Type 1 IFN-independent activation of a subset of interferon stimulated genes in West Nile virus Eg101-infected mouse cells

Abstract

Although infection of mouse embryofibroblasts (MEFs) with WNV Eg101 induced interferon (IFN) beta production and STAT1 and STAT2 phosphorylation, these transcription factors (TFs) were not detected in the nucleus or on the promoters of four IRF-3-independent interferon stimulated genes (ISGs): Oas1a and Irf7 (previously characterized as IFN/ISGF3-dependent), Oas1b and Irf1. These ISGs were upregulated in WNV Eg101-infected STAT1−/−, STAT2−/−, and IFN alpha/beta receptor −/− MEFs. Although either IRF-3 or IRF-7 could amplify/sustain Oas1a and Oas1b upregulation at later times after infection, these factors were not required for the initial gene activation. The lack of upregulation of these ISGs in WNV Eg101-infected IRF-3/9−/− MEFs suggested the involvement of IRF-9. Activation of Irf1 in infected MEFs did not depend on any of these IRFs. The data indicate that additional alternative activation mechanisms exist for subsets of ISGs when a virus infection has blocked ISG activation by the canonical IFN-mediated pathway.

Increased Early RNA Replication by Chimeric West Nile Virus W956IC Leads to IPS-1-Mediated Activation of NF-κB and Insufficient Virus-Mediated Counteraction of the Resulting Canonical Type I Interferon Signaling

“…3). As previously reported, STAT2 was located in the cytoplasm in cells infected with Eg101 at 8, 12, and 16 h after infection (18). In contrast, some STAT2 was observed both in the cytoplasm and nucleus in some cells infected with W956IC at 8 h after infection (Fig.…”

“…Since a number of known and yet to be identified IPS-1 downstream molecules are expected to cross talk in mediating the signal to IFN-␤ and ISGs, further studies are needed to fully elucidate the mechanisms of induction of the antiviral state in WNV-infected cells. The results of a recent study from our lab showed that the initial upregulation of a set of ISGs in MEFs infected with the natural WNV strain Eg101 was only partially dependent on IPS-1 and that upregulation of these ISGs was independent of IFN-␤ both at early and late times of infection (18). The present study revealed a role for IPS-1 in mediating NF-B activation and IFN-␤ induction in cells infected with W956IC virus.…”

“…However, a previous study from our lab reported that even though STAT1 and STAT2 were phosphorylated in WNV-infected MEFs and even though phospho-STAT1/STAT2 translocation to the nucleus was efficiently blocked, IFN-independent upregulation of the ISGs, Oas1a, Oas1b, Irf7, and Irf1, was still observed (18). To determine whether the higher early IFN induction resulted in increased ISG upregulation, Oas1a, Irf7, and Irf1 mRNA levels were analyzed by real-time qRT-PCR in C3H/He MEFs infected with Eg101 or W956IC at 16, 24, and 48 h after infection.…”

“…Phosphorylation of STAT1 and STAT2 proteins was previously shown to be blocked in human cells infected with natural strains of WNV (30)(31)(32), but STAT1 and STAT2 phosphorylation were not inhibited in the mouse cells ( Fig. 2E) (18). Phosphorylation of STAT1 on Tyr701 was therefore next compared in A549 human cells infected with Eg101 or W956IC at an MOI of 1.…”

“…PKR has also been reported to play a role in both NF-B and type I IFN signaling in response to viral infections (17). Our lab previously reported that although infections in MEFs with WNV Eg101 and other natural WNV strains induce IFN-␤ production and phosphorylation of signal transducer and activator of transcription 1 and 2 (STAT1 and STAT2, respectively), these TFs were not detected in the nuclei or on the promoters of four IRF-3-independent ISGs in infected MEFs at any of the times after infection analyzed (18).…”