Differential expression of interferon (IFN) regulatory factors and IFN-stimulated genes at early times after West Nile virus infection of mouse embryo fibroblasts

Abstract

Although lineage I West Nile virus (WNV) strain Eg101 induced beta interferon (IFN-beta) production as early as 12 h after infection in primary mouse embryo fibroblasts and did not inhibit the JAK-STAT signaling pathway, it was still able to replicate efficiently. To gain insights about possible viral countermeasures used by this virus to suppress the host response, the cell transcriptional profile and the kinetics of IFN regulatory factor (IRF) expression and activation were examined at early times after infection. By 12 h after WNV infection, the majority of the up-regulated genes were ones involved in IFN pathways. However, comparison of IFN-stimulated gene (ISG) expression levels in mock-infected, IFN-treated, and virus-infected cells indicated that WNV infection suppressed the up-regulation of a subset of ISGs, including genes involved in transcriptional regulation, apoptosis, and stress responses, prior to 24 h after infection. Analysis of mRNA and protein levels for representative genes indicated that suppression was at the transcriptional and posttranscriptional levels. Translocation of IRF-3 to the nucleus was observed beginning at 8 h, IRF-7 expression was detected by 12 h, but IRF-1 expression was not detected until 24 h after infection. Virus-induced gene suppression was sufficient to overcome the effect of exogenous IFN pretreatment for 1 h but not for 4 h prior to infection. These data indicate that WNV can selectively counteract the host response at early times after infection by previously unreported mechanisms.

FIG. 1.

Kinetics of WNV replication in primary He MEFs with or without IFN pretreatment. (A) Confluent monolayers of He MEFs were infected with WNV at an MOI of 10. (B) Confluent monolayers of He MEFs were treated as follows and then infected with WNV at an MOI of 10: untreated, incubated with 1,000 IU/ml of universal type I IFN for 1 or 4 h prior to infection, or treated with IFN for 1 h and then incubated with medium without IFN for 3 h prior to infection. Culture fluid samples harvested at the indicated times after infection were titrated for infectivity by plaque assay. Each data point is the average of duplicate titrations from two experiments. Error bars indicate standard deviations.

FIG. 2.

Hierarchical clustering of genes showing altered expression levels after WNV infection or IFN treatment in primary He MEFs. Total cellular RNA from mock-infected (M), WNV-infected (W), or IFN-treated (I) He MEFs was extracted and analyzed on 430A microarray chips as described in Materials and Methods. Only genes with about or more than a twofold increase in their expression levels in WNV-infected or IFN-treated cells compared to mock-infected MEFs were included in the hierarchical clustering shown. The color scale on the right indicates normalized expression values from the highest (5.0) to the lowest (0.0) levels.

FIG. 3.

Analysis of changes in the expression levels of selected genes after IFN treatment or WNV infection by real-time qRT-PCR. Primary He MEFs were mock infected (M), infected with WNV at an MOI of 10, or incubated with 1,000 IU/ml of universal type I IFN for 1 h followed by a 2-h incubation in medium without IFN (I). Changes in intracellular mRNA levels of the Zc3hdc1, Oas1g, Trim21, Rsad2, Oas-like 1, Tyki, B2m, Daxx, and Socs1 genes are shown. Infected cell samples were harvested 12, 24, or 48 h after infection (W12, W24, or W48, respectively). The mRNA level for each gene is expressed in RQ units as the log₁₀ relative change in expression compared to the level of the same mRNA present in mock-infected He cells or, in the case of the Oas-like 1 gene, in 12-h WNV-infected cells. Error bars represent the SE (n = 3) and are based on an RQ_Min/Max of the 95% confidence level. If the error bars for any two samples do not overlap, the expression levels of these samples are significantly different (P value of <0.05).

FIG. 4.

Real-time qRT-PCR and Western blot analysis of Casp11 expression in response to IFN treatment and/or WNV infection. Primary He MEFs were mock infected for 1 h (M), infected with WNV at an MOI of 10 (W), incubated with 1,000 IU/ml of universal type I IFN for 1 h and then either incubated with medium without IFN for 2 h (I) or infected with WNV for 1 h and incubated with medium for 1 h (I/W2). Replicate infected cultures were harvested at the indicated times after infection. (A) Relative quantification of intracellular Casp11 mRNA by real-time qRT-PCR. RNA levels are expressed in RQ units as the log₁₀ relative change in expression compared to the level of this mRNA in mock-infected cells. Each mRNA was normalized to GAPDH mRNA. Error bars represent the SE (n = 3). (B) Caspase 11 proteins in cell lysates were detected by Western blotting as described in Materials and Methods. The blots shown are representative of three independent experiments. (C) Primary He MEFs were treated with 10, 100, or 1,000 U/ml of mouse IFN-β, and intracellular mRNA levels expressed from the Casp11, Irf1, and Daxx genes were quantified by qRT-PCR.

FIG. 5.

Analysis of expression and activation of IRF-3 in WNV-infected He MEFs. (A) Primary He MEFs were mock infected for 1 h (M) or infected with WNV at an MOI of 10 (W). Replicate infected cultures were harvested at the indicated times after mock or WNV infection. IRF-3 dimers were detected by Western blotting after separation by electrophoresis on 7.5% nondenaturing (ND) polyacrylamide gels (left and right upper panels). Total IRF-3 and phosphoSer396 IRF-3 were detected by Western blotting after separation in 10% SDS-PAGE (middle panels). Actin was used as a cell protein control. (B) He MEFs were mock infected or infected with WNV for 8, 12, or 16 h. The cells were fixed, permeabilized, and incubated with anti-IRF-3 antibody at a dilution of 1:50, followed by incubation with secondary fluorescein isothiocyanate-conjugated antibody and visualized by confocal microscopy. Representative images are shown for each time point.

FIG. 6.

Analysis of IRF-1, IRF-7, and STAT5 expression and STAT5 and CrkL phosphorylation in WNV-infected He MEFs. Primary He MEFs were mock infected for 1 h (M), infected with WNV at an MOI of 10 (W), incubated with 1,000 IU/ml of universal type I IFN for 1 h and then incubated with medium without IFN for 2 h (I), or incubated with 1,000 IU/ml of universal type I IFN for 1 h and then infected with WNV (MOI of 10) for 2 h (I/W2). (A) Relative quantification of intracellular Irf7 mRNA by real-time qRT-PCR. RNA levels are expressed in RQ units as the log₁₀ relative change in expression compared to the level of this mRNA in mock-infected cells. Error bars represent the SE (n = 3). (B) Relative quantification of intracellular Irf7 mRNA by real-time qRT-PCR. Each mRNA was normalized to GAPDH mRNA. (C) IRF-1 and IRF-7 expression detected by Western blotting after separation by 10% SDS-PAGE. (D) Phosphorylated forms of STAT5 and CrkL as well as total STAT5 were detected by Western blotting after separation by 10% SDS-PAGE. Actin or eIF4E were used as cell protein controls. The blots shown are representative of three independent experiments.

FIG. 7.

Effect of different lengths of IFN pretreatment on IRF-1 expression and STAT1 phosphorylation after WNV infection. Primary He MEFs were mock infected for 1 h (M) or untreated (C) or incubated with 1,000 IU/ml of universal type I IFN for 1 h or 4 h and then either incubated with medium without IFN (M2 to M16) or infected with WNV (W2 to W16) for the indicated times. IRF-1 (A and B) or phospho-STAT1 (C and D) protein expression was detected by Western blotting after protein separation by 10% SDS-PAGE. eIF4E was used as a protein control. The blots shown are representative of three independent experiments.