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 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 Antibodies to gp350/220 Enhance the Ability of Epstein-Barr Virus To Infect Epithelial Cells

2006 ◽  
Vol 80 (19) ◽  
pp. 9628-9633 ◽  
Author(s):  
Susan M. Turk ◽  
Ru Jiang ◽  
Liudmila S. Chesnokova ◽  
Lindsey M. Hutt-Fletcher
Keyword(s):  
B Cells ◽  
Nasopharyngeal Carcinoma ◽  
Epithelial Cells ◽  
Epstein Barr Virus ◽  
Lytic Cycle ◽  
Virus Envelope ◽  
Barr Virus ◽  
High Risk Populations ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) is a persistent, orally transmitted herpesvirus that replicates in B cells and epithelial cells and is associated with lymphoid and epithelial malignancies. The virus binds to CD21 on B cells via glycoprotein gp350/220 and infects efficiently. Infection of cultured epithelial cells has not typically been efficient but can occur in the absence of gp350/220 and CD21 and in vivo is thought to be important to the development of nasopharyngeal carcinoma. We report here that antibodies to gp350/220, which inhibit EBV infection of B cells, enhance infection of epithelial cells. The effect is not mediated by Fc receptor binding but is further enhanced by antibody cross-linking, which may patch gp350/220 in the virus envelope. Saliva from EBV-seropositive individuals has similar effects that can be reversed by depletion of antibody. The results are consistent with a model in which gp350/220 interferes with the access of other important players to the epithelial cell surface. The results may have implications for the development of nasopharyngeal carcinoma in high-risk populations in which elevated titers of antibody to EBV lytic cycle proteins are prognostic.

scholarly journals Molecular virology of Epstein–Barr virus

2001 ◽  
Vol 356 (1408) ◽  
pp. 437-459 ◽  
Author(s):  
Georg W. Bornkamm ◽  
Wolfgang Hammerschmidt
Keyword(s):  
B Cell ◽  
Epstein Barr Virus ◽  
Descriptive Analysis ◽  
Genetic System ◽  
Lytic Cycle ◽  
Barr Virus ◽  
Cell Immortalization ◽  
Epstein Barr

Epstein–Barr virus (EBV) interacts with its host in three distinct ways in a highly regulated fashion: (i) EBV infects human B lymphocytes and induces proliferation of the infected cells, (ii) it enters into a latent phase in vivo that follows the proliferative phase, and (iii) it can be reactivated giving rise to the production of infectious progeny for reinfection of cells of the same type or transmission of the virus to another individual. In healthy people, these processes take place simultaneously in different anatomical and functional compartments and are linked to each other in a highly dynamic steady–state equilibrium. The development of a genetic system has paved the way for the dissection of those processes at a molecular level that can be studied in vitro , i.e. B–cell immortalization and the lytic cycle leading to production of infectious progeny. Polymerase chain reaction analyses coupled to fluorescent–activated cell sorting has on the other hand allowed a descriptive analysis of the virus–host interaction in peripheral blood cells as well as in tonsillar B cells in vivo . This paper is aimed at compiling our present knowledge on the process of B–cell immortalization in vitro as well as in vivo latency, and attempts to integrate this knowledge into the framework of the viral life cycle in vivo .

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 Terminal Differentiation into Plasma Cells Initiates the Replicative Cycle of Epstein-Barr Virus In Vivo

