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

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. Nonstructural protein 5 (NS5) is a multifunctional protein es-sential 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 RNA-dependent 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 surveil-lance (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)