virus (EBV) SM protein is an essential lytic cycle protein with multiple posttranscriptional mechanisms of action. SM binds RNA and increases accumulation of specific EBV transcripts. Previous studies using microarrays and PCR have shown that SM-null mutants fail to accumulate several lytic cycle mRNAs and proteins at wild-type levels. However, the complete effect of SM on the EBV transcriptome has been incompletely characterized. Here we precisely identify the effects of SM on all EBV transcripts by high-throughput RNA sequencing, quantitative PCR (qPCR), and Northern blotting. The effect of SM on EBV mRNAs was highly skewed and was most evident on 13 late genes, demonstrating why SM is essential for infectious EBV production. EBV DNA replication was also partially impaired in SM mutants, suggesting additional roles for SM in EBV DNA replication. While it has been suggested that SM specificity is based on recognition of either RNA sequence motifs or other sequence properties, no such unifying property of SM-responsive targets was discernible. The binding affinity of mRNAs for SM also did not correlate with SM responsiveness. These data suggest that while target RNA binding by SM may be required for its effect, specific activation by SM is due to differences in inherent properties of individual transcripts. We therefore propose a new model for the mechanism of action and specificity of SM and its homologs in other herpesviruses: that they bind many RNAs but only enhance accumulation of those that are intrinsically unstable and poorly expressed.IMPORTANCEThis study examines the mechanism of action of EBV SM protein, which is essential for EBV replication and infectious virus production. Since SM protein is not similar to any cellular protein and has homologs in all other human herpesviruses, it has potential importance as a therapeutic target. Here we establish which EBV RNAs are most highly upregulated by SM, allowing us to understand why it is essential for EBV replication. By comparing and characterizing these RNA transcripts, we conclude that the mechanism of specific activity is unlikely to be based simply on preferential recognition of a target motif. Rather, SM binding to its target RNA may be necessary but not sufficient for enhancing accumulation of the RNA. Preferential effects of SM on its most responsive RNA targets may depend on other inherent characteristics of these specific mRNAs that require SM for efficient expression, such as RNA stability.

scholarly journals Epstein-Barr Virus SM Protein Functions as an Alternative Splicing Factor

2008 ◽  
Vol 82 (14) ◽  
pp. 7180-7188 ◽  
Author(s):  
Dinesh Verma ◽  
Sankar Swaminathan
Keyword(s):  
Gene Expression ◽  
Alternative Splicing ◽  
Viral Protein ◽  
Epstein Barr Virus ◽  
Regulatory Control ◽  
Splicing Factor ◽  
Barr Virus ◽  
Alternative Mrna Splicing ◽  
Epstein Barr ◽  
Sm Protein

ABSTRACT Alternative splicing of RNA increases the coding potential of the genome and allows for additional regulatory control over gene expression. The full extent of alternative splicing remains to be defined but is likely to significantly expand the size of the human transcriptome. There are several examples of mammalian viruses regulating viral splicing or inhibiting cellular splicing in order to facilitate viral replication. Here, we describe a viral protein that induces alternative splicing of a cellular RNA transcript. Epstein-Barr virus (EBV) SM protein is a viral protein essential for replication that enhances EBV gene expression by enhancing RNA stability and export. SM also increases cellular STAT1 expression, a central mediator of interferon signal transduction, but disproportionately increases the abundance of the STAT1β splicing isoform, which can act as a dominant-negative suppressor of STAT1α. SM induces splicing of STAT1 at a novel 5′ splice site, resulting in a STAT1 mRNA incapable of producing STAT1α. SM-induced alternative splicing is dependent on the presence of an RNA sequence to which SM binds directly and which can confer SM-dependent splicing on heterologous RNA. The cellular splicing factor ASF/SF2 also binds to this region and inhibits SM-RNA binding and SM-induced alternative splicing. These results suggest that viruses may regulate cellular gene expression at the level of alternative mRNA splicing in order to facilitate virus replication or persistence in vivo.

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

1999 ◽  
Vol 73 (8) ◽  
pp. 6872-6881 ◽  
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.

scholarly journals Identification of a Lytic-Cycle Epstein-Barr Virus Gene Product That Can Regulate PKR Activation

2003 ◽  
Vol 77 (1) ◽  
pp. 228-236 ◽  
Author(s):  
Jeremy Poppers ◽  
Matthew Mulvey ◽  
Cesar Perez ◽  
David Khoo ◽  
Ian Mohr
Keyword(s):  
Gene Product ◽  
Epstein Barr Virus ◽  
Viral Gene Expression ◽  
Lytic Cycle ◽  
Viral Gene ◽  
Barr Virus ◽  
Epstein Barr ◽  
Pkr Kinase ◽  
Sm Protein ◽  
Hsv 1

