Inhibition of Interferon Signaling by the New York 99 Strain and Kunjin Subtype of West Nile Virus Involves Blockage of STAT1 and STAT2 Activation by Nonstructural Proteins

Abstract

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...

Genetic and Phenotypic Properties of Vero Cell-Adapted Japanese Encephalitis Virus SA14-14-2 Vaccine Strain Variants and a Recombinant Clone, Which Demonstrates Attenuation and Immunogenicity in Mice

“…Previously, it was shown that a single JEV E protein mutation (E138K) increased virus sensitivity to type I IFN, but this phenotype was coupled with altered levels of virus replication in tissue culture, 60 unlike the E protein mutations in JEV SA14-14-2 variants 2 IFN and C. However, the JEV NS4A protein and the NS2B and NS3 proteins of genetically related West Nile virus Kunjin are known to function as type I IFN-antagonists. 61,62 Therefore, it is likely that one or more unique non-structural protein mutations in JEV SA14-14-2 variants C, 2 IFN and 5 IFN modulated sensitivity to type I IFN in Vero cells.…”

Live Attenuated Chimeric Yellow Fever Dengue Type 2 (ChimeriVax™-DEN2) Vaccine: Phase I Clinical Trial for Safety and Immunogenicity: Effect of Yellow Fever Pre-immunity in Induction of Cross Neutralizing Antibody Responses to All

“…The anamnestic response among YF-immune subjects could have great implications for the development of an immunization strategy for a tetravalent vaccine by avoiding the potential interference problem that presenting a single YF-DEN chimeric virus poses to the host. In this instance, the induction of interferon occurs in concert with virus replication and is modulated effectively by the YF nonstructural proteins 18,19 to allow sufficient replication required for immunization. Since the initial immunizing agent (YF) is incapable of sensitizing the subject to DHF, there will be no danger that the first (priming) inoculation would leave the subject vulnerable to this disease if the second injection was delayed or not administered.…”

Inhibition of Interferon-Stimulated JAK-STAT Signaling by a Tick-Borne Flavivirus and Identification of NS5 as an Interferon Antagonist

“…However, use of IFN-␣/␤ in clinical trials for treatment of flavivirus infection has not been uniformly successful (34,50). This may be explained by the ability of some flaviviruses, including dengue virus serotype 2 (DEN-2), Japanese encephalitis virus, West Nile virus (WNV), and Kunjin virus (KUN) (16,30,33,38,39), to inhibit the JAK-STAT (Janus kinasesignal transducer and activator of transcription) pathway in response to IFN-␣/␤ and/or IFN-␥. Efforts to determine the specific gene products of these viruses responsible for IFN antagonism have implicated the nonstructural proteins.…”