The interferon (IFN) response is the first line of defense against viral infections, and the majority of viruses have developed different strategies to counteract IFN responses in order to ensure their survival in an infected host. In this study, the abilities to inhibit IFN signaling of two closely related West Nile viruses, the New York 99 strain (NY99) and Kunjin virus (KUN), strain MRM61C, were analyzed using reporter plasmid assays, as well as immunofluorescence and Western blot analyses. We have demonstrated that infections with both NY99 and KUN, as well as transient or stable transfections with their replicon RNAs, inhibited the signaling of both alpha/beta IFN (IFN-␣/␤) and gamma IFN (IFN-␥) by blocking the phosphorylation of STAT1 and its translocation to the nucleus. In addition, the phosphorylation of STAT2 and its translocation to the nucleus were also blocked by KUN, NY99, and their replicons in response to treatment with IFN-␣. IFN-␣ signaling and STAT2 translocation to the nucleus was inhibited when the KUN nonstructural proteins NS2A, NS2B, NS3, NS4A, and NS4B, but not NS1 and NS5, were expressed individually from the pcDNA3 vector. The results clearly demonstrate that both NY99 and KUN inhibit IFN signaling by preventing STAT1 and STAT2 phosphorylation and identify nonstructural proteins responsible for this inhibition.The interferons (IFNs) are a large family of multifunctional secreted cytokines involved in antiviral defense, cell growth regulation, and immune activation. IFNs are produced by the majority of cells and include 14 different species of alpha IFN (IFN-␣) and one species of beta IFN (IFN-␤); these IFNs are involved primarily in antiviral and antiproliferative responses (7,16,17,28). Gamma IFN (IFN-␥) is IFN that is usually produced by specific cells of the immune system, including CD8 ϩ T cells, and has potent antiviral and immunomodulating activities (7,16,17,28). The binding of IFNs to corresponding receptors on cell surfaces triggers a cascade of different signaling pathways that eventually lead to the transcriptional activation of a large number of IFN-stimulated genes (ISGs), which can establish antiviral, antiproliferative, and/or immunoregulatory states in host cells. The best-studied IFN signaling pathways are based on IFN receptor-Janus Kinase (JAK)/signal transducer and activator of transcription (STAT) activation (7, 16). The binding of IFN-␣ and IFN-␤ to the IFN-␣/␤ receptor, which consists of IFNAR1 and IFNAR2 molecules, leads to the activation of JAK1 and Tyk-2 kinases via tyrosine phosphorylation. Activated Tyk-2 phosphorylates IFNAR1, which then serves as a binding site for STAT2. STAT2 is then phosphorylated by Tyk-2 and serves as a binding site for STAT1, which is subsequently phosphorylated by JAK1. The phosphorylated STAT2-STAT1 heterodimers then dissociate from the receptor and associate with p48/IRF-9 to form an ISGF3 complex that translocates to the nucleus, where it initiates the transcription of ISGs via binding to the IFN-stimulated response element (ISRE...

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The establishment of persistent noncytopathic replication by replicon RNAs of a number of positive-strand RNA viruses usually leads to generation of adaptive mutations in nonstructural genes. Some of these adaptive mutations (e.g., in hepatitis C virus) increase the ability of RNA replication to resist the antiviral action of alpha/beta interferon (IFN-␣/␤); others (e.g., in Sindbis virus) may also lead to more efficient IFN production. Using puromycin-selectable Kunjin virus (KUN) replicon RNA, we identified two adaptive mutations in the NS2A gene (producing Ala30-to-Pro and Asn101-to-Asp mutations in the gene product; for simplicity, these will be referred to hereafter as Ala30-to-Pro and Asn101-to-Asp mutations) that, when introduced individually or together into the original wild-type (wt) replicon RNA, resulted in ϳ15-to 50-fold more efficient establishment of persistent replication in hamster ( (29), with one long open reading frame coding for 3,433 amino acids in three structural proteins (C, prM, and E) and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (7). The gene order of KUN genome RNA is 5Ј-C-(pr)M-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-3Ј. We previously constructed the first flavivirus replicons based on KUN cDNA by deleting the majority of the genomic region including structural genes (30) and used them extensively for the development of a gene expression system (2,19,28,30,44,45). KUN replicons were also used extensively in RNA replication and complementation studies (23)(24)(25)(26)(27)32) that contributed substantially to generating a comprehensive model for the formation and operation of the flavivirus RNA replication complex (47,48).The establishment of persistent noncytopathic replication by replicon RNAs of a number of positive-strand RNA viruses was shown to lead to the generation of adaptive mutations in nonstructural genes that either decreased (alphavirus replicons) or enhanced (hepatitis C replicons) RNA replication efficiency (1,4,13,14,34,39,41,50). Some of these adaptive mutations (e.g., in the NS5A protein of the hepatitis C replicon) were shown to increase the ability of RNA replication to resist the antiviral action of alpha/beta interferon (IFN-␣/␤) (41), while others (e.g., in the nsP2 protein of Sindbis virus) were shown to lead to more efficient IFN production (14). IFN response is the first line of cell defense against viral infections, and a majority of viruses have developed various strategies to overcome it, by either inhibiting IFN production or blocking IFN signaling (15,22,35,43). Recent studies of dengue virus have indicated that flaviviruses may interfere with early steps of IFN signaling and have implied roles for the small nonstructural proteins NS2A, NS4A, and NS4B in this process (37).In this study, we describe the identification of adaptive mutations in KUN replicon RNA that confirm an advantage in establishing persistent replication in a number of different cell lines. We also demonstrate that induction of IFN-␤ promoter-* Correspondi...