Japanese Encephalitis Virus exploits the microRNA-432 to regulate the expression of Suppressor of Cytokine Signaling (SOCS) 5

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 Jak-STAT 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.

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 genes 12 . 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 SOCS5 13 . 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 response 14 . 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 Jak2 15 . 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 carcinoma 16 . Chen et al. has also demonstrated tumor suppressive role of miR-432 in lung cancer cells 17 . 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 miR-432 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 Jak1 15 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′ UTR 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 pathway 18 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 stage 19 . Interferon secreted upon JEV infection binds to IFN-α /β receptor 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 activity. The increased levels of IL-6 and TNF-α were found in miR-432 overexpressing cells after JEV infection ( Supplementary Fig. 3A,C). The viral RNA titre was found to be lower in miR-432 overexpressing cells which indicated that miR-432 suppressed the viral replication (Fig. 4A). Additionally, the viral NS1 protein was also checked through western blotting which demonstrated the decreased viral protein levels (Fig. 4C). The viral RNA 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 C t 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 μ m). 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 < 0.05, ** denotes P < 0.01, *** denotes P < 0.001. was isolated from culture supernatant and viral copies were determined to check the effect of miR-432 on release of viral particles. We found decrease in viral copies in miR-432 overexpressing cells as compared to scramble  Fig. 2) and transfected along with miR-432 mimic. The mutant clone did not display any reduction in luciferase activity. Non-targeting mimic miR-146a was also used as negative control. β -galactosidase activity was used for normalization. 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 < 0.05, ** denotes P < 0.01, *** denotes P < 0.001. Scientific RepoRts | 6:27685 | DOI: 10.1038/srep27685 transfected cells (Fig. 4B). We found enhanced viral replication in antimiR-432 transfected cells due to increased levels of SOCS5 in antimR-432 transfected cells (Fig. 4D). The increased expression of miR-432 suppressed SOCS5 levels and created strong anti-viral environment in the cell, which led the reduced viral replication. 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 < 0.05, ** denotes P < 0.01, ***denotes P < 0.001. The data are shown as mean ± S.E from three independent experiments. The fold change is significant where * denotes P < 0.05, ** denotes P < 0.01, *** denotes P < 0.001. Scientific RepoRts | 6:27685 | DOI: 10.1038/srep27685 SOCS5 knock-down supresses viral replication. To further decipher the specific role of SOCS5 during JEV infection, we used siRNA mediated approach to knock-down of SOCS5 gene. 10 nM of siRNA was transfected along with scramble sequence and knock down was confirmed by western blotting (Fig. 5A). The SOCS5 silenced cells were infected by JEV to demonstrate its effect on JEV replication. We found that SOCS5 knock down enhanced the STAT1 phosphorylation which confirmed the negative regulatory effect of SOCS5 on Jak-STAT pathway (Fig. 5B). To analyse the downstream effect of SOCS5 knock down, the ISRE activity was checked in SOCS5 knocked down cells enhanced ISRE activity was found in SOCS5 knockdown cells compared to scramble transfected cells upon JEV infection (Fig. 5F). SOCS5 knockdown suppressed viral replication in CHME3 cells and resulted into decreased in viral RNA (Fig. 5D) as well as viral NS1 protein in SOCS5 knockdown cells (Fig. 5C). The release of virus in culture supernatant was also suppressed in SOCS5 knockdown cells (Fig. 5E). The SOCS5 gene was cloned in eukaryotic vector pcDNA 3.1 and SOCS5 was overexpressed in CHME3 cells prior to JEV infection. SOCS5 overexpressing cells displayed reduced expression of pro-inflammatory cytokines (IL-6 and TNF-α ) ( Supplementary Fig. 3B,D) and higher JEV replication ( Supplementary Fig. 1B,C). The higher copies of JEV RNA as well as NS1 protein expression was observed in SOCS5 overexpressing cells compared to empty vector transfected cells. This explains that the suppressive effect of miR-432 on JEV replication is specifically due to SOCS5 downregulation in miR-432 overexpressing cells and SOCS5 has a direct regulatory effect on anti-viral Jak-STAT pathway.

