A Crystal Structure of the Dengue Virus NS5 Protein Reveals a Novel Inter-domain Interface Essential for Protein Flexibility and Virus Replication

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 the 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 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 NS5 from DENV3 resembles that of NS5 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 that may be required for its function. While the inter-domain region is mostly disordered in NS5 from JEV, the NS5 structure from DENV3 reveals a well-ordered linker region comprising a short 310 helix that may act as a swivel. Solution Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS) analysis reveals an increased mobility of the thumb subdomain of RdRp in the context of the full length NS5 protein which correlates well with the analysis of the crystallographic temperature factors. 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, suggesting that inter-domain cross-talk in NS5 regulates virus replication. Collectively, a picture for the molecular origin of NS5 flexibility is emerging with profound implications for flavivirus replication and for the development of therapeutics targeting NS5.

Flexibility of NS5 Methyltransferase-Polymerase Linker Region Is Essential for Dengue Virus Replication

“…2A), and assessed for their enzymatic activities as previously described (15). Interestingly, the four mutant NS5 proteins displayed dramatic differences in the coupled de novo initiation/elongation activity, even though their elongation activities were comparable ( Fig.…”

“…Interestingly, an Nterminally extended RdRp spanning residues from 265 to 900 improved thermostability and RdRp activity compared with a shorter RdRp spanning residues 273 to 900 (14). More recently, the structures of the full-length NS5 protein from DENV3 (15) and Japanese encephalitis virus (JEV) full-length NS5 (PDB code 4K6M) (16) have been resolved. Extensive comparisons between the two structures have been reported (15).…”

“…More recently, the structures of the full-length NS5 protein from DENV3 (15) and Japanese encephalitis virus (JEV) full-length NS5 (PDB code 4K6M) (16) have been resolved. Extensive comparisons between the two structures have been reported (15). Notably, unlike JEV NS5, the DENV NS5 structure revealed a hydrophilic interdomain interface and a linker region composed of a 3 10 -helix (amino acids [aa] 263 to 266) followed by a short loop (aa 267 to 272) ( Fig.…”

Zika Virus Methyltransferase: Structure and Functions for Drug Design Perspectives

“…The interface between the MTase and the RdRp domains has been reported to be essential for the replication regulation of dengue virus (36)(37)(38) or Japanese encephalitis virus (39). It is also known that the MTase domain contributes to an efficient initiation and elongation of the RNA polymerization by the dengue virus RdRp (40).…”

Interactions between the Dengue Virus Polymerase NS5 and Stem-Loop A

“…The C-terminal domain possesses RdRp activity, including the active site for RNA synthesis. Crystal structures of DENV and JEV NS5 have been determined (17)(18)(19). These structures showed different interdomain interactions within the NS5 monomer and indicated that NS5 can adopt a number of conformations with different relative orientations of the MTase and RdRp domains.…”