Role of the homologous MTase-RdRp interface of flavivirus intramolecular NS5 on duck tembusu virus

Flavivirus nonstructural protein 5 (NS5) harbors the N-terminal methyltransferase (MTase) and C-terminal polymerase RNA-dependent RNA polymerase (RdRp). The intramolecular NS5 features an integral MTase and RdRp interface with two components: a six-residue hydrophobic network and a GTR linker. Herein, the determinants of the MTase-RdRp interface and flavivirus substituted GTR linker were explored in TMUV replication and proliferation. First, the NanoLuc® Binary Technology (NanoBiT) and coimmunoprecipitation assays (Co-IP) methods confirmed the interaction between the MTase and RdRp domains of TMUV NS5. To screen for an optimal orientation for reporter gene fusion to the protein of interest, the signal activity of eight combinations of MTase and RdRp was explored. Intriguingly, all the combinations with the reporter gene fused to the C-terminal of MTase (1.1 C/2.1 C MTase) could barely detect any positive signal, suggesting a role for the GTR linker of the MTase C-terminal in MTase-RdRp affinity. Based on the flavivirus NS5 homologous interplay, we introduced alanine mutations into the MTase-RdRp interface of TMUV NS5. However, no single or pairwise mutation was found to abort the NS5 intramolecular interaction. Then, a mutated replicon and infectious clone were constructed to analyze the replication ability and properties of the recombinant virus. The mutant replicons of MTase F113A and M115A replicated to comparable extent as the wild type (WT). However, the replication level of the mutant MTase W121A was impaired without an obvious decrease in proliferation and virulence. Both the RdRp F351A and P585A mutants could replicate and proliferate well. Notably, the RdRp F467A virus was attenuated and did not strikingly impair the MTase-RdRp interaction. Furthermore, the TMUV was specifically compatible with the substituted NS5 with a Japanese encephalitis virus (JEV) GTR linker. Compensatory mutations were observed in the context of a defective MTase-RdRp interface after several passages of the rescued mutants in BHK-21 cells. A greater understanding of the molecular mechanism of the NS5 protein controlling duck TMUV replication will facilitate the design of novel therapies.