Molecular basis for specific viral RNA recognition and 2′-O-ribose methylation by the dengue virus nonstructural protein 5 (NS5)

Zhao, Yongxiang; Soh, Tingjin Sherryl; Lim, Siew Pheng; Chung, Ka Yan; Swaminathan, Kunchithapadam; Vasudevan, Subhash G.; Shi, Pei–Yong; Lescar, Julien; Luo, Dahai

doi:10.1073/pnas.1514978112

Cited by 90 publications

(90 citation statements)

References 45 publications

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“…The RNA polymerase structures from WNV (PDB #2HFZ)34, JEV (PDB #4K6M)35, DENV (PDB #5DTO)36 and HCV (PDB #4WTG)37 share 72, 70, 68, and 25% sequence identity, respectively, with the orthologous ZIKV enzyme. Despite its relatively low sequence identity to ZIKV, the HCV enzyme structure is complexed with sofosbuvir, and the amino acids residues that interact with the drug are highly conserved (approximately 80%) among the members of the Flaviviridae family37.…”

Section: Resultsmentioning

confidence: 99%

The clinically approved antiviral drug sofosbuvir inhibits Zika virus replication

Sacramento¹,

Melo²,

Freitas³

et al. 2017

Sci Rep

185

172

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Zika virus (ZIKV) is a member of the Flaviviridae family, along with other agents of clinical significance such as dengue (DENV) and hepatitis C (HCV) viruses. Since ZIKV causes neurological disorders during fetal development and in adulthood, antiviral drugs are necessary. Sofosbuvir is clinically approved for use against HCV and targets the protein that is most conserved among the members of the Flaviviridae family, the viral RNA polymerase. Indeed, we found that sofosbuvir inhibits ZIKV RNA polymerase, targeting conserved amino acid residues. Sofosbuvir inhibited ZIKV replication in different cellular systems, such as hepatoma (Huh-7) cells, neuroblastoma (SH-Sy5y) cells, neural stem cells (NSC) and brain organoids. In addition to the direct inhibition of the viral RNA polymerase, we observed that sofosbuvir also induced an increase in A-to-G mutations in the viral genome. Together, our data highlight a potential secondary use of sofosbuvir, an anti-HCV drug, against ZIKV.

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Section: Resultsmentioning

confidence: 99%

The clinically approved antiviral drug sofosbuvir inhibits Zika virus replication

Sacramento¹,

Melo²,

Freitas³

et al. 2017

Sci Rep

185

172

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“…The sulfate molecule bound via a cluster formed by arginines Arg43, Arg47, Arg63, and Arg90 (Fig. 2D) is closely located in between two phosphodiester groups of bound RNA in the structure described by Zhao et al (25) (namely P3 U and P4 U from chain B, with distances for S-P3 of 3 Å and for S-P4 of 4 Å). In this cluster formed by arginines Arg43, Arg47, Arg63, and Arg90, the main chain of ZIKV MTase (chain A) and DENV MTase (chain B [23]) structures overlap perfectly.…”

Section: Discussionmentioning

confidence: 77%

“…The homologous arginine residues were already reported to play a key role in the recruitment of RNA (25). Especially, residues corresponding to ZIKV MTase Arg63 and Arg90 (Arg57 and Arg84 in DENV, respectively) bind to the phosphate groups of the RNA during the 2=-O-methylation (25). The sulfate molecule bound via a cluster formed by arginines Arg43, Arg47, Arg63, and Arg90 (Fig.…”

Section: Discussionmentioning

confidence: 94%

“…The MTase core structure closely resembles the catalytic domain of other viral and cellular SAMdependent MTases with a conserved SAM binding site in close vicinity of the conserved K-D-K-E catalytic tetrad, the latter being a signature of 2=-O-MTases. In addition, structures determined in the presence of cap analogues, GTP or RNA reveal a cap binding pocket where the cap guanosine is held during 2=-O-methylation by stacking interactions with a phenylalanine (F24 residue in the DENV NS5 sequence) and an RNA binding zone where the RNA chain downstream of the cap structure is accommodated by a basic groove (4,6,7,(22)(23)(24)(25).…”

mentioning

confidence: 99%

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Zika Virus Methyltransferase: Structure and Functions for Drug Design Perspectives

