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received: 30 January 2016 accepted: 20 May 2016 Published: 10 June 2016
Japanese Encephalitis Virus exploits the microRNA-432 to regulate the expression of Suppressor of Cytokine Signaling (SOCS) 5 Nikhil Sharma1, Kanhaiya L. Kumawat2, Meghana Rastogi3, Anirban Basu2 & Sunit K. Singh3 Japanese encephalitis virus (JEV) is a plus strand RNA virus, which infects brain. MicroRNAs are regulatory non-coding RNAs which regulate the expression of various genes in cells. Viruses modulate the expression of various microRNAs to suppress anti-viral signaling and evade the immune response. SOCS (Suppressor of cytokine signalling) family of proteins are negative regulators of anti-viral JakSTAT pathway. In this study, we demonstrated the regulatory role of SOCS5 in Jak-STAT signaling and its exploitation by JEV through a microRNA mediated mechanism. JEV infection in human brain microglial cells (CHME3) downregulated the expression of miR-432, and upregulated SOCS5 levels. SOCS5 was validated as a target of miR-432 by using 3′UTR clone of SOCS5 in luciferase vector along with miR-432 mimic. The overexpression of miR-432 prior to JEV infection enhanced the phosphorylation of STAT1 resulting into increased ISRE activity and cellular inflammatory response resulting into diminished viral replication. The knockdown of SOCS5 resulted into increased STAT1 phosphorylation and suppressed viral replication. JEV infection mediated downregulation of miR-432 leads to SOCS5 upregulation, which helps the virus to evade cellular anti-viral response. This study demonstrated that JEV utilizes this microRNA mediated strategy to manipulate cellular immune response promoting JEV pathogenesis. Japanese encephalitis virus (JEV) is a mosquito borne plus strand RNA virus which infects CNS and resides in neuronal and microglial cells. JEV is a neurotropic virus causing neuroinflammation and neuronal damage1. It mainly infects children between 1–5 years of age and leads to the permanent neuronal damage, motor deficits and memory loss2. JEV has adopted a zoonotic life cycle between pigs, water birds and Culex mosquitoes. Human are dead end host for the virus3. In spite of vaccines available against JEV, three million deaths have been estimated due to lack of effective anti-viral drug against JEV4. JEV is able to modulate the anti-viral immune signalling pathways to dampen the cellular anti-viral response. MicroRNAs are small RNAs (19–24 nucleotides) regulating the expression of about 60% of human genes by binding through their seed region to the complementary sites present in 3′UTR of target gene5. Viruses have been reported to modulate the expression pattern of cellular microRNAs either to suppress immune response or to increase the rate of replication6,7. Viral proteins have also been reported to modulate miRNA expression. Bakre et al.8, reported that respiratory syncytial virus (RSV) NS1 protein interacts with KLF6 transcription factor to modulate miR-24 expression which facilitates viral replication8. JEV has been reported to induce the expression of miR-15b which targets ring finger protein RNF125, a negative regulator of RIG-1 pathway9. We have also reported the miR-146a mediated regulation of anti-viral Jak-STAT pathway, by JEV to promote its survival10. The expression of interferon (IFN) during viral Infections triggers the anti-viral Jak-STAT signaling (JakJanus kinases; STAT- Signal transducer and activator of transcription) in the infected cells. Interferon binds to IFN-α/βreceptors, which recruits Janus kinases (Jak). Jak autophosphorylates itself and activates the STATs (Signal Transducer and Activator of Transcription) by phosphorylation11. STATs undergo phosphorylation and 1
Laboratory of Neurovirology and Inflammation Biology, CSIR-Centre for Cellular and Molecular Biology (CCMB), Uppal Road, Hyderabad-500007, India. 2National Brain Research Centre Manesar, Haryana-122050, Haryana, India. 3 Laboratory of Human Molecular Virology & Immunology, Molecular Biology Unit, Faculty of Medicine, Institute of Medical Sciences (IMS), Banaras Hindu University (BHU), Varanasi-221005, India. Correspondence and requests for materials should be addressed to S.K.S. (email:
[email protected]) Scientific Reports | 6:27685 | DOI: 10.1038/srep27685
