Functional analysis of two cavities in flavivirus NS5 polymerase.

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.

Crystal structure of the dengue virus methyltransferase bound to a 5'-capped octameric RNA.

The N-terminal domain of the flavivirus NS5 protein functions as a methyltransferase (MTase). It sequentially methylates the N7 and 2'-O positions of the viral RNA cap structure (GpppA→(7me)GpppA→(7me)GpppA(2'-O-me)). The same NS5 domain could also have a guanylyltransferase activity (GTP+ppA-RNA→GpppA). The mechanism by which this protein domain catalyzes these three distinct functions is currently unknown. Here we report the crystallographic structure of DENV-3 MTase in complex with a 5'-capped RNA octamer (G(ppp)AGAACCUG) at a resolution of 2.9 A. Two RNA octamers arranged as kissing loops are encircled by four MTase monomers around a 2-fold non-crystallography symmetry axis. Only two of the four monomers make direct contact with the 5' end of RNA. The RNA structure is stabilised by the formation of several intra and intermolecular base stacking and non-canonical base pairs. The structure may represent the product of guanylylation of the viral genome prior to the subsequent methylation events that require repositioning of the RNA substrate to reach to the methyl-donor sites. The crystal structure provides a structural explanation for the observed trans-complementation of MTases with different methylation defects.