title: Functional analysis of two cavities in flavivirus NS5 polymerase.
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 cavity B, 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.
authors.affiliation: Novartis Institute for Tropical Diseases, Singapore 138670.
authors.forename: Gang
authors.initials: G
authors.lastname: Zou
authors.forename: Yen-Liang
authors.initials: YL
authors.lastname: Chen
authors.forename: Hongping
authors.initials: H
authors.lastname: Dong
authors.forename: Chin Chin
authors.initials: CC
authors.lastname: Lim
authors.forename: Li Jian
authors.initials: LJ
authors.lastname: Yap
authors.forename: Yin Hoe
authors.initials: YH
authors.lastname: Yau
authors.forename: Susana Geifman
authors.initials: SG
authors.lastname: Shochat
authors.forename: Julien
authors.initials: J
authors.lastname: Lescar
authors.forename: Pei-Yong
authors.initials: PY
authors.lastname: Shi
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002522:Chlorocebus aethiops; D006224:Cricetinae; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry; D015195:Drug Design; D004798:Enzymes / Q000737:chemistry; D005416:Flavivirus / Q000201:enzymology*; D008239:Lysine / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D025941:Protein Interaction Mapping; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D014364:Tryptophan / Q000737:chemistry; D014709:Vero Cells; D017361:Viral Nonstructural Proteins / Q000737:chemistry*; D014779:Virus Replication
chemical_list: D004798:Enzymes; C096041:NS5 protein, flavivirus; D017361:Viral Nonstructural Proteins; D014364:Tryptophan; D012324:RNA-Dependent RNA Polymerase; D012321:DNA-Directed RNA Polymerases; D008239:Lysine