Abstract
Interferons (IFNs) play critical roles in host defense by modulating the expression of various genes via tyrosine phosphorylation of STAT transcription factors. IFN-alpha/beta activates another important transcription factor, nuclear factor-kappaB (NF-kappaB), but its role in IFN-mediated activity is poorly understood. Sensitivity to the antiviral and gene-inducing effects of IFN was examined in normal fibroblasts and in NF-kappaB knockout fibroblasts from p50- and p65-null mice. Antiviral assays demonstrated that NF-kappaB knockout fibroblasts were sensitized to the antiviral action of IFN. Moreover, analysis of IFN-stimulated gene expression by real-time PCR demonstrated selective effects of NF-kappaB on gene expression. Our results demonstrate that a subset of IFN-stimulated genes is regulated through an NF-kappaB-dependent pathway and that NF-kappaB may regulate the sensitivity of cells to IFN-mediated antiviral activity.
Highlights
Interferons (IFNs)1 are a family of multifunctional cytokines that block viral infection, inhibit cell proliferation, and modulate cell differentiation
Our results demonstrate that a subset of IFN-stimulated genes is regulated through an nuclear factor-B (NF-B)-dependent pathway and that NF-B may regulate the sensitivity of cells to IFN-mediated antiviral activity
To determine whether IFNs promote NF-B activation, fibroblasts derived from wild-type and p50 and p65 double knockout (p50:p65 KO) mice were stimulated with IFN-, and NF-B activation was examined by EMSA
Summary
Biological Reagents and Cell Culture—Recombinant Chinese hamster ovary cell-expressed rat IFN- was obtained from Biogen Idec, Inc. [6]. Assays for the Antiviral Activity of IFN—To determine cellular sensitivity to the ability of IFN to reduce virus titer, cell cultures were preincubated overnight with IFN, followed by infection with VSV for 1.5 h at 0.1 plaque-forming unit/cell. To determine the sensitivity to protection against the cytopathic effect of VSV, fibroblasts were cultured in 96-well plates, incubated with serial 2-fold dilutions of IFN for 24 h, infected with VSV (0.1 plaque-forming unit/cell), and scored for viability at 24 – 48 h postinfection by uptake of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium dye (Promega, Madison, WI). RNA Preparation and Microarray Analysis—Total cellular RNA from untreated and IFN-treated (2500 units/ml for 5 h) MEFs was extracted with TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. The raw expression data for six arrays (performed on RNAs prepared from three individual untreated and three IFN-treated fibroblast cultures) are given in Supplemental Table I. Proteins were transferred to polyvinylidene difluoride membranes; immunoblotted with anti-Isg, anti-nmi, or anti-actin antibody; and visualized by chemiluminescence with ECL reagent (Amersham Biosciences)
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