Comparative mechanistic studies of de novo RNA synthesis by flavivirus RNA-dependent RNA polymerases

Selisko, Barbara; Dutartre, Hélène; Guillemot, Jean‐Claude; Debarnot, Claire; Benarroch, Delphine; Khromykh, Alexander A.; Desprès, Philippe; Egloff, Marie‐Pierre; Canard, Bruno

doi:10.1016/j.virol.2006.03.026

Cited by 112 publications

(161 citation statements)

References 48 publications

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“…Using a poly(C) RNA as template, the recombinant NS5pol showed high RNA synthesis activity, in agreement with previous reports (Selisko et al 2006). RNA homopolymers, like poly(C), are efficient but nonspecific templates for many viral RdRps.…”

Section: An Rna Element Present At the 5ј End Of DV Genome Promotes Ssupporting

confidence: 91%

A 5′ RNA element promotes dengue virus RNA synthesis on a circular genome

Filomatori¹,

Lodeiro²,

Álvarez³

et al. 2006

Genes Dev.

331

430

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The mechanisms of RNA replication of plus-strand RNA viruses are still unclear. Here, we identified the first promoter element for RNA synthesis described in a flavivirus. Using dengue virus as a model, we found that the viral RdRp discriminates the viral RNA by specific recognition of a 5 element named SLA. We demonstrated that RNA-RNA interactions between 5 and 3 end sequences of the viral genome enhance dengue virus RNA synthesis only in the presence of an intact SLA. We propose a novel mechanism for minus-strand RNA synthesis in which the viral polymerase binds SLA at the 5 end of the genome and reaches the site of initiation at the 3 end via long-range RNA-RNA interactions. These findings provide an explanation for the strict requirement of dengue virus genome cyclization during viral replication.[Keywords: Flavivirus; RNA-dependent RNA polymerase; RNA cyclization; viral RNA synthesis; AFM] Supplemental material is available at http://www.genesdev.org.

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Section: An Rna Element Present At the 5ј End Of DV Genome Promotes Ssupporting

confidence: 91%

A 5′ RNA element promotes dengue virus RNA synthesis on a circular genome

Filomatori¹,

Lodeiro²,

Álvarez³

et al. 2006

Genes Dev.

331

430

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“…RdRp activity of WNV KUN NS5 was monitored by a test using a homopolymeric template poly(rC) and tritium-labelled GTP followed by filter-binding and liquid scintillation counting. Reactions were set up by preparing premixes which contained RdRp buffer and recombinant WNV KUN NS5 RdRp domain as described by Selisko et al (2006). For inhibition assays, supernatants of mAb hybridoma cultures (serum-free medium, Gibco; stabilized with 0.02 % azide) or dilutions thereof (1 : 5, 1 : 10 or 1 : 25) were added to the premixes.…”

Section: Methodsmentioning

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%

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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“…Their RTPase function is provided by nonstructural protein NS3 (Bartelma & Padmanabhan, 2002; Benarroch et al, 2004b), and a viral GTase has not yet been identified. Interestingly, both N7-MTase and 29O-MTase activities are located in the same protein, the N-terminal domain of protein NS5 (Egloff et al, 2002;Ray et al, 2006;Zhou et al, 2007), which also bears the RNA-dependent RNA polymerase function in the C-terminal domain (Selisko et al, 2006;Yap et al, 2007). Many flavivirus MTase domains have been characterized structurally (Assenberg et al, 2007;Bollati et al, 2009;Egloff et al, 2002;Geiss et al, 2009;Mastrangelo et al, 2007;Zhou et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

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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Comparative mechanistic studies of de novo RNA synthesis by flavivirus RNA-dependent RNA polymerases

Cited by 112 publications

References 48 publications

A 5′ RNA element promotes dengue virus RNA synthesis on a circular genome

A 5′ RNA element promotes dengue virus RNA synthesis on a circular genome

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

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