Lim, Siew Pheng, Shi, Pei-Yong, Soh, Sherryl, Zhao, Yongqian, Zheng , Jie , Phoo , Wint Wint , Swaminathan, Kunchithapadam , Cornvik , Tobias C., Lescar , Julien , Vasudevan , Subhash G. and Luo , Dahai (2013) A crystal structure of the dengue virus NS5 protein reveals a novel inter-domain interface essential for protein flexibility and virus replication. PLOS Pathogens.

Abstract

Flavivirus RNA replication occurs within a replication complex (RC) that assembles on ER membranes and comprises both non-structural (NS) viral proteins and host cofactors. As the largest protein component within flavivirus RC, NS5 plays key enzymatic roles through its N-terminal methyltransferase (MTase) and C-terminal RNA-dependent-RNA polymerase (RdRp) domains and constitutes a major target for antivirals. We determined a crystal structure of the full-length NS5 protein (NS5FL) from Dengue virus serotype 3 (DENV3) at a resolution of 2.3 Å in the presence of bound SAH and GTP. Although the overall molecular shape of NS5FL from DENV3 resembles that of NS5FL from Japanese Encephalitis Virus (JEV), the relative orientation between the MTase and RdRp domains differs between the two structures, providing direct evidence for the existence of a set of discrete stable molecular conformations. While the inter-domain region is mostly disordered in NS5FL from JEV, the NS5FL structure from DENV3 reveals a well-ordered linker region comprising a short 310 helix that is likely to act as a conformational switch. Solution Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS) analysis showed that the thumb subdomain of RdRp is more dynamic in NS5FL compared to NS5-RdRp, suggesting that the MTase domain regulates the structural dynamics of the RdRp domain. Site-directed mutagenesis targeting the mostly polar interface between the MTase and RdRp domains identified several evolutionarily conserved residues that are important for viral replication in a sub-genomic replicon, suggesting an inter-domain cross-talk. A picture for the molecular origin of NS5 flexibility now emerges which has profound implications for flavivirus replication and for the developing therapeutics targeting NS5.

Item Type:	Article
Date Deposited:	13 Oct 2015 13:12
Last Modified:	13 Oct 2015 13:12
URI:	https://oak.novartis.com/id/eprint/23748