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.

scholarly journals Multiple Roles of Epstein-Barr Virus SM Protein in Lytic Replication

2007 ◽  
Vol 81 (8) ◽  
pp. 4058-4069 ◽  
Author(s):  
Zhao Han ◽  
Elessa Marendy ◽  
Yong-Dong Wang ◽  
Jing Yuan ◽  
Jeffery T. Sample ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Replication ◽  
Epstein Barr Virus ◽  
Lytic Replication ◽  
Late Gene ◽  
Late Gene Expression ◽  
Barr Virus ◽  
Ebv Dna ◽  
Epstein Barr ◽  
Sm Protein

ABSTRACT The effect of Epstein-Barr virus (EBV) SM protein on EBV gene expression was examined using a recombinant EBV strain with the SM gene deleted and DNA microarrays representing all known EBV coding regions. Induction of lytic EBV replication in the absence of SM led to expression of approximately 40% of EBV genes, but a block in expression of over 50% of EBV genes. Contrary to previous findings, several early genes were SM dependent, and lytic EBV DNA replication did not occur in the absence of SM. Notably, two genes essential for lytic EBV DNA replication, BSLF1 and BALF5, encoding EBV DNA primase and polymerase, respectively, were SM dependent. Lytic DNA replication was partially rescued by ectopic expression of EBV primase and polymerase, but virion production was not. Rescue of DNA replication only enhanced expression of a subset of late genes, consistent with a direct requirement for SM for late gene expression in addition to its contribution to DNA replication. Therefore, while SM is essential for most late gene expression, the proximate block to virion production by the EBV SM deletion strain is an inability to replicate linear DNA. The block to DNA replication combined with the direct effect of SM on late gene expression leads to a global deficiency of late gene expression. SM also inhibited BHRF1 expression during productive replication in comparison to that of cells induced into lytic replication in the absence of SM. Thus, SM plays a role in multiple steps of lytic cycle EBV gene expression and that it is transcript-specific in both activation and repression functions.

scholarly journals Functional Analysis of Epstein-Barr Virus SM Protein: Identification of Amino Acids Essential for Structure, Transactivation, Splicing Inhibition, and Virion Production

2004 ◽  
Vol 78 (1) ◽  
pp. 340-352 ◽  
Author(s):  
Vivian Ruvolo ◽  
Liang Sun ◽  
Karilynn Howard ◽  
Seung Sung ◽  
Henri-Jacques Delecluse ◽  
...  
Keyword(s):  
Gene Expression ◽  
Amino Acids ◽  
Epstein Barr Virus ◽  
Protein Identification ◽  
Lytic Replication ◽  
Viral Gene ◽  
Barr Virus ◽  
Virion Production ◽  
Epstein Barr ◽  
Sm Protein

ABSTRACT The Epstein-Barr virus (EBV) SM protein is a posttranscriptional regulator of cellular and viral gene expression that binds and stabilizes target mRNAs and shuttles from nucleus to cytoplasm. SM enhances expression of several EBV genes required for lytic replication and is essential for virion production. SM increases accumulation of specific mRNAs but also inhibits expression of several intron-containing transcripts. The mechanism by which SM inhibits gene expression is poorly understood. The experiments described here had several aims: to determine whether specific domains of SM were responsible for activation or inhibition function; whether these functions could be separated; and whether one or more of these functions were essential for virion production. A mutational analysis of SM was performed, focusing on amino acids in SM that are evolutionarily conserved among SM homologs in other herpesviruses. Mutation of the carboxy-terminal region of SM revealed a region that is likely to be structurally important for SM protein conformation. In addition, several amino acids were identified that are critical for activation and inhibition function. A specific mutation of a highly conserved cysteine residue revealed that it was essential for gene inhibition but not for transactivation, indicating that these two functions operate through independent mechanisms. Furthermore, the ability of wild-type SM and the inability of the mutant to inhibit gene expression were shown to correlate with the ability to inhibit splicing of a human target gene and thereby prevent accumulation of its processed mRNA. Surprisingly, some mutations which preserved both activation and inhibition functions in vitro nevertheless abolished virion production, suggesting that other SM functions or protein-protein interactions are also required for lytic replication.

