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.

Although infections with "natural" West Nile virus (WNV) and the chimeric W956IC WNV infectious clone virus produce comparable peak virus yields in type I interferon (IFN) response-deficient BHK cells, W956IC infection produces higher levels of "unprotected" viral RNA at early times after infection. Analysis of infections with these two viruses in IFN-competent cells showed that W956IC activated NF-κB, induced higher levels of IFN-ß, and produced lower virus yields than WNV strain Eg101. IPS-1 was required for both increased induction of IFN-ß and decreased yields of W956IC. In Eg101-infected cells, phospho-STAT1/STAT2 nuclear translocation was blocked at all times analyzed, while some phospho-STAT1/STAT2 nuclear translocation was still detected at 8 h after infection in W956IC-infected mouse embryonic fibroblasts (MEFs), and early viral protein levels were lower in these cells. A set of additional chimeras was made by replacing various W956IC gene regions with the Eg101 equivalents. As reported previously, for three of these chimeras, the low early RNA phenotype of Eg101 was restored in BHK cells. Analysis of infections with two of these chimeric viruses in MEFs detected lower early viral RNA levels, higher early viral protein levels, lower early IFN-ß levels, and higher virus yields similar to those seen after Eg101 infection. The data suggest that replicase protein interactions directly or indirectly regulate genome switching between replication and translation at early times in favor of translation to minimize NF-κB activation and IFN induction by decreasing the amount of unprotected viral RNA, to produce sufficient viral protein to block canonical type I IFN signaling, and to efficiently remodel cell membranes for exponential genome amplification.

Identification of novel host cell binding partners of Oas1b, the protein conferring resistance to flavivirus-induced disease in mice.

Oas1b was previously identified as the product of the Flv(r) allele that confers flavivirus-specific resistance to virus-induced disease in mice by an uncharacterized, RNase L-independent mechanism. To gain insights about the mechanism by which Oas1b specifically reduces the efficiency of flavivirus replication, cellular protein interaction partners were identified and their involvement in the Oas1b-mediated flavivirus resistance mechanism was analyzed. Initial difficulties in getting the two-hybrid assay to work with full-length Oas1b led to the discovery that this Oas protein uniquely has a C-terminal transmembrane domain that targets it to the endoplasmic reticulum (ER). Two peptides matching to oxysterol binding protein-related protein 1L (ORP1L) and ATP binding cassette protein 3, subfamily F (ABCF3), were identified as Oas1b interaction partners in yeast two-hybrid assays, and both in vitro-transcribed/translated peptides and full-length proteins in mammalian cell lysates coimmunoprecipitated with Oas1b. Knockdown of a partner involved in Oas1b-mediated antiflavivirus activity would be expected to increase flavivirus replication but not that of other types of viruses. However, RNA interference (RNAi) knockdown of ORP1L decreased the replication of the flavivirus West Nile virus (WNV) as well as that of other types of RNA viruses. This virus-nonspecific effect may be due to the recently reported dysregulation of late endosome movement by ORP1L knockdown. Knockdown of ABCF3 protein levels increased the replication of WNV but not that of other types of RNA viruses, and this effect on WNV replication was observed only in Oas1b-expressing cells. The results suggest that Oas1b is part of a complex located in the ER and that ABCF3 is a component of the Flv(r)-mediated resistance mechanism.

West nile virus infections suppress early viral RNA synthesis and avoid inducing the cell stress granule response.

West Nile virus (WNV) recently became endemic in the United States and is a significant cause of human morbidity and mortality. Natural WNV strain infections do not induce stress granules (SGs), while W956IC (a lineage 2/1 chimeric WNV infectious clone) virus infections produce high levels of early viral RNA and efficiently induce SGs through protein kinase R (PKR) activation. Additional WNV chimeric viruses made by replacing one or more W956IC genes with the lineage 1 Eg101 equivalent in the W956IC backbone were analyzed. The Eg-NS4b+5, Eg-NS1+3+4a, and Eg-NS1+4b+5 chimeras produced low levels of viral RNA at early times of infection and inefficiently induced SGs, suggesting the possibility that interactions between viral nonstructural proteins and/or between viral nonstructural proteins and cell proteins are involved in suppressing early viral RNA synthesis and membrane remodeling during natural WNV strain infections. Detection of exposed viral double-stranded RNA (dsRNA) in W956IC-infected cells suggested that the enhanced early viral RNA synthesis surpassed the available virus-induced membrane protection and allowed viral dsRNA to activate PKR.