Dengue Virus Nonstructural Protein 5 (NS5) Assembles into a Dimer with a Unique Methyltransferase and Polymerase Interface

Abstract

Flavivirus nonstructural protein 5 (NS5) consists of methyltransferase (MTase) and RNA-dependent RNA polymerase (RdRp) domains, which catalyze 5’-RNA capping/methylation and RNA synthesis, respectively, during viral genome replication. Although the crystal structure of flavivirus NS5 is known, no data about the quaternary organization of the functional enzyme are available. We report the crystal structure of dengue virus full-length NS5, where eight molecules of NS5 are arranged as four independent dimers in the crystallographic asymmetric unit. The relative orientation of each monomer within the dimer, as well as the orientations of the MTase and RdRp domains within each monomer, is conserved, suggesting that these structural arrangements represent the biologically relevant conformation and assembly of this multi-functional enzyme. Essential interactions between MTase and RdRp domains are maintained in the NS5 dimer via inter-molecular interactions, providing evidence that flavivirus NS5 can adopt multiple conformations while preserving necessary interactions between the MTase and RdRp domains. Furthermore, many NS5 residues that reduce viral replication are located at either the inter-domain interface within a monomer or at the inter-molecular interface within the dimer. Hence the X-ray structure of NS5 presented here suggests that MTase and RdRp activities could be coordinated as a dimer during viral genome replication.

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).…”

Host-Virus Interaction of ZIKA Virus in Modulating Disease Pathogenesis

“…NS5 is a highly conserved 103 kDa protein and plays a crucial role in viral replication (Lu and Gong, 2013; Li et al, 2014; Klema et al, 2016). It has N-terminal RNA cap-processing activity (homology with the S-adenosyl-methionine (SAM)-dependent methyltransferases) (Klema et al, 2016), GTP-binding activity (Benarroch et al, 2004), and C-terminal RdRP activity (Malet et al, 2007; Zhang et al, 2008).…”

“…It has N-terminal RNA cap-processing activity (homology with the S-adenosyl-methionine (SAM)-dependent methyltransferases) (Klema et al, 2016), GTP-binding activity (Benarroch et al, 2004), and C-terminal RdRP activity (Malet et al, 2007; Zhang et al, 2008). The interaction of NS5 protein stimulates NS3 NTPase activity (Feito et al, 2008) and creates an importin binding site.…”

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.…”

“…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. Additionally, interdomain interactions between monomers in an NS5 dimer suggest the coordination of RdRp and MTase activities across the NS5 monomer and dimer (17). Data from small-angle X-ray scattering (SAXS) studies indicate that NS5 proteins of all four DENV serotypes can adopt a more elongated shape than that in the crystal structure (20,21), suggesting that flexibility between the MTase and RdRp domains is likely essential for the viral replication process.…”