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.
DENV causes widespread mosquito-borne viral infections worldwide and nearly 40% of the world’s population is at risk of being infected. Currently, no licensed vaccines or specific drugs are available to treat severe infections by DENV. NS5 is a large protein of 900 amino acids composed of two domains with several key enzymatic activities for viral RNA replication in the host cell and constitutes a prime target for the design of antiviral inhibitors. We succeeded in trapping a stable conformation of the full-length NS5 protein and report its crystal structure at a resolution of 2.3 Å. This conformation reveals the entire inter-domain region and clarifies the determinants of NS5 flexibility. The inter-domain interface is stabilized by several polar contacts between residues projecting from the MTase and RdRp domains of NS5. Several evolutionarily conserved residues at the interface play a crucial role for virus replication as shown by reverse genetics, although the analogous mutations mostly do not abolish the in vitro enzymatic activities of the recombinant proteins.