2005 ◽  
Vol 79 (2) ◽  
pp. 1296-1307 ◽  
Author(s):  
Lauri L. Laichalk ◽  
David A. Thorley-Lawson
Keyword(s):  
Viral Replication ◽  
Epstein Barr Virus ◽  
Plasma Cells ◽  
Acute Stress ◽  
Cytotoxic T Lymphocyte ◽  
Lytic Cycle ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT In this paper we demonstrate that the cells which initiate replication of Epstein-Barr virus (EBV) in the tonsils of healthy carriers are plasma cells (CD38hi, CD10−, CD19+, CD20lo, surface immunoglobulin negative, and cytoplasmic immunoglobulin positive). We further conclude that differentiation into plasma cells, and not the signals that induce differentiation, initiates viral replication. This was confirmed by in vitro studies showing that the promoter for BZLF1, the gene that begins viral replication, becomes active only after memory cells differentiate into plasma cells and is also active in plasma cell lines. This differs from the reactivation of BZLF1 in vitro, which occurs acutely and is associated with apoptosis and not with differentiation. We suggest that differentiation and acute stress represent two distinct pathways of EBV reactivation in vivo. The fraction of cells replicating the virus decreases as the cells progress through the lytic cycle such that only a tiny fraction actually release infectious virus. This may reflect abortive replication or elimination of cells by the cellular immune response. Consistent with the later conclusion, the cells did not down regulate major histocompatibility complex class I molecules, suggesting that this is not an immune evasion tactic used by EBV and that the cells remain vulnerable to cytotoxic-T-lymphocyte attack.

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 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 CCAAT/Enhancer Binding Protein α Binds to the Epstein-Barr Virus (EBV) ZTA Protein through Oligomeric Interactions and Contributes to Cooperative Transcriptional Activation of the ZTA Promoter through Direct Binding to the ZII and ZIIIB Motifs during Induction of the EBV Lytic Cycle

2004 ◽  
Vol 78 (9) ◽  
pp. 4847-4865 ◽  
Author(s):  
Frederick Y. Wu ◽  
Shizhen Emily Wang ◽  
Honglin Chen ◽  
Ling Wang ◽  
S. Diane Hayward ◽  
...  
Keyword(s):  
Protein Expression ◽  
Epstein Barr Virus ◽  
Leucine Zipper ◽  
Binding Protein ◽  
Lytic Cycle ◽  
Barr Virus ◽  
Enhancer Binding Protein ◽  
Epstein Barr

ABSTRACT The Epstein-Barr virus (EBV)-encoded ZTA protein interacts strongly with and stabilizes the cellular CCAAT/enhancer binding protein α (C/EBPα), leading to the induction of p21-mediated G1 cell cycle arrest. Despite the strong interaction between these two basic leucine zipper (bZIP) family proteins, the ZTA and C/EBPα subunits do not heterodimerize, as indicated by an in vitro cross-linking assay with in vitro-cotranslated 35S-labeled C/EBPα and 35S-labeled ZTA protein. Instead, they evidently form a higher-order oligomeric complex that competes with C/EBPα binding but not with ZTA binding in electrophoretic mobility shift assays (EMSAs). Glutathione S-transferase affinity assays with mutant ZTA proteins revealed that the basic DNA binding domain and the key leucine zipper residues required for homodimerization are all required for the interaction with C/EBPα. ZTA is known to bind to two ZRE sites within the ZTA promoter and to positively autoregulate its own expression in transient cotransfection assays, but there is conflicting evidence about whether it does so in vivo. Examination of the proximal ZTA upstream promoter region by in vitro EMSA analysis revealed two high-affinity C/EBP binding sites (C-2 and C-3), which overlap the ZII and ZIIIB motifs, implicated as playing a key role in lytic cycle induction. A chromatin immunoprecipitation assay confirmed the in vivo binding of both endogenous C/EBPα and ZTA protein to the ZTA promoter after lytic cycle induction but not during the latent state in EBV-infected Akata cells. Reporter assays revealed that cotransfected C/EBPα activated the ZTA promoter even more effectively than cotransfected ZTA. However, synergistic activation of the ZTA promoter was not observed when ZTA and C/EBPα were cotransfected together in either HeLa or DG75 cells. Mutagenesis of either the ZII or the ZIIIB sites in the ZTA promoter strongly reduced C/EBPα transactivation, suggesting that these sites act cooperatively. Furthermore, the introduction of exogenous C/EBPα into EBV-infected HeLa-BX1 cells induced endogenous ZTA mRNA and protein expression, as demonstrated by both reverse transcription-PCR and immunoblotting assays. Finally, double-label immunofluorescence assays suggested that EAD protein expression was activated even better than ZTA expression in latently infected C/EBPα-transfected Akata cells, perhaps because of the presence of a strong B-cell-specific repressed chromatin conformation on the ZTA promoter itself during EBV latency.

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