Nuclear localization of duck Tembusu virus NS5 protein attenuates viral replication in vitro and NS5-NS2B3 interaction

Duck Tembusu virus (TMUV) belongs to the flavivirus genus whose genome replication involved in capping and RNA synthesis dominating by nonstructural protein 5 (NS5). Flaviviral replication has been well documented to occur in the cytoplasm, but the effect of NS5 to gain access to the nucleus remains controversial. Here, TMUV NS5 was observed to localize within the cytoplasm of transfected and infected cells and co-localized with the endoplasmic reticulum. We introduced two arginine mutations into the N390 and Q392 (N390R and Q392R) of the NS5 bipartite nuclear localization sequence (alpha/beta NLS) and designated that mutagenesis as NS5(NLSmut), which has shown the ability to access the nucleus and hence attenuates viral replication and production in vitro. Additionally, there was no significant difference between the recovered wild-type TMUV (rTMUV-WT) and engineered mutant (rTMUV-NS5(NLSmut)) on plaque morphology, survival rate of infected duck embryos or virus copies in tissues. Considering that NS5(NLSmut) is mainly located in the cytoplasm of rTMUV-NS5(NLSmut) infected cells at the early stage of infection. We further confirmed that NS5(NLSmut) attenuated its interaction with nonstructural NS2B-NS3 (NS2B3) following transfection and infection. Meanwhile, the rTMUV-NS5(NLSmut) tended to stimulate more interferon beta (IFN beta) than rTMUV-WT. However, preliminary study on transient NS5 and NS5(NLSmut) detected the same levels of IFN beta mRNA mediated by RIG-I detection of NS5 RNA polymerase activity in cell. In summary, these results provide further insights into the relationship between the viral property and subcellular localization of flavivirus NS5 in terms of the NS5-NS2B3 interaction.