Discussion
Viruses have evolved various strategies to evade cellular immune response by exploiting the host cellular machinery. Various viral proteins interact with different anti-viral proteins to modulate their functions. Many viruses and viral proteins adopt microRNA mediated regulation of anti-viral pathways to alleviate their pathogenesis. Influenza A virus utilizes miR-302c to target NF-κ B kinase activity in order to modulate IFN-β pathway 20 . Hepatitis B virus X protein has been reported to induce the expression of miR-21 to promote hepatocellular carcinoma in hepatocytes 21 . Few viral proteins of RNA viruses have been reported to localize into nucleus and interact with histones to modify gene expression 22,23 .
Wang et al. 24 has reported the crucial role of Shp-2 (a PTP) for controlling the replication of respiratory syncytial virus (RSV) in A549 cells. They reported that the blocking of Shp-2 led to the impaired STAT1 phosphorylation 24 . JEV NS5 has been reported to block STAT1 phosphorylation by activation of phospho tyrosine phosphatases PTPs 25 . SOCS family of proteins have been well characterized as negative regulators of Jak-STAT pathway 26,27 . RSV non-structural proteins have been reported to induce the expression of SOCS1 and SOCS3 proteins ultimately leading to the suppression of IFN signaling 28 . Kundu et al. 29 has reported the upregulation of SOCS1 and SOCS3 in JEV infected mouse macrophages 29 . Li et al. 30 has reported the upregulation of SOCS3 in JEV infected mice brain 30 . The upregulation of these SOCS proteins alleviate viral survival in host cells. Kario et al. 31 has reported the SOCS5 as a negative regulator of EGFR signalling 31 . Zhuang et al. 32 demonstrated targeting of SOCS5 by miR-9 in vascular endothelial cells co-cultured with tumour cells which promoted endothelial cell migration 32 . However, the role of SOCS5 has not been well understood during viral infection.
JEV adopts various strategies to facilitate its replication in the host cells. JEV infection leads to the activation of microglial cells resulting into the release of pro-inflammatory cytokines 33,34 . JEV infection triggers antiviral Jak-STAT pathway by increasing the STAT1 phosphorylation in human microglial cells in early course of infection. The downregulation of phospho STAT1 levels was observed at the late time point (24 hours). In our previous study, we reported the decreased ISRE activity as well as reduced levels of expression of Interferon stimulated genes (ISG-56, ISG-54) during late time points of infection 10 . A similar immunosuppressive pattern of STAT1 phosphorylation was observed in JEV infected mice brain tissue. The phosphorylation of STAT1 and expression of IFIT1 and IFIT2 was reduced in 4 day post infected tissue as compared to 2 day infected tissue (Supplementary Fig. 2C-E). Hence we hypothesize that virus tries to suppress antiviral signalling at different time points of infection to evade immune response through various strategies.
We demonstrated that JEV downregulates miR-432 in CHME3 cells, which leads to the increased expression of SOCS5. To validate the SOCS5 targeting by miR-432, the UTR clone of SOCS5 in luciferase vector along with miR-432 mimic was transfected in HeLa cells, which resulted into reduced luciferase activity. To further study the specificity of the targeting, the recognition sequences of miR-432 present in 3′ UTR of SOCS5 were deleted and cloned in to luciferase vector. The mutated UTR clone did not display reduction in luciferase activity which confirmed the SOCS5 as a target of miR-432. Since SOCS5 inhibits auto-phosphorylation of Jaks, the effect of miR-432 overexpression was analysed on Jak-STAT signalling. miR-432 overexpression suppressed SOCS5 levels causing enhanced Y-701 STAT1 phosphorylation. This resulted into elevated ISRE activity and upregulated expression of pro-inflammatory cytokines. miR-432 overexpression caused enhanced production of pro-inflammatory cytokines (IL-6 and TNF-α ) ( Supplementary Fig. 3A,C). Hence, miR-432 overexpression created a potent antiviral milieu in the cell, which further led to the reduced viral replication in the cells as well as release of viral particles in culture supernatant. Anti-miR-432 reduced STAT1 phosphorylation and lowered ISRE activity upon JEV infection, which led to the enhanced viral replication.
