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 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 Epstein-Barr Virus Exploits the Secretory Pathway to Release Virions

2020 ◽  
Vol 8 (5) ◽  
pp. 729
Author(s):  
Asuka Nanbo
Keyword(s):  
Epstein Barr Virus ◽  
Secretory Pathway ◽  
Lytic Cycle ◽  
Rab Gtpase ◽  
Rab Gtpases ◽  
Extracellular Milieu ◽  
Barr Virus ◽  
Intracellular Compartments ◽  
Epstein Barr

Herpesvirus egress mechanisms are strongly associated with intracellular compartment remodeling processes. Previously, we and other groups have described that intracellular compartments derived from the Golgi apparatus are the maturation sites of Epstein-Barr virus (EBV) virions. However, the mechanism by which these virions are released from the host cell to the extracellular milieu is poorly understood. Here, I adapted two independent induction systems of the EBV lytic cycle in vitro, in the context of Rab GTPase silencing, to characterize the EBV release pathway. Immunofluorescence staining revealed that p350/220, the major EBV glycoprotein, partially co-localized with three Rab GTPases: Rab8a, Rab10, and Rab11a. Furthermore, the knockdown of these Rab GTPases promoted the intracellular accumulation of viral structural proteins by inhibiting its distribution to the plasma membrane. Finally, the knockdown of the Rab8a, Rab10, and Rab11a proteins suppressed the release of EBV infectious virions. Taken together, these findings support the hypothesis that mature EBV virions are released from infected cells to the extracellular milieu via the secretory pathway, as well as providing new insights into the EBV life cycle.

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 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 Marked Variation in Response of Consensus Binding Elements for the Rta Protein of Epstein-Barr Virus

2005 ◽  
Vol 79 (15) ◽  
pp. 9635-9650 ◽  
Author(s):  
Lee-Wen Chen ◽  
Pey-Jium Chang ◽  
Henri-Jacques Delecluse ◽  
George Miller
Keyword(s):  
Dna Sequences ◽  
Transcriptional Activation ◽  
Mammalian Cells ◽  
Epstein Barr Virus ◽  
Lytic Cycle ◽  
Maximal Response ◽  
Binding Affinities ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT The R transactivator (Rta) protein activates Epstein-Barr virus (EBV) lytic-cycle genes by several distinct mechanisms that include direct binding to viral promoters, synergy with BamHI Z EBV replication activator (ZEBRA), and activation of cellular signaling pathways. In the direct and synergistic mechanisms of action, Rta binds to specific DNA sequences that are present in the promoters of responsive genes. It has been difficult to demonstrate the capacity of Rta expressed in mammalian cells to bind DNA in vitro in order to study the relative affinities of Rta binding elements. We discovered that a short C-terminal region of Rta inhibits the ability of Rta to bind DNA in vitro. C-terminally truncated versions of Rta bind DNA efficiently and thus facilitate a comparison of consensus Rta binding elements (CRBEs) found in promoters of five Rta-responsive genes: BMLF1, BHLF1, BMRF1, BaRF1, and BLRF2. All CRBEs in the promoters of the five genes conform to the proposed recognition sequence GNCCN9GGNG, where N is any nucleotide and N9 represents a sequence of nine nucleotides. Nonetheless, CRBEs varied markedly in their abilities to bind Rta in electrophoretic mobility shift assays. Not all CRBEs bound or responded to Rta. Binding affinities of the CRBEs and the capacity to be activated by Rta in reporter assays were strongly correlated. The CRBEs from the BMLF1 and BHLF1 promoters conferred the greatest response. The response of the BMRF1, BaRF1, and BLRF2 CRBEs was less robust. By creation of chimeras, inversions, and point mutations, differences in binding affinities and transcriptional activation levels could be attributed to N9 sequence variation. The length of N9 was also critical for a maximal response. In Raji and BZLF1-knockout cells, the mRNAs of the five Rta-responsive lytic-cycle genes differed dramatically in kinetics of expression, abundance, and synergistic responses to ZEBRA and Rta. Affinities of Rta response elements for Rta are likely to play an important role in temporal regulation and the level of lytic-cycle EBV gene expression.

scholarly journals Epstein–Barr virus origin of lytic replication mediates association of replicating episomes with promyelocytic leukaemia protein nuclear bodies and replication compartments

2006 ◽  
Vol 87 (5) ◽  
pp. 1133-1137 ◽  
Author(s):  
Wolfgang Amon ◽  
Robert E. White ◽  
Paul J. Farrell
Keyword(s):  
Gene Expression ◽  
Epstein Barr Virus ◽  
Lytic Replication ◽  
Nuclear Bodies ◽  
Lytic Cycle ◽  
Promyelocytic Leukaemia ◽  
Late Gene ◽  
Barr Virus ◽  
Pml Nuclear Bodies ◽  
Epstein Barr