ABSTRACT The Epstein-Barr virus (EBV) SM protein is a posttranscriptional regulator of viral gene expression. Like many transactivators encoded by herpesviruses, SM transports predominantly unspliced viral mRNA cargo from the nucleus to the cytosol, where it is subsequently translated. This activity likely involves a region of the protein that has homology to the herpes simplex virus type 1 (HSV-1) ICP27 gene product, the first member of this class of regulators to be discovered. However, SM also contains a repetitive segment rich in arginine and proline residues that is dispensable for its effects on RNA transport and splicing. This portion of SM, comprised of RXP triplet repeats, shows homology to the carboxyl-terminal domain of Us11, a double-stranded RNA (dsRNA) binding protein encoded by HSV-1 that inhibits activation of the cellular PKR kinase. To evaluate the intrinsic ability of SM to regulate PKR, we expressed and purified several SM protein derivatives and examined their activity in a variety of biochemical assays. The full-length SM protein bound dsRNA, associated physically with PKR, and prevented PKR activation. Removal of the 37-residue RXP domain significantly compromised all of these activities. Furthermore, the SM RXP domain was itself sufficient to inhibit PKR activation and interact with the kinase. Relative to its Us11 counterpart, the SM RXP segment bound dsRNA with reduced affinity and responded differently to single-stranded competitor polynucleotides. Thus, SM represents the first EBV gene product expressed during the lytic cycle that can prevent PKR activation. In addition, the RXP repeat segment appears to be a conserved herpesvirus motif capable of associating with dsRNA and modulating activation of the PKR kinase, a molecule important for the control of translation and the cellular antiviral response.

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 Negative Autoregulation of Epstein-Barr Virus (EBV) Replicative Gene Expression by EBV SM Protein

2009 ◽  
Vol 83 (16) ◽  
pp. 8041-8050 ◽  
Author(s):  
Dinesh Verma ◽  
Chen Ling ◽  
Eric Johannsen ◽  
Tirumuru Nagaraja ◽  
Sankar Swaminathan
Keyword(s):  
Epstein Barr Virus ◽  
Feedback Regulation ◽  
Lytic Replication ◽  
Lytic Cycle ◽  
Immediate Early ◽  
Mrna Levels ◽  
Barr Virus ◽  
Infected Cells ◽  
Epstein Barr ◽  
Sm Protein

ABSTRACT The Epstein-Barr virus (EBV) SM protein is essential for lytic EBV DNA replication and virion production. When EBV replication is induced in cells infected with an SM-deleted recombinant EBV, approximately 50% of EBV genes are expressed inefficiently. When EBV replication is rescued by transfection of SM, SM enhances expression of these genes by direct and indirect mechanisms. While expression of most EBV genes is either unaffected or enhanced by SM, expression of several genes is decreased in the presence of SM. Expression of BHRF1, a homolog of cellular bcl-2, is particularly decreased in the presence of SM. Investigation of the mechanism of BHRF1 downregulation revealed that SM downregulates expression of the immediate-early EBV transactivator R. In EBV-infected cells, R-responsive promoters, including the BHRF1 and SM promoters, were less active in the presence of SM, consistent with SM inhibition of R expression. SM decreased spliced R mRNA levels, supporting a posttranscriptional mechanism of R inhibition. R and BHRF1 expression were also found to decrease during later stages of EBV lytic replication in EBV-infected lymphoma cells. These data indicate that feedback regulation of immediate-early and early genes occurs during the lytic cycle of EBV regulation.

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 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 Cellular RNA Helicase DHX9 Interacts with the Essential Epstein-Barr Virus (EBV) Protein SM and Restricts EBV Lytic Replication

2018 ◽  
Vol 93 (4) ◽  
Author(s):  
Wenmin Fu ◽  
Dinesh Verma ◽  
Ashlee Burton ◽  
Sankar Swaminathan
Keyword(s):  
Gene Expression ◽  
Epstein Barr Virus ◽  
Rna Binding ◽  
Virus Production ◽  
Lytic Replication ◽  
Late Gene Expression ◽  
Barr Virus ◽  
Virion Production ◽  
Epstein Barr ◽  
Sm Protein

ABSTRACTEpstein-Barr virus (EBV) SM protein is an RNA-binding protein that has multiple posttranscriptional gene regulatory functions essential for EBV lytic replication. In this study, we identified an interaction between SM and DHX9, a DExH-box helicase family member, by mass spectrometry and coimmunoprecipitation. DHX9 participates in many cellular pathways involving RNA, including transcription, processing, transport, and translation. DHX9 enhances virus production or infectivity of a wide variety of DNA and RNA viruses. Surprisingly, an increase in EBV late gene expression and virion production occurred upon knockdown of DHX9. To further characterize the SM-DHX9 interaction, we performed immunofluorescence microscopy of EBV-infected cells and found that DHX9 partially colocalized with SM in nuclear foci during EBV lytic replication. However, the positive effect of DHX9 depletion on EBV lytic gene expression was not confined to SM-dependent genes, indicating that the antiviral effect of DHX9 was not mediated through its effects on SM. DHX9 enhanced activation of innate antiviral pathways comprised of several interferon-stimulated genes that are active against EBV. SM inhibited the transcription-activating function of DHX9, which acts through cAMP response elements (CREs), suggesting that SM may also act to counteract DHX9’s antiviral functions during lytic replication.IMPORTANCEThis study identifies an interaction between Epstein-Barr virus (EBV) SM protein and cellular helicase DHX9, exploring the roles that this interaction plays in viral infection and host defenses. Whereas most previous studies established DHX9 as a proviral factor, we demonstrate that DHX9 may act as an inhibitor of EBV virion production. DHX9 enhanced innate antiviral pathways active against EBV and was needed for maximal expression of several interferon-induced genes. We show that SM binds to and colocalizes DHX9 and may counteract the antiviral function of DHX9. These data indicate that DHX9 possesses antiviral activity and that SM may suppress the antiviral functions of DHX9 through this association. Our study presents a novel host-pathogen interaction between EBV and the host cell.

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