An interaction between the methyltransferase and RNA dependent RNA polymerase domains of the West Nile virus NS5 protein

Tan, Cindy S. E.; Hobson‐Peters, Jody; Stoermer, Martin J.; Fairlie, David P.; Khromykh, Alexander A.; Hall, Roy A.

doi:10.1099/vir.0.054395-0

Cited by 17 publications

(18 citation statements)

References 20 publications

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“…Genetic interaction between NS5-MTase and NS5-Pol has been reported for DENV ( 21 ) and WNV ( 22 ). In addition, the interaction between recombinant WNV NS5-MTase and NS5-Pol was demonstrated in vitro ( 23 ). The NS5-Pol domain was shown to enhance the internal RNA methylation activity of the NS5-MTase domain in the context of the full-length NS5 proteins of DENV and WNV ( 24 ).…”

Section: Introductionmentioning

confidence: 99%

The methyltransferase domain of dengue virus protein NS5 ensures efficient RNA synthesis initiation and elongation by the polymerase domain

Potisopon

Priet

Collet

et al. 2014

Nucleic Acids Research

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Viral RNA-dependent RNA polymerases (RdRps) responsible for the replication of single-strand RNA virus genomes exert their function in the context of complex replication machineries. Within these replication complexes the polymerase activity is often highly regulated by RNA elements, proteins or other domains of multi-domain polymerases. Here, we present data of the influence of the methyltransferase domain (NS5-MTase) of dengue virus (DENV) protein NS5 on the RdRp activity of the polymerase domain (NS5-Pol). The steady-state polymerase activities of DENV-2 recombinant NS5 and NS5-Pol are compared using different biochemical assays allowing the dissection of the de novo initiation, transition and elongation steps of RNA synthesis. We show that NS5-MTase ensures efficient RdRp activity by stimulating the de novo initiation and the elongation phase. This stimulation is related to a higher affinity of NS5 toward the single-strand RNA template indicating NS5-MTase either completes a high-affinity RNA binding site and/or promotes the correct formation of the template tunnel. Furthermore, the NS5-MTase increases the affinity of the priming nucleotide ATP upon de novo initiation and causes a higher catalytic efficiency of the polymerase upon elongation. The complex stimulation pattern is discussed under the perspective that NS5 adopts several conformations during RNA synthesis.

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Section: Introductionmentioning

confidence: 99%

The methyltransferase domain of dengue virus protein NS5 ensures efficient RNA synthesis initiation and elongation by the polymerase domain

Potisopon

Priet

Collet

et al. 2014

Nucleic Acids Research

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“…Earlier crystal structures of NS5 individual domains suggested that the evolutionarily conserved R262, whose guanidinium group hydrogen bonds with the main chain carbonyl oxygen of V97, defines the C terminus of the MTase domain ( Fig. 1C ; S3 Fig in S1 Text ) [ 1 , 28 , 32 , 57 , 60 ]. Residues H263-V264-N265-A266, which are located after the C-terminal end of the MTase domain, are the least evolutionarily conserved in the interface region ( Fig.…”

Section: Resultsmentioning

confidence: 99%

A Crystal Structure of the Dengue Virus NS5 Protein Reveals a Novel Inter-domain Interface Essential for Protein Flexibility and Virus Replication

et al. 2015

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Flavivirus RNA replication occurs within a replication complex (RC) that assembles on ER membranes and comprises both non-structural (NS) viral proteins and host cofactors. As the largest protein component within the flavivirus RC, NS5 plays key enzymatic roles through its N-terminal methyltransferase (MTase) and C-terminal RNA-dependent-RNA polymerase (RdRp) domains, and constitutes a major target for antivirals. We determined a crystal structure of the full-length NS5 protein from Dengue virus serotype 3 (DENV3) at a resolution of 2.3 Å in the presence of bound SAH and GTP. Although the overall molecular shape of NS5 from DENV3 resembles that of NS5 from Japanese Encephalitis Virus (JEV), the relative orientation between the MTase and RdRp domains differs between the two structures, providing direct evidence for the existence of a set of discrete stable molecular conformations that may be required for its function. While the inter-domain region is mostly disordered in NS5 from JEV, the NS5 structure from DENV3 reveals a well-ordered linker region comprising a short 310 helix that may act as a swivel. Solution Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS) analysis reveals an increased mobility of the thumb subdomain of RdRp in the context of the full length NS5 protein which correlates well with the analysis of the crystallographic temperature factors. Site-directed mutagenesis targeting the mostly polar interface between the MTase and RdRp domains identified several evolutionarily conserved residues that are important for viral replication, suggesting that inter-domain cross-talk in NS5 regulates virus replication. Collectively, a picture for the molecular origin of NS5 flexibility is emerging with profound implications for flavivirus replication and for the development of therapeutics targeting NS5.

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“…In a previous study, substitution of amino acids 46, 47 and 49 in the αA3 motif of the MTase in a dengue infectious clone severely reduced virus replication but this mutant was rescued by a spontaneous second site mutation at amino acid 512 in the palm of the RdRP suggesting intra-domain interaction between these residues [177]. However, although co-expressed WNV MTase and RdRp domains could be co-immunoprecipitated confirming interactions between these domains, none of the αA3 mutations affected the efficiency of the MTase-RdRp interaction [182]. In the structure of the full length Japanese encephalitis virus NS5, the MTase domain was found to interact with the backside of the RdRp domain and to only partially cover the top-right corner of the NTP entry channel instead of being located close to the dsRNA channel as previously predicted [181].…”

Section: Viral Nonstructural Proteinsmentioning

confidence: 99%

Replication Cycle and Molecular Biology of the West Nile Virus

Brinton

2013

Viruses

125

128

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West Nile virus (WNV) is a member of the genus Flavivirus in the family Flaviviridae. Flaviviruses replicate in the cytoplasm of infected cells and modify the host cell environment. Although much has been learned about virion structure and virion-endosomal membrane fusion, the cell receptor(s) used have not been definitively identified and little is known about the early stages of the virus replication cycle. Members of the genus Flavivirus differ from members of the two other genera of the family by the lack of a genomic internal ribosomal entry sequence and the creation of invaginations in the ER membrane rather than double-membrane vesicles that are used as the sites of exponential genome synthesis. The WNV genome 3' and 5' sequences that form the long distance RNA-RNA interaction required for minus strand initiation have been identified and contact sites on the 5' RNA stem loop for NS5 have been mapped. Structures obtained for many of the viral proteins have provided information relevant to their functions. Viral nonstructural protein interactions are complex and some may occur only in infected cells. Although interactions between many cellular proteins and virus components have been identified, the functions of most of these interactions have not been delineated.

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An interaction between the methyltransferase and RNA dependent RNA polymerase domains of the West Nile virus NS5 protein

Cited by 17 publications

References 20 publications

The methyltransferase domain of dengue virus protein NS5 ensures efficient RNA synthesis initiation and elongation by the polymerase domain

The methyltransferase domain of dengue virus protein NS5 ensures efficient RNA synthesis initiation and elongation by the polymerase domain

A Crystal Structure of the Dengue Virus NS5 Protein Reveals a Novel Inter-domain Interface Essential for Protein Flexibility and Virus Replication

Replication Cycle and Molecular Biology of the West Nile Virus

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