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www.nature.com/scientificreports/ dimerization and bind to IRF-9 to form ISGF-3 complex (Interferon Stimulated Gene Factor-3) which enters in nucleus to bind ISRE elements present at promoter region of Interferon Stimulated genes12. This anti-viral mechanism of the infected cells is negatively regulated by SOCS (Suppressor of Cytokine Signaling) family of proteins which include various members like SOCS1, SOCS2, SOCS3, and SOCS513. SOCS1 and SOCS3 are well studied inhibitory proteins of Jak-STAT pathway. Viruses have been reported to induce the expression of SOCS proteins to suppress cellular anti-viral response14. Linossi et al. demonstrated that SOCS5 interacts with Jak (Janus Kinases) via its Jak interacting region (JIR) and inhibits the auto-phosphorylation of Jak1 and Jak215. This leads to the inhibition of the kinase activity of Jak and hence, SOCS5 negatively regulate Jak-STAT signalling. However, the function of SOCS5 has not been well studied with respect to the viral infections. miR-432 has been reported to activate Wnt/β-catenin signalling and its downregulation in hepatocytes promoted hepatocellular carcinoma16. Chen et al. has also demonstrated tumor suppressive role of miR-432 in lung cancer cells17. The role of miR-432 in modulating immune signalling pathways during viral infections has not been well elucidated. In our study, we found downregulated expression of miR-432 in our microRNA profiling data in JEV infected CHME3 cells. Bioinformatics tools predicted SOCS5 as a potential target of miR-432 so we elucidated the role of microRNA mediated regulation of SOCS5 during JEV infection. We have demonstrated downregulation of miR432 by JEV leads to SOCS5 upregulation in JEV infected CHME3 cells as well as in JEV infected mice brain. We validated SOCS5 as a target of miR-432 and demonstrated the effect of overexpression and silencing of miR-432 on ISRE activity, production of pro-inflammatory cytokines and its effect on viral replication.
Results
JEV infection downregulates miR-432 expression. JEV infection modulates cellular microRNA
expression pattern, which further alters the expression of various genes. The miR-432 was found to be downregulated in CHME3 cells upon JEV infection (Fig. 1A). Replication of JEV in CHME3 cells was confirmed by real time PCR (Supplementary Fig. 2A). The validation of miR-432 downregulation upon JEV infection was also done in brain tissue of JEV infected mice in-vivo (Fig. 1B). Replication of JEV in mice brain was confirmed by real time PCR (Supplementary Fig. 2B). Since SOCS5 is a potential target of miR-432, therefore the levels of SOCS5 expression was analysed post JEV infection. JEV induced the SOCS5 expression levels in CHME3 cells (Fig. 1C). The seed region for mature miR-432 was found to be conserved in both human and mouse. Therefore SOCS-5 expression levels were checked in mice brain tissue by western blotting and found to be upregulated (Fig. 1D). In addition, we analysed SOCS5 expression in JEV infected mice brain by immunohistochemistry and found enhanced levels of SOCS5 in JEV infected mice brain tissue (Fig. 1E). This demonstrated that JEV downregulated miR-432 levels in mouse brain, which led SOCS5 upregulation. SOCS5 has been reported to inhibit auto-phosphorylation of Jak115 which inhibits Jak-STAT pathway downstream.
miR-432 targets SOCS5. To confirm the targeting of SOCS5 UTR by miR-432, mimic sequence of miR-432
was overexpressed in CHME3 cells along with scramble control. The overexpression and silencing of miR-432 was confirmed by real time PCR. The SOCS5 was downregulated upon miR-432 overexpression (Fig. 2A). To further delineate the effect of miR-432 on SOCS5, antimiR-432 was overexpressed in CHME3 cells, which resulted into increased levels of SOCS5 upon antimiR-432 overexpression (Fig. 2C). To further validate the targeting of 3′U TR of SOCS5 by miR-432 mimic, luciferase vector containing 3′UTR of SOCS5 was transfected along with miR-432 mimic. The decreased luciferase activity was observed due to targeting of SOCS5 3′UTR by miR-432 (Fig. 2D). A mutant was generated by deleting the targeting site of miR-432 present in 3′UTR of SOCS5 and cloned in the luciferase vector. The mutated UTR cloned in luciferase vector did not display any reduction of luciferase activity, when transfected along with miR-432 mimic. This confirmed the targeting of 3′UTR of SOCS5 by miR-432.
