Yeast-based assays for the high-throughput screening of inhibitors of coronavirus RNA cap guanine-N7-methyltransferase

Sun, Yan; Wang, Zidao; Tao, Jiali; Wang, Yi; Wu, Andong; Yang, Ziwen; Wang, Kaimei; Shi, Liqiao; Chen, Yu; Guo, Deyin

doi:10.1016/j.antiviral.2014.02.002

Cited by 38 publications

(55 citation statements)

References 53 publications

(66 reference statements)

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“…This observation is corroborated by former biochemical data showing that the N7-methyl guanosine of cap structures is recognized by the eukaryotic translation initiation factor 4E (eIF4E) and participates in the initiation of viral mRNA translation into proteins (Case et al, 2016;Cougot et al, 2004). Accordingly, inhibitors blocking nsp14 N7-MTase activity have been identified by yeast based screening assay on SARS-CoV, and induced a potent antiviral effect demonstrating that nsp14 MTase activity is an attractive antiviral target (Sun et al, 2014). Whereas N7-MTase mutants are replication defective, 2 0 O-MTase mutants show limited effect on virus replication in cell culture but have an attenuated phenotype in animal models Menachery et al, 2014;Zhang et al, 2014;Züst et al, 2013).…”

Section: Introductionsupporting

confidence: 62%

Toward the identification of viral cap-methyltransferase inhibitors by fluorescence screening assay

et al. 2017

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Two highly pathogenic human coronaviruses associated with severe respiratory syndromes emerged since the beginning of the century. The severe acute respiratory syndrome SARS-coronavirus (CoV) spread first in southern China in 2003 with about 8000 infected cases in few months. Then in 2012, the Middle East respiratory syndrome (MERS-CoV) emerged from the Arabian Peninsula giving a still on-going epidemic associated to a high fatality rate. CoVs are thus considered a major health threat. This is especially true as no vaccine nor specific therapeutic are available against either SARS- or MERS-CoV. Therefore, new drugs need to be identified in order to develop antiviral treatments limiting CoV replication. In this study, we focus on the nsp14 protein, which plays a key role in virus replication as it methylates the RNA cap structure at the N7 position of the guanine. We developed a high-throughput N7-MTase assay based on Homogenous Time Resolved Fluorescence (HTRF) and screened chemical libraries (2000 compounds) on the SARS-CoV nsp14. 20 compounds inhibiting the SARS-CoV nsp14 were further evaluated by IC determination and their specificity was assessed toward flavivirus- and human cap N7-MTases. Our results reveal three classes of compounds: 1) molecules inhibiting several MTases as well as the dengue virus polymerase activity unspecifically, 2) pan MTases inhibitors targeting both viral and cellular MTases, and 3) inhibitors targeting one viral MTase more specifically showing however activity against the human cap N7-MTase. These compounds provide a first basis towards the development of more specific inhibitors of viral methyltransferases.

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Section: Introductionsupporting

confidence: 62%

Toward the identification of viral cap-methyltransferase inhibitors by fluorescence screening assay

et al. 2017

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“…Biochemical assays demonstrated that ATA inhibits both nsp14 N7-MTase and nsp16 2 -O-MTase activities . Recently, Sun et al (2014) developed a yeast-based high-throughput antiviral screening system to find novel anti-coronavirus drugs. This study confirmed that sinefungin significantly suppresses the growth of yeast cells complemented with nsp14 N7-MTase gene.…”

Section: The 2 -O-methyltransferase (2 -O-mtase)mentioning

confidence: 99%

“…This study confirmed that sinefungin significantly suppresses the growth of yeast cells complemented with nsp14 N7-MTase gene. However, sinefungin is not a specific inhibitor as it also inhibits yeast and human MTases (Sun et al, 2014).…”

Section: The 2 -O-methyltransferase (2 -O-mtase)mentioning

confidence: 99%

Insights into RNA synthesis, capping, and proofreading mechanisms of SARS-coronavirus

et al. 2014

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The successive emergence of highly pathogenic coronaviruses (CoVs) such as the Severe Acute Respiratory Syndrome (SARS-CoV) in 2003 and the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) in 2012 has stimulated a number of studies on the molecular biology. This research has provided significant new insight into functions and activities of the replication/transcription multi-protein complex. The latter directs both continuous and discontinuous RNA synthesis to replicate and transcribe the large coronavirus genome made of a single-stranded, positive-sense RNA of ∼30 kb. In this review, we summarize our current understanding of SARS-CoV enzymes involved in RNA biochemistry, such as the in vitro characterization of a highly active and processive RNA polymerase complex which can associate with methyltransferase and 3'-5' exoribonuclease activities involved in RNA capping, and RNA proofreading, respectively. The recent discoveries reveal fascinating RNA-synthesizing machinery, highlighting the unique position of coronaviruses in the RNA virus world.

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“…ATA was also shown to limit SARS-CoV replication in infected cells (He et al, 2004). In the yeast-△MTase trans-complementation assay mentioned earlier, micromolar concentrations of sinefungin were reported to effectively suppress the nsp14 N7-MTase activity of SARS-CoV, MHV, transmissible gastroenteritis virus (TGEV), and IBV (Sun et al, 2014). However, other compounds, such as ATA and AdoHcy, did not exert an inhibitory effect in the context of yeast cells.…”

Section: The Nsp14 N7-methyl Transferasementioning

confidence: 95%

The Nonstructural Proteins Directing Coronavirus RNA Synthesis and Processing

2016

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Coronaviruses are animal and human pathogens that can cause lethal zoonotic infections like SARS and MERS. They have polycistronic plus-stranded RNA genomes and belong to the order Nidovirales, a diverse group of viruses for which common ancestry was inferred from the common principles underlying their genome organization and expression, and from the conservation of an array of core replicase domains, including key RNA-synthesizing enzymes. Coronavirus genomes (~26-32 kilobases) are the largest RNA genomes known to date and their expansion was likely enabled by acquiring enzyme functions that counter the commonly high error frequency of viral RNA polymerases. The primary functions that direct coronavirus RNA synthesis and processing reside in nonstructural protein (nsp) 7 to nsp16, which are cleavage products of two large replicase polyproteins translated from the coronavirus genome. Significant progress has now been made regarding their structural and functional characterization, stimulated by technical advances like improved methods for bioinformatics and structural biology, in vitro enzyme characterization, and site-directed mutagenesis of coronavirus genomes. Coronavirus replicase functions include more or less universal activities of plus-stranded RNA viruses, like an RNA polymerase (nsp12) and helicase (nsp13), but also a number of rare or even unique domains involved in mRNA capping (nsp14, nsp16) and fidelity control (nsp14). Several smaller subunits (nsp7-nsp10) act as crucial cofactors of these enzymes and contribute to the emerging "nsp interactome." Understanding the structure, function, and interactions of the RNA-synthesizing machinery of coronaviruses will be key to rationalizing their evolutionary success and the development of improved control strategies.

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Yeast-based assays for the high-throughput screening of inhibitors of coronavirus RNA cap guanine-N7-methyltransferase

Cited by 38 publications

References 53 publications

Toward the identification of viral cap-methyltransferase inhibitors by fluorescence screening assay

Toward the identification of viral cap-methyltransferase inhibitors by fluorescence screening assay

Insights into RNA synthesis, capping, and proofreading mechanisms of SARS-coronavirus

The Nonstructural Proteins Directing Coronavirus RNA Synthesis and Processing

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