Abstract
The transient control of diverse biological responses that occurs in response to varied forms of stress is often a highly regulated process. During the interferon (IFN) response, translational repression due to phosphorylation of eukaryotic initiation factor 2alpha, eIF2alpha, by the double-stranded RNA-dependent protein kinase, PKR, constitutes a means of inhibiting viral replication. Here we show that the transient nature of the IFN response against acute viral infections is regulated, at least in part, by RNase L. During the IFN antiviral response in RNase L-null cells, PKR mRNA stability was enhanced, PKR induction was increased, and the phosphorylated form of eIF2alpha appeared with extended kinetics compared with similarly treated wild type cells. An enhanced IFN response in RNase L-null cells was also demonstrated by monitoring inhibition of viral protein synthesis. Furthermore, ectopic expression of RNase L from a plasmid vector prevented the IFN induction of PKR. These results suggest a role for RNase L in the transient control of the IFN response and possibly of other cytokine and stress responses.
Highlights
Negative feedback mechanisms are essential for maintaining transient responses such as those of the immune response by attenuating undesirable outcomes resulting from prolonged responses
PKR Activity in the Presence and Absence of RNase L—During the course of experiments aimed at studying the role of RNase L in controlling protein synthesis during acute viral infections, regulation of PKR was monitored in RNase Lϩ/ϩ and RNase LϪ/Ϫ mouse embryonic fibroblast (MEF) cell lines by different methods
The analysis revealed that the half-lives of PKR mRNA in RNase LϪ/Ϫ MEFs was 6.7 h compared with only 0.8 h in RNase Lϩ/ϩ cells (Fig. 6, A and B)
Summary
WISH cell line (HeLa markers) was obtained from the American Type Culture Collection (ATCC, Rockville, MD) and cultured in RPMI 1640 supplemented with 10% FBS and antibiotics. Confluent MEFs (2 ϫ 105 cells per well) in 24-well plates (Linbro, Flow Laboratories) were incubated in maintenance medium (DMEM plus 2% FBS) in the absence or presence of IFN-␣ at 1000 units/ml for 20 h. Protein (200 g) prepared from frozen (Ϫ70 °C) spleens and thymuses were separated by SDS-PAGE, transferred to membrane, and probed with rabbit anti-human PKR (crossreactive with mouse PKR) and goat anti-rabbit IgG-horseradish peroxidase (HRP, Invitrogen, Carlsbad, CA) and visualized (ECL, Amersham Biosciences). RNA signals were measured with a densitometer (Bio-Rad, Hercules, CA) and quantitated by ImageMaster image analysis software (Amersham Biosciences)
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