To further ascertain the negative regulatory effect of SOCS5 on Jak-STAT pathway, we specifically knocked down SOCS5 by using siRNA. SOCS5 knock down led to the enhanced phosphorylation of STAT1 and increased ISRE activity. siRNA mediated SOCS5 knock down strengthened the cellular anti-viral response, which led to the reduction in viral replication in knock down cells as well as diminished release of viral particles in culture supernatant. Watanabe et al. had deciphered the role of SOCS5 overexpressing T-cells in augmenting the release of pro-inflammatory cytokines from macrophages during septic peritonitis 35 . We also analysed the effect of JEV infection in SOCS5 overexpressing microglial cells and found reduced levels of pro-inflammatory cytokines (IL-6 and TNF-α ) post JEV infection (Supplementary Fig. 3B,D). This resulted into enhanced viral replication in SOCS5 overexpressing cells (Supplementary Fig. 1B,C). In addition, we also checked the levels of miR-432 in Scientific RepoRts | 6:27685 | DOI: 10.1038/srep27685 JEV infected mice brain and we found decreased levels of miR-432 in brain of 2 day and 4 day infected mice. The seed sequence present in mature miR-432 of mouse and human are conserved, therefore we checked the levels of SOCS5 in JEV infected mice brain by western blotting and immunohistochemistry. We observed elevated levels of SOCS5 upon JEV infection in mice brain tissue. These findings support that JEV downregulates miR-432 in The data are shown as mean ± S.E from three independent experiments. The fold change is significant where *denotes P < 0.05, **denotes P < 0.01, ***denotes P < 0.001.
Scientific RepoRts | 6:27685 | DOI: 10.1038/srep27685 order to induce SOCS5 expression, to negatively regulate the Jak-STAT pathway to aid the replication and survival of the virus.
Cells trigger various anti-viral mechanisms to combat viral infection. The effect of JEV on the expression pattern of miR-432 is time dependent. Further study is required to understand the expression of miR-432 in the in vivo conditions at later time points. The successful establishment of JEV in the CNS takes place due to various strategies employed by the virus for suppressing the cellular immune response further resulting into neuroinflammation. The study of microRNAs in modulating expression of immune regulatory genes will render a better understanding of viral pathogenesis. Overexpression and silencing of Anti-miR-432. CHME3 cells were transfected in 6 well plates with 100 pmol of miR-432 seed sequence mimic (Bioserve, Hyderabad, India) by using Lipofectamine 2000 (#11668-019; Invitrogen) according to manufacturer's protocol. Scrambled seed sequence of miR-432 was transfected as control. miR-432 oligo and scramble sequence have been mentioned in Table 1. To silence miR-432, 100 pmol of anti-miR-432 (# AM17000, Ambion) was transfected and JEV infection was given 24 hours post transfection. 100 pmol Cy3 labelled scrambled Anti-miR (#AM17011; Ambion) was used as negative control. The overexpression and silencing of miR-432 was confirmed by real time PCR using miR-432 specific TaqMan primer and probe (# 4427975, Ambion). The cells were harvested after 48 hours post transfection. SOCS5 knock down. 10   TaqMan probe (# 4427975, Ambion) and universal PCR master mix (#4324018; Applied Biosystems). The expression of miR-432 was normalized by RNU24 expression as endogenous control.

