Abstract
Double-stranded RNA (dsRNA) molecules generated during virus infection can initiate a host antiviral response to limit further infection. Such a response involves induction of antiviral gene expression by the dsRNA-activated protein kinase (PKR) and the NF-kappaB transcription factor. In addition, dsRNA can also induce apoptosis by an incompletely understood mechanism that may serve to further limit viral replication. Here we demonstrate a novel role for the RelA subunit of NF-kappaB in inhibiting dsRNA-induced cell death. dsRNA treatment resulted in caspase 3 activation and apoptotic morphological transformations in mouse embryonic fibroblasts (MEFs) derived from RelA-/- mice but not from RelA+/+ mice. Such dsRNA-induced killing could be inhibited by expression of either a dominant-negative mutant of PKR or wild-type RelA. Interestingly, caspase 3 activated following dsRNA treatment of RelA-/- MEFs was essential for apoptotic nuclear changes but dispensable for cytotoxicity. A broader specificity caspase inhibitor was also unable to inhibit dsRNA-induced cytotoxicity, suggesting that caspase activation is not essential for the induction of cell death by dsRNA in MEFs. However, combined inhibition of caspase 3 and reactive oxygen species production resulted in complete inhibition of dsRNA-induced cytotoxicity. These results demonstrate an essential role for NF-kappaB in protecting cells from dsRNA-induced apoptosis and suggest that NF-kappaB may inhibit both caspase-dependent and reactive oxygen species-dependent cytotoxic pathways.
Highlights
Virus replication within infected cells results in generation of double-stranded RNA1 molecules that can trigger host antiviral responses [1]
The RelA Subunit of NF-B Is Required for Double-stranded RNA (dsRNA)-induced Gene Expression—Our recent studies have revealed a critical role for the RelA subunit of NF-B in the regulation of TNF␣ and LPS-induced Fas expression [26]
RelAϩ/Ϫ mouse embryonic fibroblasts (MEFs) [19] were treated with dsRNA for 2 h, after which nuclear extracts were tested for B-site binding activity by Electrophoretic mobility shift assay (EMSA)
Summary
Fetal liver macrophages, and 3T3 fibroblasts were derived as described previously [21]. Human TNF␣ was obtained from R & D Systems and used at concentration of 10 ng/ml in all experiments. DsRNA poly(I-C) was purchased from Sigma and used at a final concentration of 100 g/ml. Actinomycin D and antioxidant butylated hydroxyanisole (BHA) were obtained from Sigma and used at 2 g/ml and 100 M, respectively. PLPC expression vector was a gift from Dr S. Lowe (Cold Spring Harbor Laboratory, New York). PRelA was constructed by cloning the mouse RelA cDNA into pLPC. PPKRDN, which has a six-amino acid deletion as described previously [29], was a gift from Dr A. GarcıaSastre (Mount Sinai Medical Center, New York)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
