Resistance to flavivirus-induced disease is conferred by a single gene that encodes oligoadenylate synthetase (Oas) 1b (Oas1b). Oas1b is not a functional synthetase suggesting its anti-flavivirus mechanism is RNase L-independent and that it may be mediated by interactions with other host cell protein(s). A yeast two-hybrid screen was used to identify host cell binding partners of Oas1b. Candidate partners were confirmed by yeast co-transformation and co-immunoprecipitation analyses. Oxysterol binding protein-related 1L (ORP1L) and ATP binding cassette subfamily F 3 (ABCF3) were found to interact with Oas1b. RNAi knockdown studies suggested that ORP1L and ABCF3 form a tripartite complex with Oas1b that is critical for the flavivirus-induced disease resistance mechanism. Stresses including oxidation, nutrient starvation, and viral infections often induce the formation of stress granules (SGs) in eukaryotic cells. In response to stress, eIF2α kinases phosphorylate eIF2α leading to stalled 48S pre-initiation complexes and SG formation. West Nile virus (WNV) Eg101 infections were previously shown not to induce the formation of SGs. Infections with viruses of other natural WNV strains, as well as a WNV lineage 1/2-based infectious clone (W956IC) were analyzed and only W956IC infections were found to induce SGs. eIF2α kinase knockout MEFs were used to show that the W956IC-induced SGs were PKR-dependent. WNV chimeras were made by inserting Eg101 genes into the W956IC backbone. Chimeras replacing NS5 or NS1 and NS5 or NS1 and NS3 and NS4a reduced SG formation as well as early viral RNA synthesis similar to Eg101 infections. W956IC infections but not Eg101 infections were shown to produce exposed viral dsRNA at early times after infection. The data suggest that natural WNV infections evade the cell SG response by suppressing the amplification of viral RNA until cytoplasmic membranes have been remodeled to protect replication complexes from detection. It was previously reported that WNV Eg101 infections inhibited the formation of arsenite-induced SGs. The ability of other natural WNV strain infections to inhibit SG formation by arsenite (HRI), DTT (PERK), W956IC co-infection (PKR), and heat shock treatments was assessed. WNV infections only inhibited arsenite-induced SG formation suggesting that WNV infections specifically suppress the response to oxidative intermediates.

Two cellular innate responses, the dsRNA protein kinase (PKR) pathway and the 2'-5' oligoadenylate synthetase (OAS)/RNase L pathway, are activated by dsRNAs produced by viruses and reduce translation of host and viral mRNAs. PKR activation results in eIF2a phosphorylation. As a consequence of eIF2a phosphorylation, stress granules (SGs) are formed by the aggregation of stalled SG proteins with pre-initiation complexes and mRNA. West Nile virus (WNV) infections do not induce eIF2a phosphorylation despite upregulation of PKR mRNA and protein suggesting an active suppression of PKR activation. Assessment of the mechanism of suppression of PKR activation in WNV-infected cells indicated that WNV infections do not induce PKR phosphorylation so that active suppression is not required. In contrast to infections with "natural" strains of WNV, infections with the chimeric W956 infectious clone (IC) virus efficiently induce SGs in infected cells. After two serial passages, the IC virus generated a mutant (IC-P) that does not induce SGs efficiently but does induce the formation of NS3 granules that persist throughout the infection. This mutant was characterized. 2'-5' oligoadenylate synthetases (OAS) are activated by viral dsRNA to produce 2-5A oligos that activate RNase L to digest viral and cellular RNAs. Resistance to flavivirus-induced disease in mice is conferred by the full-length 2'-5' oligoadenylate synthetase 1b (Oas1b) protein. Oas1b is an inactive synthetase that is able to suppress the in vitro synthetase activity of the active synthetase Oas1a. The ability of Oas1b to inhibit Oas1a synthetase activity in vivo and to form a heteromeric complex with Oas1a was investigated. Oas1b suppressed 2-5A production in vivo. Oas1a and Oas1b overexpressed in mammalian cells co-immunoprecipitated indicating the formation of heteromeric complexes by these proteins. Unlike mice, humans encode a single OAS1 gene that generates alternatively spliced transcripts encoding different isoforms. Synthetase activity has previously been reported for only three of the isoforms. The in vitro synthetase activity of additional OAS1 isoforms was analyzed. All tested isoforms synthesized higher order 2-5A oligos. However, p44A only produced 2-5A dimers which inhibit RNase L.

The induction of type I interferon (IFN) and subsequent activation of interferon stimulated genes (ISGs) represent a first line of defense against viral infection. Typically type I IFN signaling leads to the phosphorylation of the STAT1 and STAT2 transcription factors (TFs) which then form a trimetric complex with IRF-9 and translocate to the nucleus to induce ISG expression. However, the results of this study showed that IFN-mediated upregulation of the ISG Oas1b, the product of which confers resistance to flavivirus induced disease, can be induced in a STAT1-independent manner. Since numerous ISGs have antiviral functions, many viruses have evolved strategies to disrupt the type I IFN-signaling pathway. In cases when STAT1 activation is blocked by a viral infection, STAT1-independent upregulation of ISGs provides an additional strategy for the cell to mount an effective antiviral response. Infection of mouse embryofibroblasts (MEFs) with West Nile virus (WNV) induced the production of IFN beta and STAT1 and STAT2 phosphorylation but blocked nuclear translocation and binding of these TFs to the promoters of the ISGs, Oas1a, Oas1a, Irf7 and Irf1. However, each of these antiviral ISGs was efficiently upregulated in infected cells and IRF-9 was shown to be crucial for the upregulation of Oas1a, Oas1b and Irf-7. IRF-3 or IRF-7 was needed to maintain the upregulation of these genes at later times of infection. In contrast, the upregulation of Irf1 by WNV infection did not depend on the tested IRFs but was reduced by inhibition of the p38 or NF-kappa B pathways. Although Irf1 mRNA was efficiently upregulated in WNV-infected cells IRF-1 protein synthesis was blocked. The precise mechanism of the IRF-1 translational suppression is not yet known, but the suppression was shown not to be due to increased proteasomal degradation of IRF-1 nor to alternative splicing of Irf1 mRNA. Preliminary results suggest miRNAs may play an indirect role in regulating IRF-1 translation. The results of this study expand knowledge about the strategies evolved by viruses to evade host cell antiviral responses and also provide valuable insights about alternative mechanisms utilized by the host cell to counteract viral infections.