Viral noncoding RNAs have been shown to play an important role in virus-host interplay to facilitate virus replication. We report that members of the genus Flavivirus, a large group of medically important encephalitic RNA viruses, produce a unique and highly structured noncoding RNA of 0.3-0.5 kb derived from the 3' untranslated region of the viral genome. Using West Nile virus as a model, we show that this subgenomic RNA is a product of incomplete degradation of viral genomic RNA by cellular ribonucleases. Highly conserved RNA structures located at the beginning of the 3' untranslated region render this RNA resistant to nucleases, and the resulting subgenomic RNA product is essential for virus-induced cytopathicity and pathogenicity. Thus, flaviviruses evolved a unique strategy to generate a noncoding RNA product that allows them to kill the host more efficiently.

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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