The substitution at residue 218 of the NS5 protein methyltransferase domain of Tembusu virus impairs viral replication and translation and may triggers RIG-I-like receptor signaling

Flavivirus RNA cap-methylation plays an important role in viral infection, proliferation, and escape from innate immunity. The methyltransferase (MTase) of the flavivirus NS5 protein catalyzes viral RNA methylation. The E218 amino acid of the NS5 pro-tein MTase domain is one of the active sites of flavivirus methyltransferase. In flaviviruses, the E218A mutation abolished 2'-O methylation activity and significantly reduced N-7 methylation activity. Tembusu virus (TMUV, genus Flavivirus) was a pathogen that caused neurological symptoms in ducklings and decreased egg production in laying ducks. In this study, we focused on a comprehensive understanding of the effects of the E218A mutation on TMUV characteristics and the host immune response. E218A mutation reduced TMUV rep-lication and proliferation, but did not affect viral adsorp-tion and entry. Based on a TMUV replicon system, we found that the E218A mutation impaired viral transla-tion. In addition, E218A mutant virus might be more readily recognized by RIG-I-like receptors to activate the corresponding antiviral immune signaling than WT virus. Together, our data suggest that the E218A muta-tion of TMUV MTase domain impairs viral replication and translation and may activates RIG-I-like receptor signaling, ultimately leading to a reduction in viral proliferation.