The Epstein–Barr virus nuclear protein SM is both a post-transcriptional inhibitor and activator of gene expression

Ruvolo, Vivian; Wang, Eryu; Boyle, Sarah; Swaminathan, Sankar

doi:10.1073/pnas.95.15.8852

Cited by 66 publications

(163 citation statements)

References 53 publications

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“…SM binds EBV RNA and affects RNA stability (15)(16)(17)(18)(19). Although it preferentially enhances accumulation of some EBV mRNAs, SM action likely depends on inherent characteristics of inefficiently expressed RNAs, such as stability or nuclear exportability (8,9).…”

Section: Discussionmentioning

confidence: 99%

Spironolactone blocks Epstein–Barr virus production by inhibiting EBV SM protein function

Verma

Thompson

Swaminathan

2016

Proc. Natl. Acad. Sci. U.S.A.

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Clinically available drugs active against Epstein-Barr virus (EBV) and other human herpesviruses are limited to those targeting viral DNA replication. To identify compounds directed against other steps in the viral life cycle, we searched for drugs active against the EBV SM protein, which is essential for infectious virus production. SM has a highly gene-specific mode of action and preferentially enhances expression of several late lytic cycle EBV genes. Here we demonstrate that spironolactone, a mineralocorticoid receptor antagonist approved for clinical use, inhibits SM function and infectious EBV production. Expression of EBV viral capsid antigen is highly SM dependent, and spironolactone inhibits viral capsid antigen synthesis and capsid formation, blocking EBV virion production at a step subsequent to viral DNA replication. In addition, spironolactone inhibits expression of other SM-dependent genes necessary for infectious virion formation. We further demonstrate that molecules structurally related to spironolactone with similar antimineralocorticoid blocking activity do not inhibit EBV production. These findings pave the way for development of antiherpesvirus drugs with new mechanisms of action directed against SM and homologous essential proteins in other herpesviruses. . EBV infects the majority of humans worldwide and establishes a persistent, lifelong latent infection in memory B cells. Occasional reactivation from latency occurs, and EBV enters a lytic phase of replication, during which a tightly regulated series of lytic genes is expressed, culminating in lysis of the host cell and release of infectious viral progeny. All herpesviruses share these abilities to establish asymptomatic latent infection and reactivate intermittently.All herpesviruses also express a regulatory protein early in lytic replication that is essential for efficient gene expression and infectious virion production. The EBV member of this family, which includes herpes simplex virus ICP27, cytomegalovirus (CMV) UL69, and Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57, is known as SM (2). SM acts posttranscriptionally to enhance accumulation of many lytic EBV transcripts. SM activity is highly gene-specific and preferentially enhances expression of several late lytic transcripts, including those encoding the major viral capsid antigen (VCA) and gp350, which is required for infection of B lymphocytes (3-5). SM and its homologs in other herpesviruses may also enhance transcription and translation of specific viral genes (6).Currently, all available antiherpesvirus drugs target viral DNA polymerases and are usually highly effective, but toxicity and development of resistance limits their use (7). To discover potential novel therapeutic compounds, we applied a cell-based screening assay to identify compounds that would specifically target SM function (8). Using this assay, spironolactone (SPR), a drug in clinical use for over 50 y, was identified as inhibiting SM function and EBV virion production. SPR is an aldosterone antagonis...

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Section: Discussionmentioning

confidence: 99%

Spironolactone blocks Epstein–Barr virus production by inhibiting EBV SM protein function

Verma

Thompson

Swaminathan

2016

Proc. Natl. Acad. Sci. U.S.A.

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Section: Methodsmentioning

confidence: 99%

“…SM cDNA cloned in the cytomegalovirus (CMV) promoter-driven vector pCDNA3 has been described previously (40). The cDNA cloned in reverse orientation in pCDNA3 (aSM) used as a control plasmid in some transfections has also been described previously (40). The bait plasmid for the yeast two-hybrid assay was constructed by insertion of SM cDNA lacking RXP repeat domains (SM⌬RXP) (37) in frame with the DNA binding domain of GAL4 in the pAS2 vector (Clontech).…”

Section: Methodsmentioning

confidence: 99%

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Epstein-Barr Virus (EBV) SM Protein Induces and Recruits Cellular Sp110b To Stabilize mRNAs and Enhance EBV Lytic Gene Expression

