Biochemical and genetic characterization of dengue virus methyltransferase

Dong, Hansong; Chang, David C.; Xie, Xuping; Toh, Ying Xiu; Chung, Ka Yan; Zou, Gang; Lescar, Julien; Lim, Siew Pheng; Shi, Pei Yong

doi:10.1016/j.virol.2010.06.039

Cited by 98 publications

(94 citation statements)

References 33 publications

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“…This difference between WNV and DENV is also supported by a recent report showing that both viruses have different NS5 requirements. In this regard, chimeric DENVs containing the WNV MTase, RdRp, or full-length NS5 were found to be impaired in their replication, indicating that these proteins are not exchangeable (35). These recent observations together with our studies support the idea that, although flaviviruses share general FIGURE 7.…”

Section: Discussionsupporting

confidence: 84%

RNA Sequences and Structures Required for the Recruitment and Activity of the Dengue Virus Polymerase

Filomatori¹,

Iglesias²,

Villordo

et al. 2011

Journal of Biological Chemistry

146

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Dengue virus RNA-dependent RNA polymerase specifically binds to the viral genome by interacting with a promoter element known as stem-loop A (SLA). Although a great deal has been learned in recent years about the function of this promoter in dengue virus-infected cells, the molecular details that explain how the SLA interacts with the polymerase to promote viral RNA synthesis remain poorly understood. Using RNA binding and polymerase activity assays, we defined two elements of the SLA that are involved in polymerase interaction and RNA synthesis. Mutations at the top of the SLA resulted in RNAs that retained the ability to bind the polymerase but impaired promoter-dependent RNA synthesis. These results indicate that protein binding to the SLA is not sufficient to induce polymerase activity and that specific nucleotides of the SLA are necessary to render an active polymerase-promoter complex for RNA synthesis. We also report that protein binding to the viral RNA induces conformational changes downstream of the promoter element. Furthermore, we found that structured RNA elements at the 3 end of the template repress dengue virus polymerase activity in the context of a fully active SLA promoter. Using assays to evaluate initiation of RNA synthesis at the viral 3-UTR, we found that the RNA-RNA interaction mediated by 5-3-hybridization was able to release the silencing effect of the 3-stem-loop structure. We propose that the long range RNA-RNA interactions in the viral genome play multiple roles during RNA synthesis. Together, we provide new molecular details about the promoter-dependent dengue virus RNA polymerase activity. Dengue virus (DENV)3 is a member of the Flavivirus genus in the Flaviviridae family, together with other important human pathogens such as yellow fever virus, West Nile virus (WNV), Saint Luis encephalitis virus, and Japanese encephalitis virus (1). DENV is the most significant mosquito-borne human viral pathogen worldwide and is responsible for the highest rates of disease and mortality among the members of the Flavivirus genus. The lack of vaccines and antivirals against DENV leaves two billion people at risk, mainly in poor countries.DENV genome is a single-stranded RNA molecule of positive polarity of ϳ11 kb in length that encodes a long polyprotein that is co-and post-translationally processed by host and viral proteases to yield three structural proteins (C, prM, and E), and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The coding sequence is flanked by highly structured 5Ј-and 3Ј-UTRs. In recent years, a number of cis-acting RNA elements have been identified in the viral 5Ј-and 3Ј-UTRs that are essential for viral RNA amplification (for review see Ref.2). A model for DENV RNA synthesis has been proposed previously (3). This model involves binding of the viral RNA-dependent RNA polymerase (RdRp) to an RNA element present at the 5Ј end of the genome known as stemloop A (SLA). This element of 70 nucleotides folds into a Yshaped structure that is conserved among diff...

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Section: Discussionsupporting

confidence: 84%

RNA Sequences and Structures Required for the Recruitment and Activity of the Dengue Virus Polymerase

Filomatori¹,

Iglesias²,

Villordo

et al. 2011

Journal of Biological Chemistry

146

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“…The RdRp domain of NS5 binds specifically to the DENV 5Ј UTR (17,21). In addition, the MTase domain recognizes the cap structure present at the 5Ј end of the genome (12,13). Thus, we hypothesized that NS5 could bind capped 5Ј UTR molecules with higher affinity than uncapped RNAs.…”

Section: Resultsmentioning

confidence: 99%

“…The MTase is responsible for methylation of the cap structure present at the 5Ј end of the viral genome. This process involves methylation in two positions, guanine N-7 and ribose 2Ј-O (12,13,28,38). The RdRp domain has primer independent (de novo) RNA synthesis activity (1,26).…”

mentioning

confidence: 99%

The F1 Motif of Dengue Virus Polymerase NS5 Is Involved in Promoter-Dependent RNA Synthesis

