Functional analysis of two cavities in flavivirus NS5 polymerase

Gang Zou, Yen Liang Chen, Hongping Dong, Chin Chin Lim, Li Jian Yap, Yin Hoe Yau, Susana Geifman Shochat, Julien Lescar, Pei-Yong Shi

Research output: Contribution to journalArticle

72 Citations (Scopus)

Abstract

Flavivirus NS5 protein encodes methyltransferase and RNA-dependent RNA polymerase (RdRp) activities. Structural analysis of flavivirus RdRp domains uncovered two conserved cavities (A and B). Both cavities are located in the thumb subdomains and represent potential targets for development of allosteric inhibitors. In this study, we used dengue virus as a model to analyze the function of the two RdRp cavities. Amino acids from both cavities were subjected to mutagenesis analysis in the context of genome-length RNA and recombinant NS5 protein; residues critical for viral replication were subjected to revertant analysis. For cavity A, we found that only one (Lys-756) of the seven selected amino acids is critical for viral replication. Alanine substitution of Lys-756 did not affect the RdRp activity, suggesting that this residue functions through a nonenzymatic mechanism. For cavityB,all four selected amino acids (Leu-328, Lys-330, Trp-859, and Ile-863) are critical for viral replication. Biochemical and revertant analyses showed that three of the four mutated residues (Leu-328, Trp-859, and Ile-863) function at the step of initiation of RNA synthesis, whereas the fourth residue (Lys-330) functions by interacting with the viral NS3 helicase domain. Collectively, our results have provided direct evidence for the hypothesis that cavity B, but not cavity A, from dengue virus NS5 polymerase could be a target for rational drug design.

Original languageEnglish (US)
Pages (from-to)14362-14372
Number of pages11
JournalJournal of Biological Chemistry
Volume286
Issue number16
DOIs
StatePublished - Apr 22 2011
Externally publishedYes

Fingerprint

RNA Replicase
Flavivirus
Functional analysis
Dengue Virus
Viruses
Amino Acids
Protein Methyltransferases
RNA
Recombinant proteins
Mutagenesis
Drug Design
Thumb
Methyltransferases
Recombinant Proteins
Structural analysis
Alanine
Substitution reactions
Genes
Genome
Pharmaceutical Preparations

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

Zou, G., Chen, Y. L., Dong, H., Lim, C. C., Yap, L. J., Yau, Y. H., ... Shi, P-Y. (2011). Functional analysis of two cavities in flavivirus NS5 polymerase. Journal of Biological Chemistry, 286(16), 14362-14372. https://doi.org/10.1074/jbc.M110.214189

Functional analysis of two cavities in flavivirus NS5 polymerase. / Zou, Gang; Chen, Yen Liang; Dong, Hongping; Lim, Chin Chin; Yap, Li Jian; Yau, Yin Hoe; Shochat, Susana Geifman; Lescar, Julien; Shi, Pei-Yong.

In: Journal of Biological Chemistry, Vol. 286, No. 16, 22.04.2011, p. 14362-14372.

Research output: Contribution to journalArticle

Zou, G, Chen, YL, Dong, H, Lim, CC, Yap, LJ, Yau, YH, Shochat, SG, Lescar, J & Shi, P-Y 2011, 'Functional analysis of two cavities in flavivirus NS5 polymerase', Journal of Biological Chemistry, vol. 286, no. 16, pp. 14362-14372. https://doi.org/10.1074/jbc.M110.214189
Zou G, Chen YL, Dong H, Lim CC, Yap LJ, Yau YH et al. Functional analysis of two cavities in flavivirus NS5 polymerase. Journal of Biological Chemistry. 2011 Apr 22;286(16):14362-14372. https://doi.org/10.1074/jbc.M110.214189
Zou, Gang ; Chen, Yen Liang ; Dong, Hongping ; Lim, Chin Chin ; Yap, Li Jian ; Yau, Yin Hoe ; Shochat, Susana Geifman ; Lescar, Julien ; Shi, Pei-Yong. / Functional analysis of two cavities in flavivirus NS5 polymerase. In: Journal of Biological Chemistry. 2011 ; Vol. 286, No. 16. pp. 14362-14372.
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