Protein Kinase Regulated by dsRNA Downregulates the Interferon Production in Dengue Virus- and dsRNA-Stimulated Human Lung Epithelial Cells

Abstract

BackgroundDengue virus (DENV) is found in the tropical and subtropical regions and affects millions of people annually. Currently, no specific vaccine or antiviral treatment against dengue virus is available. Innate immunity has been shown to be important for host resistance to DENV infection. Although protein kinase regulated by double-stranded RNA (PKR) has been found to promote the innate signaling in response to infection by several viruses, its role in the innate response to DENV infection is still unclear. Our study aimed to investigate the role of PKR in DENV-induced innate immune responses.Methodology/Principal FindingsBy RNAi, silencing of PKR significantly enhanced the expression of interferon (IFN)-β in DENV infected human lung epithelial A549 cells. Western blot and immunofluorescence microscopy data showed that PKR knockdown upregulated the activation of innate signaling cascades including p38 and JNK mitogen-activated protein kinases (MAPKs), interferon regulatory factor-3 and NF-κB, following DENV2 infection. Likewise, a negative regulatory effect of PKR on the IFN production was also observed in poly(IC) challenged cells. Moreover, the PKR knockdown-mediated IFN induction was attenuated by RIG-I or IPS-1 silencing. Finally, overexpression of a catalytically inactive PKR mutant (K296R), but not of a mutant lacking dsRNA binding activity (K64E) or the double mutant (K64EK296R), reversed the IFN induction mediated by PKR knockdown, suggesting that the dsRNA binding activity is required for PKR to downregulate IFN production.Conclusions/SignificancePKR acts as a negative regulator of IFN induction triggered by DENVs and poly(IC), and this regulation relies on its dsRNA binding activity. These findings reveal a novel regulatory role for PKR in innate immunity, suggesting that PKR might be a promising target for anti-DENV treatments.

Human Genome-Wide RNAi Screen Identifies an Essential Role for Inositol Pyrophosphates in Type-I Interferon Response

“…The analytic parameters for the siRNA screen including the signal intensity and Z-score distributions are given in the Figures S2A–D. We re-identified several previously known regulators of RIG-I pathway such as RIG-I, IRF3, TRIM21, PRKR and MAVS as hits, validating the ability of the screen to discover proteins that regulate the interferon response [35], [36]. More importantly, the RNAi screen led to the identification of a total of 226 additional novel regulators of RIG-I mediated IFNβ response (Table S1).…”

Interplay between Inflammation and Cellular Stress Triggered by Flaviviridae Viruses

“…PKR silencing resulted in decreased IFN-β production in hepatoma cells infected with DENV, but not in fibroblast nor macrophages (Li Y. et al, 2013). In addition, decreased protein synthesis induced by PKR activation might, in fact, inhibit the synthesis of ISG proteins, in spite of increased mRNA expression triggered by the RNA sensor (Garaigorta and Chisari, 2009).…”

IPS-1 plays an essential role in stress granule formation induced by dsRNA through interacting with PKR and mediating its activation

“…After transfection with siRNAs for 48 h, cells were transfected with 1 mg poly(I:C) by using Lipofectamine 2000 (Invitrogen, CA) for 6 h. Wholecell extracts were prepared in the presence of 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1% (v/v) protease inhibitor cocktail (Sigma, MO) as described previously (Li et al, 2013). Proteins were fractionated by electrophoresis on sodium dodecyl sulphate-10% polyacrylamide gels, transferred to nitrocellulose membranes, blocked and then probed with an appropriate dilution of primary antibody in PBS containing 3% (w/v) skimmed milk.…”