scholarly journals The Epstein-Barr Virus BDLF4 Gene Is Required for Efficient Expression of Viral Late Lytic Genes

2015 ◽  
Vol 89 (19) ◽  
pp. 10120-10124 ◽  
Author(s):  
Takahiro Watanabe ◽  
Yohei Narita ◽  
Masahiro Yoshida ◽  
Yoshitaka Sato ◽  
Fumi Goshima ◽  
...  
Keyword(s):  
Gene Expression ◽  
Epstein Barr Virus ◽  
Lytic Replication ◽  
Late Gene ◽  
Late Gene Expression ◽  
Lytic Gene ◽  
Barr Virus ◽  
Efficient Expression ◽  
Epstein Barr ◽  
Lytic Genes

Epstein-Barr virus (EBV) is a gammaherpesvirus, associated with infectious mononucleosis and various types of malignancy. We focused here on the BDLF4 gene of EBV and identified it as a lytic gene, expressed with early kinetics. Viral late gene expression of the BDLF4 knockout strain was severely restricted; this could be restored by an exogenous supply of BDLF4. These results indicate that BDLF4 is important for the EBV lytic replication cycle, especially in late gene expression.

scholarly journals Efficient Translation of Epstein-Barr Virus (EBV) DNA Polymerase Contributes to the Enhanced Lytic Replication Phenotype of M81 EBV

2017 ◽  
Vol 92 (6) ◽  
Author(s):  
Trenton Mel Church ◽  
Dinesh Verma ◽  
Jacob Thompson ◽  
Sankar Swaminathan
Keyword(s):  
Nasopharyngeal Carcinoma ◽  
Dna Polymerase ◽  
Epstein Barr Virus ◽  
Rna Binding ◽  
Virus Production ◽  
Protein Translation ◽  
Lytic Replication ◽  
Barr Virus ◽  
Ebv Dna ◽  
Epstein Barr

ABSTRACTEpstein-Barr virus (EBV) is linked to the development of both lymphoid and epithelial malignancies worldwide. The M81 strain of EBV, isolated from a Chinese patient with nasopharyngeal carcinoma (NPC), demonstrates spontaneous lytic replication and high-titer virus production in comparison to the prototype B95-8 EBV strain. Genetic comparisons of M81 and B95-8 EBVs were previously been performed in order to determine if the hyperlytic property of M81 is associated with sequence differences in essential lytic genes. EBV SM is an RNA-binding protein expressed during early lytic replication that is essential for virus production. We compared the functions of M81 SM and B95-8 SM and demonstrate that polymorphisms in SM do not contribute to the lytic phenotype of M81 EBV. However, the expression level of the EBV DNA polymerase protein was much higher in M81- than in B95-8-infected cells. The relative deficiency in the expression of B95-8 DNA polymerase was related to the B95-8 genome deletion, which truncates the BALF5 3′ untranslated region (UTR). Similarly, the insertion of bacmid DNA into the widely used recombinant B95-8 bacmid creates an inefficient BALF5 3′ UTR. We further showed that the while SM is required for and facilitates the efficient expression of both M81 and B95-8 mRNAs regardless of the 3′ UTR, the BALF5 3′ UTR sequence is important for BALF5 protein translation. These data indicate that the enhanced lytic replication and virus production of M81 compared to those of B95-8 are partly due to the robust translation of EBV DNA polymerase required for viral DNA replication due to a more efficient BALF5 3′ UTR in M81.IMPORTANCEEpstein-Barr virus (EBV) infects more than 90% of the human population, but the incidence of EBV-associated tumors varies greatly in different parts of the world. Thus, understanding the connection between genetic polymorphisms from patient isolates of EBV, gene expression phenotypes, and disease is important and may help in developing antiviral therapy. This study examines potential causes of the enhanced lytic replicative properties of M81 EBV isolated from a nasopharyngeal carcinoma (NPC) patient and provides new evidence for the role of the BALF5 gene 3′ UTR sequence in DNA polymerase protein expression during lytic replication. Variation in the gene structure of the DNA polymerase gene may therefore contribute to lytic virus reactivation and pathogenesis.

scholarly journals The Epstein-Barr Virus SM Protein Is Functionally Similar to ICP27 from Herpes Simplex Virus in Viral Infections