Coutard

Barral

Lichière

et al. 2017

J Virol

122

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The Flavivirus Zika virus (ZIKV) is the causal agent of neurological disorders like microcephaly in newborns or Guillain-Barre syndrome. Its NS5 protein embeds a methyltransferase (MTase) domain involved in the formation of the viral mRNA cap. We investigated the structural and functional properties of the ZIKV MTase. We show that the ZIKV MTase can methylate RNA cap structures at the N-7 position of the cap, and at the 2=-O position on the ribose of the first nucleotide, yielding a cap-1 structure. In addition, the ZIKV MTase methylates the ribose 2=-O position of internal adenosines of RNA substrates. The crystal structure of the ZIKV MTase determined at a 2.01-Å resolution reveals a crystallographic homodimer. One chain is bound to the methyl donor (S-adenosyl-L-methionine [SAM]) and shows a high structural similarity to the dengue virus (DENV) MTase. The second chain lacks SAM and displays conformational changes in the ␣X ␣-helix contributing to the SAM and RNA binding. These conformational modifications reveal a possible molecular mechanism of the enzymatic turnover involving a conserved Ser/Arg motif. In the second chain, the SAM binding site accommodates a sulfate close to a glycerol that could serve as a basis for structure-based drug design. In addition, compounds known to inhibit the DENV MTase show similar inhibition potency on the ZIKV MTase. Altogether these results contribute to a better understanding of the ZIKV MTase, a central player in viral replication and host innate immune response, and lay the basis for the development of potential antiviral drugs. IMPORTANCE The Zika virus (ZIKV) is associated with microcephaly in newborns,and other neurological disorders such as Guillain-Barre syndrome. It is urgent to develop antiviral strategies inhibiting the viral replication. The ZIKV NS5 embeds a methyltransferase involved in the viral mRNA capping process, which is essential for viral replication and control of virus detection by innate immune mechanisms. We demonstrate that the ZIKV methyltransferase methylates the mRNA cap and adenosines located in RNA sequences. The structure of ZIKV methyltransferase shows high structural similarities to the dengue virus methyltransferase, but conformational specificities highlight the role of a conserved Ser/Arg motif, which participates in RNA and SAM recognition during the reaction turnover. In addition, the SAM binding site accommodates a sulfate and a glycerol, offering structural information to initiate structure-based drug design. Altogether, these results contribute to a better understanding of the Flavivirus methyltransferases, which are central players in the virus replication.KEYWORDS methyltransferase, RNA processing, Zika virus, flavivirus, protein structure-function

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“…This is especially reflected in the low number of structures of proteins in complex with capped RNA (>1 nt) that have been determined to date. Currently, these structures include the human 2 ′ -O-ribose methyltransferase CMTr1 with a capped 4-mer that was produced by chemical coupling (Smietanski et al 2014), the 2 ′ -O-ribose methyltransferase of vaccinia virus with a capped 6-mer that was produced by in vitro transcription in the presence of a cap analog (Hodel et al 1998), the 2 ′ -O-ribose methyltransferase in the NS5 protein from dengue virus with an 8-mer cap-0 viral RNA that was produced using a cap analog (Zhao et al 2015), the dengue virus methyltransferase in complex with a 5 ′ -capped RNA 8-mer that was chemically synthesized (Yap et al 2010), and the innate immune receptor RIG-I that contains a chemically synthesized 24-nt-long capped hairpin RNA (Devarkar et al 2016). The high-resolution structural data available is thus confined to a small subset of the numerous enzymes and proteins that directly interact with the mRNA cap structure.…”

Section: Thementioning

confidence: 99%

A general method for rapid and cost-efficient large-scale production of 5′ capped RNA

Fuchs

Neu

Sprangers

2016

RNA

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The eukaryotic mRNA 5 ′ cap structure is indispensible for pre-mRNA processing, mRNA export, translation initiation, and mRNA stability. Despite this importance, structural and biophysical studies that involve capped RNA are challenging and rare due to the lack of a general method to prepare mRNA in sufficient quantities. Here, we show that the vaccinia capping enzyme can be used to produce capped RNA in the amounts that are required for large-scale structural studies. We have therefore designed an efficient expression and purification protocol for the vaccinia capping enzyme. Using this approach, the reaction scale can be increased in a cost-efficient manner, where the yields of the capped RNA solely depend on the amount of available uncapped RNA target. Using a large number of RNA substrates, we show that the efficiency of the capping reaction is largely independent of the sequence, length, and secondary structure of the RNA, which makes our approach generally applicable. We demonstrate that the capped RNA can be directly used for quantitative biophysical studies, including fluorescence anisotropy and highresolution NMR spectroscopy. In combination with 13 C-methyl-labeled S-adenosyl methionine, the methyl groups in the RNA can be labeled for methyl TROSY NMR spectroscopy. Finally, we show that our approach can produce both cap-0 and cap-1 RNA in high amounts. In summary, we here introduce a general and straightforward method that opens new means for structural and functional studies of proteins and enzymes in complex with capped RNA.

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Molecular basis for specific viral RNA recognition and 2′-O-ribose methylation by the dengue virus nonstructural protein 5 (NS5)

Cited by 90 publications

References 45 publications

The clinically approved antiviral drug sofosbuvir inhibits Zika virus replication

The clinically approved antiviral drug sofosbuvir inhibits Zika virus replication

Zika Virus Methyltransferase: Structure and Functions for Drug Design Perspectives

A general method for rapid and cost-efficient large-scale production of 5′ capped RNA

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