Cell
To estimate the viral RNA, total RNA was extracted from cells by using RNeasy mini kit (#74106, Qiagen) according to manufacturer's protocol. cDNA was synthesized by using superscript II (#11904-018, Invitrogen) according to manufacturer's protocol. Viral RNA was isolated from culture supernatant by using High pure viral RNA kit (#11858882001, Roche). The primers used in this study have been mentioned in the Table 1. To extract the RNA from mice brain tissue, the tissue was homogenized and RNA was extracted using miRNeasy kit (# 217004; Qiagen). cDNA was synthesized and miR-432 levels were determined by real time PCR. RNU-6 was used as normalization control.
Cloning of human SOCS5 gene. To overexpress SOCS5 gene in CHME3 cells, SOCS5 gene was cloned into eukaryotic vector pcDNA 3.1 vector. The primers used to amplify SOCS5 from human genomic DNA are listed in Table 1. Overexpression of SOCS5 was confirmed by western blotting.

MicroRNA target prediction and validation. Various bioinformatics tools like Pictar (MDC, Berlin,
Germany), Target Scan 5.2 (Whitehead Institute of Biomedical Research, MIT, Boston, USA) and MicroRNA. org were used to predict SOCS5 3′ UTR as potential target of miR-432. The binding site in 3′ UTR of SOCS5 was identified by using Human Target Scan.
For validation of microRNA targeting, 3′ UTR clone of SOCS5 in pEZX-MT06 luciferase vector (#HmiT054032-MT06, GeneCopoeia) was transfected into HeLa cells along with miR-432 mimic or scramble mimic to observe the effect on luciferase activity. To generate the mutant clone, the binding site of miR-432 present in SOCS5 3′ UTR (2242-2249) was deleted by using site directed mutagenesis. The mutated fragments were joined by using recombination PCR. Primers used for mutant generation and recombination PCR have been listed in Table 1. The mutated 3′ UTR was cloned in pEZX-MT06 vector and transfected into HeLa cells along with miR-432 mimic to check luciferase activity and β -Galactosidase (700 ng) vector was co-transfected as normalization control.
Luciferase assays. CHME3 cells were transfected with ISRE Luciferase reporter plasmid (1 μ g) or along with miR-432 mimic, scrambled or anti-miR-432 (100 pmol). β -Galactosidase (700 ng) vector was co-transfected as normalization control. Luciferase activity was measured 48 hours post-transfection. For infection, cells were counted 24 hours post transfection and infected with JEV (MOI 5). Cells were incubated for 24 hours after infection and lysed to measure luciferase activity. Luminescence activity was measured by lysing the cells in 1X lysis buffer provided by Luciferase Assay kit (#E4030; Promega, Madison, WI, USA) and luciferase assay reagent was added later as per manufacturer's protocol. Perkin Elmer multiplate reader (Enspire 2300 Multimode plate reader) was used to measure luciferase activity. For normalization, β -Galactosidase activity was measured by using β -Galactosidase kit (#E2000; Promega, Madison, WI, USA) as per manufacturer's protocol.

Immunohistochemistry. Mock and JEV infected mice were sacrificed (2 days after infection) and
their brains were excised. The brain tissue was fixed with 4% paraformaldehyde and 20 micro meter thick sections were prepared by using cryostat (Leica CM3050S) and mounted on slides. Antigen unmasking of sections was performed by boiling the sections dipped in Vector antigen unmasking solution (#H-3300, Vector Laboratories). The sections were incubated with anti-SOCS5 (#sc-5607, Santa cruz biotechnology) primary antibody at 4 o C overnight. The sections were washed and incubated with Alexa 488 conjugated secondary antibody (A 11034, Life technologies) for 1 hour and washed. The sections were then mounted with mounting medium with DAPI (H-1200, Vector Laboratories) and viewed under florescent microscope (Zeiss Axio imager).
ELISA of TNF-α. CHME3 cells were transfected by 100 pmol of miR-432 mimic. 24 hours post transfection, JEV infection was given. Scramble sequence was used as control. 24 hours post infection, the culture supernatant was collected and ELISA was performed by using human TNF-α ELISA kit (Cat No.-KHC3011, Invitrogen) according to manufacturer's protocol.

Statistical analysis.
All experiments were conducted in triplicates and one-tailed, paired Student's t-test was used to make comparison between data sets. Data was considered significant when P < 0.05; *denotes P < 0.05, **denotes P < 0.01, ***denotes P < 0.001