Epstein–Barr virus (EBV) establishes a latent persistence from which it can be reactivated to undergo lytic replication. Late lytic-cycle gene expression is linked to lytic DNA replication, as it is sensitive to the same inhibitors that block lytic replication, and it has recently been shown that the viral origin of lytic replication (ori lyt) is required in cis for late-gene expression. During the lytic cycle, the viral genome forms replication compartments, which are usually adjacent to promyelocytic leukaemia protein (PML) nuclear bodies. A tetracycline repressor DNA-binding domain–enhanced green fluorescent protein fusion was used to visualize replicating plasmids carrying a tetracycline operator sequence array. ori lyt mediated the production of plasmid replication compartments that were associated with PML nuclear bodies. Plasmids carrying ori lyt and EBV itself were visualized in the same cells and replicated in similar regions of the nucleus, further supporting the validity of the plasmids for studying late-gene regulation.

scholarly journals Epstein–Barr virus-encoded LMP1 induces a hyperproliferative and inflammatory gene expression programme in cultured keratinocytes

2008 ◽  
Vol 89 (11) ◽  
pp. 2806-2820 ◽  
Author(s):  
Mhairi A. Morris ◽  
Christopher W. Dawson ◽  
Wenbin Wei ◽  
John D. O'Neil ◽  
Suzanne E. Stewart ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Motility ◽  
Epstein Barr Virus ◽  
Terminal Differentiation ◽  
Raft Culture ◽  
Barr Virus ◽  
Lmp1 Expression ◽  
Cellular Pathways ◽  
Epstein Barr

SCC12F cells are a line of keratinocytes that retain the capacity for terminal differentiation in vitro. We showed previously that the Epstein–Barr virus (EBV)-encoded oncogene latent membrane protein 1 (LMP1) altered SCC12F morphology in vitro, downregulated cell–cell-adhesion molecule expression and promoted cell motility. In organotypic raft culture, LMP1-expressing cells failed to stratify and formed poorly organized structures which displayed impaired terminal differentiation. To understand better the mechanism(s) by which LMP1 induces these effects, we generated SCC12F cells in which LMP1 expression is inducible. Following induction, these cells exhibited phenotypic changes similar to those observed previously and allowed us to investigate the effects of LMP1 expression on cellular pathways associated with growth, differentiation and morphology. Using microarrays and a number of confirmatory techniques, we identified sets of differentially expressed genes that are characteristically expressed in inflammatory and hyperproliferative epidermis, including chemokines, cytokines and their receptors, growth factors involved in promoting epithelial cell motility and proliferation and signalling molecules that regulate actin filament reorganization and cell movement. Among the genes whose expression was differentially induced significantly by LMP1, the induction of IL-1β and IL-1α was of particular interest, as many of the LMP1-regulated genes identified are established targets of these cytokines. Our findings suggest that alterations in the IL-1 signalling network may be responsible for many of the changes in host-cell gene expression induced in response to LMP1. Identification of these LMP1-regulated genes helps to define the mechanism(s) by which this oncoprotein influences cellular pathways that regulate terminal differentiation, cell motility and inflammation.

scholarly journals An Epigenetic Journey: Epstein-Barr Virus Transcribes Chromatinized and Subsequently Unchromatinized Templates during Its Lytic Cycle

2019 ◽  
Vol 93 (8) ◽  
Author(s):  
Adityarup Chakravorty ◽  
Bill Sugden ◽  
Eric C. Johannsen
Keyword(s):  
Gene Expression ◽  
Dna Replication ◽  
Epstein Barr Virus ◽  
Dna Amplification ◽  
Lytic Cycle ◽  
Late Gene ◽  
Late Gene Expression ◽  
Viral Dna ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACTThe Epstein-Barr virus (EBV) lytic phase, like those of all herpesviruses, proceeds via an orderly cascade that integrates DNA replication and gene expression. EBV early genes are expressed independently of viral DNA amplification, and several early gene products facilitate DNA amplification. On the other hand, EBV late genes are defined by their dependence on viral DNA replication for expression. Recently, a set of orthologous genes found in beta- and gammaherpesviruses have been determined to encode a viral preinitiation complex (vPIC) that mediates late gene expression. The EBV vPIC requires an origin of lytic replication incis, implying that the vPIC mediates transcription from newly replicated DNA. In agreement with this implication, EBV late gene mRNAs localize to replication factories. Notably, these factories exclude canonical histones. In this review, we compare and contrast the mechanisms and epigenetics of EBV early and late gene expression. We summarize recent findings, propose a model explaining the dependence of EBV late gene expression on lytic DNA amplification, and suggest some directions for future study.

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.

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