Characterization of mechanism of inhibition of dengue virus NS5 methyltransferase by hit/lead compounds generated from drug discovery programs.

• Main Author: Yap, Joe Li Jian.
• Other Authors: Julien Lescar
• Format: Theses and Dissertations
• Language: English
• Published: 2012
• Subjects: DRNTU::Science::Biological sciences
• Online Access: http://hdl.handle.net/10356/50717
• Institution: Nanyang Technological University

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 → 7meGpppA → 7meGpppA2’-O-me). It is currently unknown how the MTase catalyzes these distinct sequential methylation events. We report the crystallographic structure of DENV-3 MTase in complex with a 5’-capped RNA octamer (GpppAGAACCUG) at a resolution of 2.9 Å, presented in Chapter 2. 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 stabilized by the formation of several intra and intermolecular base stacking, as well as non-canonical base pairs. This 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. We next solved the structures of ternary complexes of DENV-3 MTase with sinfungine/compounds and 7meGpppA, presented in Chapter 3. Our structures show the presence of a conserved hydrophobic pocket which is shown to be critical for cap methylation and viral replication. Therefore, this hydrophobic pocket can be harnessed for rational drug design, using S-adenosyl-L-methionine as a starting scaffold. Moieties that could extend into the hydrophobic cavity are able to exhibit improved binding and selectivity against flavivirus MTase. These findings enable the structure guided approach to develop potent anti-viral therapies specifically targeting the flavivirus MTase. Additionally, we have successfully purified the NS4B membrane protein in milligram amounts, presented in Chapter 4. The NS4B co-localized with NS3 in the perinuclear region of infected human cells; and dissociated NS3 from its bound RNA to enhance helicase activity via an in vitro unwinding assay. These results suggest that NS4B plays a role in viral replication via its interaction with NS3. To better understand the structure and function of NS4B in the context of dengue, we expressed and purified the NS4B membrane protein using bacterial expression system.