Structure and Function of Flavivirus NS5 Methyltransferase

Zhou, Yangsheng; Ray, Debashish; Zhao, Yiwei; Dong, Hansong; Ren, Suping; Li, Zhong; Guo, Yi; Bernard, Kristen A.; Shi, Pei Yong; Li, Hongmin

doi:10.1128/jvi.02704-06

Cited by 346 publications

(531 citation statements)

References 45 publications

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“…This indicated that 5H1 bound to a linear amino acid sequence (continuous epitope). This epitope was mapped to a 13 aa stretch (residues 41-53) that forms a helix-turn motif in the crystal structure of the MTase domain (Zhou et al, 2007). This region of NS5 is of particular interest in the context of the predicted structure of the full-length NS5 protein.…”

Section: Discussionmentioning

confidence: 99%

“…Thus, the structure of the full-length molecule is required to provide a more accurate model for functional analysis and drug design. While recent studies have elucidated separate crystal structures for the MTase and RdRp domains of WNV NS5 (Malet et al, 2007;Zhou et al, 2007), apparent instability of the full-length recombinant protein during purification precluded crystallization of the entire molecule. A possible solution to this dilemma is co-purification and co-crystallization of NS5 in the presence of a monoclonal antibody (mAb) that binds to both MTase and RdRp domains and stabilizes the full-length protein.…”

Section: Introductionmentioning

confidence: 99%

“…NS5 comprises an N-terminal methyltransferase (MTase) domain that catalyses both guanine N-7 and ribose 29-O methylations during viral RNA cap formation (Zhou et al, 2007) and a C-terminal domain that harbours the RNA-dependent RNA polymerase (RdRp) (Tan et al, 1996;Guyatt et al, 2001;Nomaguchi et al, 2004;Selisko et al, 2006). The crystal structure of the WNV RdRp revealed classic RdRp structure bearing palm, thumb and finger subdomains (Malet et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

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Monoclonal antibodies to the West Nile virus NS5 protein map to linear and conformational epitopes in the methyltransferase and polymerase domains

Hall

Tan

Selisko

et al. 2009

Journal of General Virology

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The West Nile virus (WNV) NS5 protein contains a methyltransferase (MTase) domain involved in RNA capping and an RNA-dependent RNA polymerase (RdRp) domain essential for virus replication. Crystal structures of individual WNV MTase and RdRp domains have been solved; however, the structure of full-length NS5 has not been determined. To gain more insight into the structure of NS5 and interactions between the MTase and RdRp domains, we generated a panel of seven monoclonal antibodies (mAbs) to the NS5 protein of WNV (Kunjin strain) and mapped their binding sites using a series of truncated NS5 proteins and synthetic peptides. Binding sites of four mAbs (5D4, 4B6, 5C11 and 6A10) were mapped to residues 354-389 in the fingers subdomain of the RdRp. This is consistent with the ability of these mAbs to inhibit RdRp activity in vitro and suggests that this region represents a potential target for RdRp inhibitors. Using a series of synthetic peptides, we also identified a linear epitope (bound by mAb 5H1) that mapped to a 13 aa stretch surrounding residues 47 and 49 in the MTase domain, a region predicted to interact with the palm subdomain of the RdRp. The failure of one mAb (7G6) to bind both N-and Cterminally truncated NS5 recombinants indicates that the antibody recognizes a conformational epitope that requires the presence of residues in both the MTase and RdRp domains. These data support a structural model of the full-length NS5 molecule that predicts a physical interaction between the MTase and the RdRp domains.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Monoclonal antibodies to the West Nile virus NS5 protein map to linear and conformational epitopes in the methyltransferase and polymerase domains

Hall

Tan

Selisko

et al. 2009

Journal of General Virology

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“…The assay was highly sensitive to inhibition, as shown by the low IC 50 values of the co-product AdoHcy and the AdoMet analogue sinefungin. Interestingly, mutational analysis of NS5MTase WNV within the viral genome has shown that the 29O-MTase activity is important for WNV reproduction (Zhou et al, 2007). Our 29O-MTase assay will be useful for screening and characterizing potential inhibitors, as already demonstrated by the identification of two flavivirus MTase inhibitors (Luzhkov et al, 2007;Milani et al, 2009).…”

mentioning

confidence: 94%

Biochemical characterization of the (nucleoside-2'O)-methyltransferase activity of dengue virus protein NS5 using purified capped RNA oligonucleotides 7MeGpppACn and GpppACn

