Biochemical and Structural Insights into the Mechanisms of SARS Coronavirus RNA Ribose 2′-O-Methylation by nsp16/nsp10 Protein Complex

Chen, Yu; Su, Ceyang; Ke, Manzhu; Jin, Xu; Xu, Lirong; Zhang, Zhou; Wu, Andong; Sun, Yan; Yang, Zhouning; Tien, Po; Ahola, Tero; Liang, Yi; Liu, Xinqi; Guo, Deyin

doi:10.1371/journal.ppat.1002294

Cited by 312 publications

(554 citation statements)

References 68 publications

(114 reference statements)

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“…CoVs replicate in the host cytoplasm and encode their own capping enzymes. Among the four capping enzymes involved in coronavirus m7GpppAm-cap formation, guanine-N7-methyltransferase (N7-MTase) was identified as nsp14 in our previous work using a yeast genetic system (Chen et al, 2009), and 2 0 -O-methyltransferase (2 0 -O-MTase) is formed by nsp16 with nsp10 as a cofactor (Chang et al, 2011;Chen et al, 2011;Decroly et al, 2011;Lugari et al, 2010). SARS-coronavirus nsp14 was previously characterized as a 3 0 -to 5 0 -exoribonuclease (ExoN) (Chen et al, 2007;Minskaia et al, 2006), and the N7-MTase domain was mapped to the carboxy-terminal part of the protein (Chen et al, 2009).…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2014

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The 5'-cap structure is a distinct feature of eukaryotic mRNAs and is important for RNA stability and protein translation by providing a molecular signature for the distinction of self or non-self mRNA. Eukaryotic viruses generally modify the 5'-end of their RNAs to mimic the cellular mRNA structure, thereby facilitating viral replication in host cells. However, the molecular organization and biochemical mechanisms of the viral capping apparatus typically differ from its cellular counterpart, which makes viral capping enzymes attractive targets for drug discovery. Our previous work showed that SARS coronavirus (SARS-CoV) non-structural protein 14 represents a structurally novel and unique guanine-N7-methyltransferase (N7-MTase) that is able to functionally complement yeast cellular N7-MTase. In the present study, we developed a yeast-based system for identifying and screening inhibitors against coronavirus N7-MTase using both 96-well and 384-well microtiter plates. The MTase inhibitors previously identified by in vitro biochemical assays were tested, and some, such as sinefungin, effectively suppressed N7-MTase in the yeast system. However, other compounds, such as ATA and AdoHcy, did not exert an inhibitory effect within a cellular context. These results validated the yeast assay system for inhibitor screening yet also demonstrated the difference between cell-based and in vitro biochemical assays. The yeast system was applied to the screening of 3000 natural product extracts, and three were observed to more potently inhibit the activity of coronavirus than human N7-MTase.

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Section: Introductionmentioning

confidence: 99%

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

et al. 2014

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“…These two polyproteins are cleaved into 16 mature replicase proteins, named as nonstructural proteins nsp1-16, which assemble the RTC located on a dedicated membrane surface (van Hemert et al, 2008). Of the 16 nsps, nsp14 was shown as exoribonuclease and (guanine-N7)-methyltransferase (N7-MTase) (Chen et al, 2007(Chen et al, , 2009Minskaia et al, 2006) and nsp16 as a 2 -O-methyltransferase (2 -O-MTase) (Chen et al, 2011;Decroly et al, 2008).…”

Section: Introductionmentioning

confidence: 99%

“…The crystal structure revealed that nsp10 belongs to a new class of zinc finger proteins and was proposed to be a viral transcription factor (Joseph et al, 2006;Su et al, 2006). It was also shown that SARS-CoV nsp16 acts as a 2 -O-MTase in complex with nsp10 to selectively 2 -O-methylate the cap-0 structure to cap-1 structure (m7GpppAm-RNA) Chen et al, 2011). Recently, we and Bruno Canard's group independently resolved the crystal structure of nsp10/nsp16 complex (Chen et al, 2011;Debarnot et al, 2011).…”

