All pathogenic flaviviruses examined thus far inhibit host interferon (IFN) responses by suppressing the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway. Both Langat virus (LGTV; a member of the tick-borne encephalitis virus serogroup) and Japanese encephalitis virus use the nonstructural protein NS5 to suppress JAK-STAT signaling. However, NS5 is also critical to virus replication, contributing methyltransferase and RNA-dependent RNA polymerase (RdRP) activities. The specific amino acid residues of NS5 involved in IFN antagonism are not known. Here, we demonstrate that the LGTV NS5 JAK-STAT inhibitory domain is contained between amino acids 355 and 735 (of 903), a range which lies within the RdRP domain. Furthermore, we identified two noncontiguous stretches of specific amino acids within the RdRP, 374 to 380 and 624 to 647, as critical for inhibition of JAK-STAT signaling. Despite considerable separation on the linear NS5 sequence, these residues localized adjacent to each other when modeled on the West Nile virus RdRP crystal structure. Due to the general conservation of RdRP structures, these results suggest that the specific residues identified act cooperatively to form a unique functional site on the RdRP responsible for JAK-STAT inhibition. This insight into the mechanism underlying flavivirus IFN evasion strategies will facilitate the design of antiviral therapeutics that potentiate the action of IFN during infection.The flaviviruses cause globally significant human diseases and include tick-borne encephalitis (TBEV), dengue (DEN), Japanese encephalitis (JEV), West Nile (WNV), and yellow fever (YFV) viruses. There is currently no specific treatment for infection with any of these viruses, although interferon (IFN) has been trialed as a potential therapeutic (22,25). IFN is a crucial element of the innate immune response to flavivirus infection, restricting virus replication, dissemination, and lethality in mouse models (1, 24). However, all flaviviruses examined thus far, including Langat virus (LGTV, a member of the TBEV serogroup) (2), JEV (14, 15), WNV (8), and DEN (10, 18), suppress IFN-mediated Janus kinase-signal transducer and activator of transcription (JAK-STAT) signal transduction (19). In response to stimulation with IFNs, these viruses compromise the phosphorylation, and hence activation, of JAKs, resulting in suppressed downstream signaling events, including STAT phosphorylation and nuclear translocation. It has recently been shown that the relative ability of WNV strains to suppress JAK-STAT signal transduction is a major virulence determinant (11). Thus, determining the precise mechanisms by which flaviviruses suppress IFN signaling is important to the understanding of flavivirus pathogenesis as well as to the development of novel antiviral therapeutics.We previously demonstrated that LGTV utilizes its nonstructural protein NS5 to inhibit IFN-mediated JAK-STAT signaling (2). Recently, JEV was also demonstrated to employ NS5 to suppress IFN-mediated JAK-STAT...