2002 ◽  
Vol 76 (18) ◽  
pp. 9420-9433 ◽  
Author(s):  
Julie L. Boyer ◽  
Sankar Swaminathan ◽  
Saul J. Silverstein
Keyword(s):  
Gene Expression ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Epstein Barr Virus ◽  
Common Mechanism ◽  
Barr Virus ◽  
Epstein Barr ◽  
Simplex Virus ◽  
Sm Protein ◽  
Hsv 1

ABSTRACT The herpes simplex virus type 1 (HSV-1) ICP27 protein is an essential RNA-binding protein that shuttles between the nucleus and cytoplasm to increase the cytoplasmic accumulation of viral late mRNAs. ICP27 homologs have been identified in each of the herpesvirus subfamilies, and accumulating evidence indicates that homologs from the gammaherpesvirus subfamily function similarly to ICP27. In particular, the Epstein-Barr virus (EBV) SM protein posttranscriptionally regulates gene expression, binds RNA in vitro and in vivo, and shuttles between the nucleus and cytoplasm. To determine if these two proteins function through a common mechanism, the ability of EBV SM to complement the growth defect of an HSV-1 ICP27-null virus was examined in a transient-expression assay. ICP27 stimulated the growth of the null mutant more efficiently than did SM, but the ability of SM to compensate for the ICP27 defects suggests conservation of common functions. To assay for complementation in the context of a viral infection, the growth properties of an HSV recombinant expressing SM in an ICP27-null background were analyzed. SM stimulated growth of the recombinant, although this growth was reduced by comparison to that of an ICP27-expressing virus. By contrast, an HSV recombinant expressing an SM mutant allele defective for transactivation activity and nucleocytoplasmic shuttling did not grow at all. These results suggest that SM and ICP27 may regulate gene expression through a common pathway that is evolutionarily conserved in herpesviruses.

Nelfinavir Activates Epstein-Barr Virus and Kaposi's Sarcoma Herpesvirus Lytic Cycle by Inducing ER Stress

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5011-5011
Author(s):  
Courtney Shirley ◽  
Nene Kalu ◽  
Richard F Ambinder
Keyword(s):  
Gene Expression ◽  
Er Stress ◽  
Kaposi's Sarcoma ◽  
Epstein Barr Virus ◽  
Primary Effusion Lymphoma ◽  
Burkitt’S Lymphoma ◽  
Burkitt's Lymphoma ◽  
Barr Virus ◽  
Virion Production ◽  
Epstein Barr

Abstract Abstract 5011 Epstein-Barr virus (EBV) and Kaposi's Sarcoma herpesvirus (KSHV) are associated with lymphomas and other malignancies. We previously demonstrated that the proteasome inhibitor, bortezomib, leads to ER stress, induction of the unfolded protein response (UPR), and activation of EBV lytic gene expression.1 Here we investigate nelfinavir, an HIV protease inhibitor that has been reported to induce the UPR.2 Nelfinavir treatment of EBV Burkitt's lymphoma (BL) and KSHV primary effusion lymphoma (PEL) cell lines resulted in changes indicative of ER stress: elevated levels of ATF4, XBP1(s), and CHOP10 (Figure 1), as well as the EBV and KSHV immediate early proteins ZTA and RTA (Figure 2), respectively. The appearance of these UPR markers preceded expression of viral lytic RNAs. Regulated knockdown of Bip, an ER-stress sensor and activator of the UPR, by shRNA inhibited viral lytic RNA induction. These effects were observed using drug levels at or just above the levels achieved with standard clinical dosing of nelfinavir. Gantt et al. have reported that nelfinavir inhibits herpes virion production, including KSHV, in vitro.3 The ability to activate viral gene expression in combination with inhibition of virion production may identify nelfinavir as an especially promising agent for virus-targeted cancer therapies.Figure 1Figure 1. Figure 2Figure 2. Analysis of nelfinavir (NFV) induced ER stress markers by RT-qPCR in Burkitt's lymphoma (Akata) and primary effusion lymphoma (BCBL-1) cells. Analysis of nelfinavir (NFV) induced lytic activation by RT-qPCR in Burkitt's lymphoma (Akata) and primary effusion lymphoma (BCBL-1) cells. Disclosures: No relevant conflicts of interest to declare.

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.

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