General and Target-Specific RNA Binding Properties of Epstein-Barr Virus SM Posttranscriptional Regulatory Protein

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

Thompson

Swaminathan

2016

Self Cite

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...

Section: Discussionmentioning

confidence: 99%

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

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

Thompson

Swaminathan

2016

Self Cite

“…The exact reasons that SM is essential for lytic replication and virus production have not been fully characterized. SM binds to RNA (7,8) and enhances accumulation of the mRNA transcripts from a wide variety of genes in cotransfection assays, but these results do not necessarily correlate with the dependence of EBV gene expression from the virus during reactivation from latency (9)(10)(11)(12). Similarly, the relative binding affinity of EBV transcripts to SM has been assessed by immunoprecipitation of SMbound mRNAs, and while preferential binding to specific transcripts was demonstrated, these have not been correlated to SM responsiveness during lytic replication (7).…”

Section: Importancementioning

confidence: 99%

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

Thompson

et al. 2016

J Virol

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Epstein-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. E pstein-Barr virus (EBV) SM protein is an essential regulatory protein expressed by EBV during the lytic phase of replication (for reviews, see references 1 and 2). SM protein is detectable after immediate early gene expression and prior to expression of other early gene products (3, 4). When the SM gene is insertionally inactivated in recombinant EBV genomes, the resultant virus is incapable of completing the lytic cycle and producing infectious virions (5, 6). The exact reasons that SM is essential for lytic replication and virus pro...

“…While ORF57 and SM appear to target specific transcripts, no clear SM/ORF57 binding site or structural motif in the mRNA has been shown to be responsible. Immunoprecipitation of SM from EBV-infected cells and qPCR analysis of SM-bound transcripts demonstrated preferential binding of SM to several EBV RNAs, but no common motif was identified (51). Similarly, specific binding of KSHV ORF57 to sites in the viral interleukin 6 (vIL-6) mRNA and PAN RNA has been demonstrated by crosslinking and immunoprecipitation of RNA targets (RNA-CLIP), but no generalizable ORF57 binding motif, such as those established for splicing factors, has been established (52,53).…”

Section: Discussionmentioning

confidence: 99%

Cell-Based Screening Assay for Antiviral Compounds Targeting the Ability of Herpesvirus Posttranscriptional Regulatory Proteins To Stabilize Viral mRNAs

Kim

Swaminathan

2013

J Virol

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Each human herpesvirus expresses a multifunctional regulatory protein that is essential for lytic viral replication. A cell-based assay targeting the function of these proteins was developed based on the finding that Epstein-Barr virus (EBV) SM and Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 stabilize specific target mRNAs. Both proteins facilitate the accumulation of lytic transcripts by incompletely characterized posttranscriptional mechanisms. SM and ORF57 exhibit target gene specificity and enhance the accumulation of certain EBV and KSHV mRNAs that are poorly expressed in their absence. Conversely, SMand ORF57-independent viral and cellular transcripts accumulate efficiently, and their expression does not respond to SM or ORF57. Fusion of an ORF57-responsive transcript to ORF57-independent transcripts demonstrated that ORF57 dependence is cis-dominant. EBV SM also enhanced the accumulation of such fused mRNA transcripts. These data suggest that the coding regions of specific viral transcripts confer instability even when fused to heterologous genes. The findings were used to develop a reporter assay that measures EBV SM function in rescuing the expression of poorly expressed transcripts by posttranscriptional mechanisms. The assay represents a method for the screening of small interfering RNAs (siRNAs) and compounds to investigate the mechanism of action of SM and its homologs and potentially to aid in the discovery of novel antiviral agents.