miR-432 overexpression enhances Jak-STAT signaling by downregulating SOCS5. SOCS5 is a negative regulator of Jak-STAT pathway18 so the effect of miR-432 overexpression was analysed on Jak-STAT pathway. Overexpression of miR-432 led to the downregulation of SOCS5 and this resulted to enhanced phosphorylation of STAT1 post JEV infection (Fig. 3A). Hence, this confirmed the negative regulatory effect of SOCS5 on Jak-STAT pathway. To further visualize the effect of miR-432 downstream STAT1 phosphorylation, the ISRE activity was visualized by luciferase vector containing ISRE sequences at promoter site. The enhanced luciferase activity was observed in miR-432 overexpressing cells post JEV infection (Fig. 3E). The increased ISRE activity was due to increased phosphorylation of STAT1. To further confirm the specificity of this effect, anti-miR-432 was transfected into cells prior to JEV infection. AntimiR-432 transfection led to the decreased phosphorylation of STAT1 (Fig. 3D) and reduced ISRE activity post-JEV infection (Fig. 3F). miR-432 downregulated SOCS5; which led to the enhanced phosphorylation of STAT1 and increased ISRE activity. miR-432 overexpression suppress viral replication. During the course of infection, JEV gradually increases its copy number by supressing the anti-viral machinery of the cell. Fan et al. has studied the kinetics of JEV replication in BHK-21 cells and found that JEV genome increases till 36 hours post infection and later attain a plateau stage19. Interferon secreted upon JEV infection binds to IFN-α/βreceptor and leads to phosphorylation and activation of STAT1. We found decrease in STAT1 phosphorylation (Y-701) at 24 hours after JEV infection as compared to 12 hours post JEV infection (Fig. 4E). The similar pattern of STAT1 phosphorylation (Y-701) was also observed in JEV infected mice brain tissue. The phosphorylation of STAT1 decreased in day-4 post infected mice as compared to day-2 post infected mice (Supplementary Fig. 2E). The expression of Interferon stimulated genes (IFIT1 and IFIT2) also reduced with progression of infection (Supplementary Fig. 2C,D). This indicated that JEV modulates the cellular machinery to suppress anti-viral response to create a favourable milieu in the cell. The overexpression of miR-432 resulted into the suppression of SOCS5 expression that ultimately led to the enhanced STAT1 phosphorylation creating strong anti-viral milieu in the cell due to enhanced ISRE Scientific Reports | 6:27685 | DOI: 10.1038/srep27685
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Figure 1. JEV infection downregulates miR-432 levels and leads to SOCS5 upregulation. Human brain microglial cells were infected by JEV (MOI 5) and cells were harvested at various time points to determine miR-432 levels. (A) Graph showing downregulation of miR-432 levels at 24 and 48 hours post infection as compared to 12 hours sample. TaqMan probe specific to miR-432 were used to determine fold change. The Ct values were normalized by RNU-24 levels. (B) Graph showing reduced levels of miR-432 in JEV infected mice brain. The RNA was isolated from brain tissue of 2 day and 4 day infected mice and mock infected mice brain was used as control. The Ct values were normalized by RNU-6 levels. (C) Western blots depicting upregulation of SOCS5 upon JEV infection. CHME3 cells were infected by JEV (MOI 5) and harvested after 24 and 48 hours. The average fold change values with respect to control have been mentioned. (D) Western blots showing upregulation of SOCS5 in JEV infected brain tissue. Mock infected mice brain tissue was used as control. Infected mice were harvested 2 day and 4 day post infection. β-tubulin was used for normalization. The average fold change values with respect to control have been mentioned. (E) Immunohistochemistry image showing increased levels of SOCS5 protein in brain of JEV infected BALB/c mice. Mock and JEV infected mice brain sections were collected 2 days post JEV infection. Enhanced levels of Alexa 488 florescence was observed in JEV infected brain section (Magnification 20x, Scale bar 100 μm). All experiments were performed in triplicates. The data are shown as mean ± S.E from three independent experiments. The fold change is significant where *denotes P
