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

Abstract

We examined the function of the conserved Val/Ile residue within the dengue virus NS5 interdomain linker (residues 263 to 272) by site-directed mutagenesis. Gly substitution or Gly/Pro insertion after the conserved residue increased the linker flexibility and created slightly attenuated viruses. In contrast, Pro substitution abolished virus replication by imposing rigidity in the linker and restricting NS5's conformational plasticity. Our biochemical and reverse genetics experiments demonstrate that NS5 utilizes conformational regulation to achieve optimum viral replication. N onstructural protein 5 (NS5) is a multifunctional protein essential for dengue virus (DENV) replication. NS5 consists of an N-terminal S-adenosyl-L-methionine (SAM)-dependent methyltransferase (MTase) domain (1) and a C-terminal RNAdependent RNA polymerase (RdRp) domain (2-5). The MTase domain catalyzes the formation of type I RNA cap, which is critical for efficient translation and subverting the host immune surveillance (1, 6-8). The MTase domain may also be responsible for guanylyltransferase activity (9, 10). The RdRp domain (NS5 RdRp) synthesizes both negative-strand RNA from the input genomic RNA (gRNA) and the progeny plus-strand gRNA (5, 11).Previous structural studies on DENV NS5 individual domains revealed electron density covering residues 6 to 262 (for the MTase domain) (PDB code 3P97) (12), and residues 273 to 882 (for RdRp) (PDB code 2J7U) (5). Limited proteolysis of full-length NS5 (NS5 FL) in combination with mass determination suggested that the residues 263 to 272 (5) form the interdomain linker, and sequence comparison across various flaviviruses showed that this region is poorly conserved (Fig. 1). Small-angle X-ray scattering (SAXS) studies suggested that NS5 could adopt different conformations permitted by a flexible linker (13). 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). 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. 1A and B). In DENV3 full-length NS5, the 3 10 -helix linker adopts a compact conformation compared to JEV full-length NS5 or DENV2 MTase (1 to 296) (PDB code 1L9K) (Fig. 1C). Functionally, the MTase domain stimulates both de novo initiation and elongation activities of the RdRp domain, which implies a critical role for an interdomain linker in controlling NS5 RNA polymerization activities (17). Despite the various conformations adopted by NS5 in solution, position 264 (in DENV3 NS5) is found to be exclusively Val in flaviviruses, except for a homologous su...

Molecular basis for specific viral RNA recognition and 2′-O-ribose methylation by the dengue virus nonstructural protein 5 (NS5)

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