Nicewonger

Suck

Bloch

et al. 2004

J Virol

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Promyelocytic leukemia protein (PML) nuclear bodies or nuclear domain 10s (ND10s) are multiprotein nuclear structures implicated in transcriptional and posttranscriptional gene regulation that are disrupted during replication of many DNA viruses. Interferon increases the size and number of PML nuclear bodies and stimulates transcription of several genes encoding PML nuclear body proteins. Moreover, some PML nuclear body proteins colocalize at sites of viral DNA synthesis and transcription. In this study, the relationship between lytic Epstein-Barr virus (EBV) replication and Sp110b, a PML nuclear body protein, was investigated. Sp110b is shown to physically and functionally interact with the EBV protein SM. SM is expressed early in the EBV replicative cycle and posttranscriptionally increases the level of target EBV lytic transcripts. SM bound to Sp110b via two distinct sites in Sp110b in an RNA-independent manner. SM also specifically induced expression of Sp110b during lytic EBV replication and in several cell types. Exogenous expression of Sp110b synergistically enhanced SM-mediated accumulation of intronless and lytic viral transcripts. This synergistic effect was shown to be promoter independent, posttranscriptional, and the result of increased stabilization of target transcripts. Finally, inhibiting Sp110b expression decreased accumulation of an SM-responsive lytic EBV transcript in EBV-infected cells. These findings imply that SM induces Sp110b expression, binds to Sp110b, and utilizes the recruited Sp110b protein to increase the stability of lytic EBV transcripts, indicating that Sp110b is a component of the cellular machinery that EBV utilizes to enhance lytic EBV replication.The Epstein-Barr virus (EBV) nuclear protein SM is expressed early after entry of EBV into the lytic cycle of replication and is essential for EBV virion production (12,19). SM increases the accumulation of several viral intronless lytic mRNA transcripts and specific cellular transcripts in both the nucleus and cytoplasm (38,40,42). SM also inhibits splicing and inhibits expression of the majority of cellular genes (38, 39). Thus, SM affects multiple cellular pathways involved in RNA processing and transport. In order to further elucidate its mechanism of action, we used the yeast two-hybrid assay to identify additional cellular proteins that interact with SM. During this investigation, we identified the promyelocytic leukemia protein (PML) nuclear body protein Sp110b, expressed from a splicing variant of Sp110 (4, 25), as a potential SM-interacting protein.PML nuclear bodies (also known as nuclear domain 10 proteins [ND10s]) are dynamic nuclear structures composed of numerous proteins, some of which localize to the PML nuclear body under specific environmental conditions (for review, see reference 13, 30). Their function is still largely unknown, but many PML nuclear body proteins, including Sp110, have been implicated in regulation of gene transcription. The number of PML nuclear bodies in the nucleus increases in response to hea...

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“…It is abundant in nuclei of KSHV-infected B cells, has persistent expression into the late viral cycle, and bears stretches of U1 [63] and a region complementary to U12 RNAs [64,65], implying that it may modulate the recognition of splice sites in spliceosome-mediated RNA splicing (Figure 4). KSHV ORF57 is a split gene (Table 1), and during lytic replication encodes a nuclear-protein that is a homologue of HSV ICP27 [66,67], HVS ORF57 [68][69][70], EBV SM protein [71,72], human CMV UL69 [73] and VZV ORF4 [74]. Among these, ICP27 is a prototype protein that inhibits RNA splicing [75][76][77] and mediates viral intronless RNA export [78].…”

Section: Mechanisms Possibly Involved In the Regulation Of Kshv Rna Smentioning

confidence: 99%

Split genes and their expression in Kaposi's sarcoma‐associated herpesvirus

Zheng

2003

Reviews in Medical Virology

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SUMMARYA split or interrupted gene is defined as a gene consisting of introns and exons. Removal (splicing) of the intron (s) from a primary transcript (pre-mRNA) is an essential process to create a mRNA. Initial assignment of a potential protein coding region in KSHV genome was based on initiation codon context and predicted protein size larger than 100 amino acids, but the gene discontinuity was disregarded. Experimental investigation of the assigned ORFs has demonstrated that there are up to 25 split genes, more than one fourth of the total KSHV genes described in KSHV genome. This includes the genes involved in all phases (latent, immediate early, early and late) of KSHV infection. The complexity of a split gene expression depends upon the availability of a proximal promoter and polyadenylation (pA) signal. Sharing a single promoter or a single pA signal by two or three genes is not uncommon in the expression of KSHV split genes and the resulting transcripts are usually polycistronic. Among those of KSHV split genes, 15 genes express a bicistronic or tricistronic RNA and 10 genes express a monocistronic RNA. Alternative RNA splicing could happen in a particular pre-mRNA due to intron or exon inclusion or skipping or the presence of an alternative 5′ ss or 3′ ss. This may, respectively, result in at least 8 species of K8 and 14 species of K15 transcripts. This appears to be related to cell differentiation and stages of the virus infection, presumably involving in viral cis elements and trans splicing factors.

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The Epstein–Barr virus nuclear protein SM is both a post-transcriptional inhibitor and activator of gene expression

Cited by 66 publications

References 53 publications

Spironolactone blocks Epstein–Barr virus production by inhibiting EBV SM protein function

Spironolactone blocks Epstein–Barr virus production by inhibiting EBV SM protein function

Epstein-Barr Virus (EBV) SM Protein Induces and Recruits Cellular Sp110b To Stabilize mRNAs and Enhance EBV Lytic Gene Expression

Split genes and their expression in Kaposi's sarcoma‐associated herpesvirus

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