Section: Introductionmentioning

confidence: 99%

Short peptides derived from the interaction domain of SARS coronavirus nonstructural protein nsp10 can suppress the 2′-O-methyltransferase activity of nsp10/nsp16 complex

Chen

et al. 2012

Virus Research

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Coronaviruses are the etiological agents of respiratory and enteric diseases in humans and livestock, exemplified by the life-threatening severe acute respiratory syndrome (SARS) caused by SARS coronavirus (SARS-CoV). However, effective means for combating coronaviruses are still lacking. The interaction between nonstructural protein (nsp) 10 and nsp16 has been demonstrated and the crystal structure of SARS-CoV nsp16/10 complex has been revealed. As nsp10 acts as an essential trigger to activate the 2'-O-methyltransferase activity of nsp16, short peptides derived from nsp10 may have inhibitory effect on viral 2'-O-methyltransferase activity. In this study, we revealed that the domain of aa 65-107 of nsp10 was sufficient for its interaction with nsp16 and the region of aa 42-120 in nsp10, which is larger than the interaction domain, was needed for stimulating the nsp16 2'-O-methyltransferase activity. We further showed that two short peptides derived from the interaction domain of nsp10 could inhibit the 2'-O-methyltransferase activity of SARS-CoV nsp16/10 complex, thus providing a novel strategy and proof-of-principle study for developing peptide inhibitors against SARS-CoV.

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“…The N7-methylation must precede the 2 0 -O-methylation that is only observed on RNA carrying cap 0 structure (Bouvet et al, 2010). The crystal structure of SARS-CoV nsp16 was solved in complex with nsp10 (Chen et al, 2011;Decroly et al, 2011). Nsp16 fold follows the typical SAMdependent 2 0 -O-MTase fold, constituted by a central b-sheet of 7 strands surrounded by 5 a-helices ( Fig.…”

Section: The Nsp16 2 0 -O-mtasementioning

confidence: 99%

SARS-CoV ORF1b-encoded nonstructural proteins 12–16: Replicative enzymes as antiviral targets

et al. 2014

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The SARS (severe acute respiratory syndrome) pandemic caused ten years ago by the SARS-coronavirus (SARS-CoV) has stimulated a number of studies on the molecular biology of coronaviruses. This research has provided significant new insight into many mechanisms used by the coronavirus replication-transcription complex (RTC). The RTC directs and coordinates processes in order to replicate and transcribe the coronavirus genome, a single-stranded, positive-sense RNA of outstanding length (∼27-32kilobases). Here, we review the up-to-date knowledge on SARS-CoV replicative enzymes encoded in the ORF1b, i.e., the main RNA-dependent RNA polymerase (nsp12), the helicase/triphosphatase (nsp13), two unusual ribonucleases (nsp14, nsp15) and RNA-cap methyltransferases (nsp14, nsp16). We also review how these enzymes co-operate with other viral co-factors (nsp7, nsp8, and nsp10) to regulate their activity. These last ten years of research on SARS-CoV have considerably contributed to unravel structural and functional details of one of the most fascinating replication/transcription machineries of the RNA virus world. This paper forms part of a series of invited articles in Antiviral Research on "From SARS to MERS: 10years of research on highly pathogenic human coronaviruses".

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Biochemical and Structural Insights into the Mechanisms of SARS Coronavirus RNA Ribose 2′-O-Methylation by nsp16/nsp10 Protein Complex

Cited by 312 publications

References 68 publications

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

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

Short peptides derived from the interaction domain of SARS coronavirus nonstructural protein nsp10 can suppress the 2′-O-methyltransferase activity of nsp10/nsp16 complex

SARS-CoV ORF1b-encoded nonstructural proteins 12–16: Replicative enzymes as antiviral targets

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