Iglesias

Filomatori

Gamarnik

2011

J Virol

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The mechanism by which viral RNA-dependent RNA polymerases (RdRp) specifically amplify viral genomes is still unclear. In the case of flaviviruses, a model has been proposed that involves the recognition of an RNA element present at the viral 5 untranslated region, stem-loop A (SLA), that serves as a promoter for NS5 polymerase binding and activity. Here, we investigated requirements for specific promoter-dependent RNA synthesis of the dengue virus NS5 protein. Using mutated purified NS5 recombinant proteins and infectious viral RNAs, we analyzed the requirement of specific amino acids of the RdRp domain on polymerase activity and viral replication. A battery of 19 mutants was designed and analyzed. By measuring polymerase activity using nonspecific poly(rC) templates or specific viral RNA molecules, we identified four mutants with impaired polymerase activity. Viral full-length RNAs carrying these mutations were found to be unable to replicate in cell culture. Interestingly, one recombinant NS5 protein carrying the mutations K456A and K457A located in the F1 motif lacked RNA synthesis dependent on the SLA promoter but displayed high activity using a poly(rC) template. Promoter RNA binding of this NS5 mutant was unaffected while de novo RNA synthesis was abolished. Furthermore, the mutant maintained RNA elongation activity, indicating a role of the F1 region in promoter-dependent initiation. In addition, four NS5 mutants were selected to have polymerase activity in the recombinant protein but delayed or impaired virus replication when introduced into an infectious clone, suggesting a role of these amino acids in other functions of NS5. This work provides new molecular insights on the specific RNA synthesis activity of the dengue virus NS5 polymerase.Dengue virus (DENV) is the single most significant arthropod-borne virus pathogen in humans. It belongs to the Flaviviridae family together with other important pathogens such as yellow fever virus (YFV), West Nile virus (WNV), Saint Louis encephalitis virus (SLEV), and Japanese encephalitis virus (JEV). The World Health Organization continues reporting dengue outbreaks every year in the Americas and Asia. In spite of the urgent medical need to control DENV infections, vaccines and antivirals are still unavailable. Although a model for DENV RNA synthesis was previously proposed (17), molecular aspects of the mechanism by which the polymerase specifically amplifies the viral genome are still unclear for DENV and other flaviviruses. To further understand this viral process, we investigated functional properties of the viral polymerase NS5.NS5 is the largest of the flavivirus proteins (105 kDa); it contains an N-terminal methyltransferase domain (MTase) and a C-terminal RNA-dependent RNA polymerase (RdRp) domain. The MTase is responsible for methylation of the cap structure present at the 5Ј end of the viral genome. This process involves methylation in two positions, guanine N-7 and ribose 2Ј-O (12, 13, 28, 38). The RdRp domain has primer independent (de novo) RNA synth...

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“…Two types of suppressor mutations restored the function of the NS2A internal cleavage site; the first occurred by insertion of amino acid residues to restore cleavability at the NS2A site, and the second occurred by a suppressor mutation in the NS3 helicase domain, which revealed a functional link between NS2A and NS3 (45). In another study, a single conserved amino acid substitution, D146A in the MT domain of D1 NS5, abolished both N-7 and 2Ј-O-methylations of G-capped RNA and was lethal for viral replication (46). Continuous passage of culture supernatants from the D146A mutant RNA-transfected cells for infection of naive Vero cells for 12 days did not yield any plaques, indicating that the mutation was lethal for replication.…”

Section: Discussionmentioning

confidence: 98%

“…Similarly, deletion of highly conserved NS5 POL motif, GDD 3 GVD, or the AdoMet binding site in the Kunjin virus NS5 MT domain (aa 78 -87) could be trans-complemented by transfection of mutant genomic RNA into BHK-21 cells stably expressing Kunjin virus replicon (47). Dengue virus type 1 chimeras containing either West Nile virus MT or POL domain or complete NS5 was replication-defective but could be trans-complemented in D1 replicon-expressing cells (46).…”

Section: Discussionmentioning

confidence: 99%

Substitution of NS5 N-terminal Domain of Dengue Virus Type 2 RNA with Type 4 Domain Caused Impaired Replication and Emergence of Adaptive Mutants with Enhanced Fitness

Teramoto

Boonyasuppayakorn

Handley

et al. 2014

Journal of Biological Chemistry

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Background:The four dengue virus serotypes, evolved from a common ancestor, are global human pathogens. Results: Interserotypic substitution of the functional domain essential for 5Ј-capping was detrimental to RNA replication. Conclusion: The chimeric RNA gradually evolves replication fitness through adaptive mutations in genes encoding two replication proteins. Significance: This study provides a possible pathway for generating attenuated dengue virus vaccine.

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Biochemical and genetic characterization of dengue virus methyltransferase

Cited by 98 publications

References 33 publications

RNA Sequences and Structures Required for the Recruitment and Activity of the Dengue Virus Polymerase

RNA Sequences and Structures Required for the Recruitment and Activity of the Dengue Virus Polymerase

The F1 Motif of Dengue Virus Polymerase NS5 Is Involved in Promoter-Dependent RNA Synthesis

Substitution of NS5 N-terminal Domain of Dengue Virus Type 2 RNA with Type 4 Domain Caused Impaired Replication and Emergence of Adaptive Mutants with Enhanced Fitness

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