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


RESULTS
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
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.
Department of Immunology, Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China.
Yuye
Y
Li
Jiong
J
Xie
Siyu
S
Wu
Jun
J
Xia
Peifen
P
Zhang
Chao
C
Liu
Ping
P
Zhang
Xi
X
Huang
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D056809:Alveolar Epithelial Cells / Q000276:immunology / Q000378:metabolism* / Q000821:virology*; D002460:Cell Line; D000071457:DEAD Box Protein 58; D053487:DEAD-box RNA Helicases / Q000235:genetics / Q000378:metabolism; D003715:Dengue / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D003716:Dengue Virus / Q000502:physiology*; D005786:Gene Expression Regulation; D055785:Gene Knockdown Techniques; D056945:Hep G2 Cells; D006801:Humans; D007113:Immunity, Innate; D050838:Interferon Regulatory Factor-3 / Q000378:metabolism; D000072640:Interferon-Induced Helicase, IFIH1; D007372:Interferons / Q000096:biosynthesis*; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D011070:Poly I-C / Q000378:metabolism; D012330:RNA, Double-Stranded / Q000378:metabolism; D011971:Receptors, Immunologic; D014779:Virus Replication; D019892:eIF-2 Kinase / Q000235:genetics / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D050838:Interferon Regulatory Factor-3; C505371:MAVS protein, human; D016328:NF-kappa B; D012330:RNA, Double-Stranded; D011971:Receptors, Immunologic; D007372:Interferons; D019892:eIF-2 Kinase; D048031:JNK Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; C452820:RIGI protein, human; C452815:IFIH1 protein, human; D000071457:DEAD Box Protein 58; D053487:DEAD-box RNA Helicases; D000072640:Interferon-Induced Helicase, IFIH1; D011070:Poly I-C