Selisko

Peyrane

Canard

et al. 2009

Journal of General Virology

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The flavivirus RNA genome contains a conserved cap-1 structure, 7Me GpppA 29OMe G, at the 59 end. Two mRNA cap methyltransferase (MTase) activities involved in the formation of the cap, the (guanine-N7)-and the (nucleoside-29O)-MTases (29O-MTase), reside in a single domain of nonstructural protein NS5 (NS5MTase). This study reports on the biochemical characterization of the 29O-MTase activity of NS5MTase of dengue virus (NS5MTase DV ) using purified, short, capped RNA substrates ( 7Me GpppAC n or GpppAC n ). NS5MTase DV methylated both types of substrate exclusively at the 29O position. The efficiency of 29O-methylation did not depend on the methylation of the N7 position. Using 7Me GpppAC n and GpppAC n substrates of increasing chain lengths, it was found that both NS5MTase DV 29O activity and substrate binding increased before reaching a plateau at n55. Thus, the cap and 6 nt might define the interface providing efficient binding of enzyme and substrate. K m values for 7Me GpppAC 5 and the co-substrate S-adenosyl-Lmethionine (AdoMet) were determined (0.39 and 3.26 mM, respectively). As reported for other AdoMet-dependent RNA and DNA MTases, the 29O-MTase activity of NS5MTase DV showed a low turnover of 3.25¾10 "4 s "1 . Finally, an inhibition assay was set up and tested on GTP and AdoMet analogues as putative inhibitors of NS5MTase DV , which confirmed efficient inhibition by the reaction product S-adenosyl-homocysteine (IC 50 0.34 mM) and sinefungin (IC 50 0.63 mM), demonstrating that the assay is sufficiently sensitive to conduct inhibitor screening and characterization assays. INTRODUCTIONMost eukaryotic and viral mRNAs are modified by the addition of a cap structure at the 59 end. The mRNA cap consists of an N7-methylguanosine linked to the first transcribed nucleotide by a 59-59 triphosphate bridge (cap-0 structure, 7Me GpppN) (Shuman, 2001). Methylation of the 29O positions of the ribose of the first or the second transcribed nucleotide leads to a cap-1 ( 7Me GpppN 29OMe ) or cap-2 ( 7Me GpppN 29OMe N 29OMe ) structure, respectively. The cap structure stabilizes mRNA and plays a vital role in its translation by controlling mRNA splicing, its transport across the nuclear membrane and its recognition by the eIF4E translation factor (Furuichi & Shatkin, 2000). In eukaryotic cells, the acquisition of the cap-0 structure is a co-transcriptional event. RNA capping implies three steps in which the 59 triphosphate end of RNA is hydrolysed to a 59 diphosphate by an RNA triphosphatase (RTPase), then capped with GMP by a guanylyltransferase (GTase) and finally methylated at the N7 position of the guanine by an S-adenosyl-L-methionine (AdoMet)-dependent (guanine-N7)-methyltransferase (N7-MTase) (Gu & Lima, 2005). In contrast to mRNAs of lower eukaryotes, which bear a cap-0 structure, the mRNAs of higher eukaryotes acquire a cap-1 structure via the action of a (nucleoside-29O)-MTase (29O-MTase) (Langberg & Moss, 1981). Compared with N7 methylation, this mRNA methylation step seems to be less important for binding, tr...

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“…By homology with other Flavivirus members, the catalytic region of the enzyme is located within the C-terminal portion of the NS5 protein. Additionally, the NS5 protein also contains methyltransferase activity in the N-terminal portion of the NS5 protein, which is involved in the capping of plus-strand genomic RNA (10)(11)(12). The overall architecture of the ZIKV RdRp most likely resembles the canonical right-hand conformation, consisting of fingers, palm, and thumb subdomains, that has been typical for all known polymerase structures and is similar to that reported for the RdRps of other members of the Flaviviridae family, including hepatitis C virus (HCV) and DFV.…”

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confidence: 99%

Analysis of Ribonucleotide 5′-Triphosphate Analogs as Potential Inhibitors of Zika Virus RNA-Dependent RNA Polymerase by Using Nonradioactive Polymerase Assays

Bluemling

Collop

et al. 2017

Antimicrob Agents Chemother

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Zika virus (ZIKV) is an emerging human pathogen that is spreading rapidly through the Americas and has been linked to the development of microcephaly and to a dramatically increased number of Guillain-Barré syndrome cases. Currently, no vaccine or therapeutic options for the prevention or treatment of ZIKV infections exist. In the study described in this report, we expressed, purified, and characterized full-length nonstructural protein 5 (NS5) and the NS5 polymerase domain (NS5pol) of ZIKV RNA-dependent RNA polymerase. Using purified NS5, we developed an in vitro nonradioactive primer extension assay employing a fluorescently labeled primertemplate pair. Both purified NS5 and NS5pol can carry out in vitro RNA-dependent RNA synthesis in this assay. Our results show that Mn 2ϩ is required for enzymatic activity, while Mg 2ϩ is not. We found that ZIKV NS5 can utilize single-stranded DNA but not double-stranded DNA as a template or a primer to synthesize RNA. The assay was used to compare the efficiency of incorporation of analog 5=-triphosphates by the ZIKV polymerase and to calculate their discrimination versus that of natural ribonucleotide triphosphates (rNTPs). The 50% inhibitory concentrations for analog rNTPs were determined in an alternative nonradioactive coupled-enzyme assay. We determined that, in general, 2=-C-methyl-and 2=-C-ethynyl-substituted analog 5=-triphosphates were efficiently incorporated by the ZIKV polymerase and were also efficient chain terminators. Derivatives of these molecules may serve as potential antiviral compounds to be developed to combat ZIKV infection. This report provides the first characterization of ZIKV polymerase and demonstrates the utility of in vitro polymerase assays in the identification of potential ZIKV inhibitors.

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Structure and Function of Flavivirus NS5 Methyltransferase

Cited by 346 publications

References 45 publications

Monoclonal antibodies to the West Nile virus NS5 protein map to linear and conformational epitopes in the methyltransferase and polymerase domains

Monoclonal antibodies to the West Nile virus NS5 protein map to linear and conformational epitopes in the methyltransferase and polymerase domains

Biochemical characterization of the (nucleoside-2'O)-methyltransferase activity of dengue virus protein NS5 using purified capped RNA oligonucleotides 7MeGpppACn and GpppACn

Analysis of Ribonucleotide 5′-Triphosphate Analogs as Potential Inhibitors of Zika Virus RNA-Dependent RNA Polymerase by Using Nonradioactive Polymerase Assays

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