Protein kinase regulated by dsRNA downregulates the interferon production in dengue virus- and dsRNA-stimulated human lung epithelial cells

Abstract

Background: Dengue 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 findings: By 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/significance: PKR 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.

Figure 1. Knockdown of PKR significantly increased IFN-β expression following DENV infection.

A549, HepG2 and THP-1 cells were transfected with siRNAs against PKR (siPKR1, and siPKR2) or control siRNA with a scramble sequence (siNC), and harvested at 48 h post transfection for Western blot (A). Transfected cells were infected with DENV2 for 16 h. Expression of IFN-β in DENV2-infected A549, HepG2 and THP-1 cells was evaluated by real-time PCR and normalized to GAPDH (B-D), ELISA was utilized to measure secreted IFN-β in A549 cells following DENV2 infection (E). Expression of IFN-β in DENV1- and DENV3- infected A549 cells was tested by real-time PCR and normalized to GAPDH (F). Data are shown as the mean ± SEM (standard error of the mean) and represent at least three independent experiments. *p<0.05,**p<0.01, ***p<0.001.

Figure 2. PKR silencing enhanced the activation of p38 and JNK MAPKs, IRF3 and NF-κB.

A549 cells were transfected with siPKR1 or siNC followed by infection with DENV2. (A), Western blot to show the phosphorylation form and total protein levels of the MAPKs p38 and JNK, IRF3 and PKR. (B), Immunoflurescence microscopy images showing the subcellular localization of NF-κB p65. (C), PicCnt 100x was used to determine the percentages of cells with nuclear staining for NF-κB. Data are shown as the mean ± SEM and represent at least three independent experiments. *p<0.05.

Figure 3. The IFN induction mediated PKR knockdown was dependent on RIG-I and IPS-1, but not MDA-5.

A549 cells were transfected with siRNA against RIG-I (siRIG-I), MDA-5 (siMDA-5) or IPS-1 (siIPS-1). Protein levels of RIG-I (A), MDA-5 (B) and IPS-I (C) were detected by Western blot. A549 cells were cotransfected with siRIG-I, siMDA-5 or siIPS-1 together with siPKR1 or siNC for 48 h followed by DENV2 infection (D, E). Total cellular RNA was analyzed for IFN-β using real-time PCR and normalized to that of GAPDH in each sample. Data are shown as the mean ± SEM and represent at least three independent experiments. *p<0.05, **p<0.01.

Figure 4. The dsRNA binding activity of PKR was important for downregulation of IFN induction.

A549 cells were transfected with pcDNA6, pcDNA6-K64E, pcDNA6-K296R or pcDNA6-K64EK296R followed by siNC or siPKR1 transfection. Cells were then harvested for Western blot analysis (A). A549 cells transfected with different PKR mutants and siPKR1 were stimulated by DENV2 infection. Total cellular RNA was harvested for real-time PCR analysis (B). Data are shown as the mean ± SEM and represent for at least three independent experiments. *p<0.05.

Figure 5. PKR depletion barely affected DENV2 replication in A549 cells.

A549 cells were transfected with siNC, siPKR1, siRIG-I and siIPS-1, followed by DENV2 infection. Cells and culture supernatants were harvested for real-time PCR and viral titration respectively. Total cellular RNA was analyzed for IFN-β (A) or DENV2 RNA (B) level by real-time PCR and normalized to that of GAPDH in each sample. Virus yields were determined by TCID50 assay (C). DENV2 RNA levels in siNC- or siPKR-transfected HepG2 and THP-1 cells were measured by real-time PCR (D). Data are shown as mean ± SEM at least three independent experiments. Phosphorylation form and total protein levels of eIF-2α in DENV2-infected A549 cells were detected by Western blot (E). *p<0.05,**p<0.01, ***p<0.001.

Figure 6. PKR knockdown enhanced IFN expression and activation of MAPKs, IRF3 and NF-κB pathways induced by poly(I:C).

A549 cells were transfected with siPKR1 or siNC for 48 h, and then transfected with poly(I:C) for 1 h. Expression levels of IFN-β (A) were tested by real-time PCR and normalized to GAPDH. Phosphorylation form and total protein of p38 and JNK MAPKs, IRF3 and PKR were detected by Western blot (B). Immunoflurescence microscopy images to show subcellular localization of NF-κB p65 (C). PicCnt 100x was used to determine the percentages of cells stained with nuclei NF-κB (D). Data are shown as mean ± SEM for at least three independent experiments. *p<0.05,**p<0.01.

Figure 7. Suppression of poly(I:C) induced IFN by PKR required RIG-I/IPS-I and dsRNA binding activity of PKR.

(A, B) A549 cells were co-transfected with siRIG-I, siMDA-5 or siIPS-1 together with siPKR1 or siNC followed by poly(I:C). Total cellular RNA levels were analyzed for IFN-β using real-time PCR and normalized to those of GAPDH in each sample. (C) Cells were transfected with pcDNA6, pcDNA6-K64E, pcDNA6-K296R or pcDNA6-K64EK296R followed by siNC or siPKR1 transfection, and were then stimulated by poly(I:C). Total cellular RNA was analyzed for IFN-β expression by real-time PCR and normalized to that of GAPDH in each sample. Data are shown as mean ± SEM at least three independent experiments. *p<0.05, **p<0.01.

Figure 8. Expression of RIG-I in different cell lines.

A549, HepG2 and THP-1 cells were harvested for real-time PCR analysis (A) or for Western blot analysis (B). Total cellular RNA was analyzed for RIG-I expression by real-time PCR and normalized to that of GAPDH in each sample. Data are shown as mean ± SEM at least three independent experiments.