Identifying a novel region in the Tembusu virus NS5 protein antagonizing type I interferon signaling

Numerous species in the genus Flavivirus have been identified to pose serious threats to public health. Most of them have developed evasion strategies to counteract antiviral activity triggered by type I interferon (IFN-I), making prevention and control challenging. Our study reveals that Tembusu virus (TMUV), a Flavivirus that mainly causes severe disease in ducks, can inhibit IFN-I signaling via its NS5 protein, efficiently preventing the nuclear translocation of IFN-activated transcription factor STATs and significantly impairing transcriptional induction of IFN-stimulated genes. Interestingly, our research shows that 37-45 amino acid (aa) residues in the N-terminal region of TMUV-NS5 constitute a functional nuclear localization signal (NLS) that interacts with karyopherins, disrupting the nuclear transport system, in contrast to the classical α/β NLS found in most flaviviruses. Moreover, through in-depth analysis, we highlight that the nuclear trafficking competence of NLS is modulated by multiple determinants, such as the species and molecular size of cargo proteins, providing critical mechanistic insights into the differential subcellular localization of Flavivirus NS5. Altogether, our findings contribute to a better understanding of how TMUV-NS5 inhibits IFN-I signaling and offer novel perspectives on the evasion of the innate immune responses by Flavivirus.IMPORTANCERecent studies have demonstrated that various flaviviruses can inhibit the innate type I interferon (IFN-I) response. Similarly, Tembusu virus (TMUV), a highly epidemic Flavivirus among ducks, has been reported to inhibit IFN-I induction. In the present study, we confirm that TMUV is also an antagonist of IFN-I signaling, and its NS5 plays a key role. However, different from α/β nuclear localization signal (NLS) in most flaviviruses, 37-45 amino acid region in N-terminus of TMUV-NS5 has been identified as a crucial area for interaction with KPNAs, thus inhibiting nuclear transport of STATs. In addition, we further discovered that the nuclear localization activity of NLS is regulated by multiple factors, such as different sizes and types of the cargos, thereby leading to the distinct subcellular distribution of Flavivirus NS5.