Mir-526a Participates In RIG-I-dependent Innate Immune Response

EV71is a positive-stranded RNA virus, which belongs to the picornavirus familyand is the causative agent of hand-foot-and-mouth disease (HFMD) in young childrenand infants. The genome of EV71is approximately7.5kb in length and contains asingle open reading frame encoding a polyprotein precursor, which is processed intostructural (VP1, VP2, VP3, and VP4) and nonstructural proteins (2A,2B,2C,3A,3B,3C, and3D) during viral infection. Despite the protective role of IFN-I on EV71infection, EV71inoculation is unable to elicit their production. Recently reports showEV71inhibit IFN-I induction by interfering with melanoma differentiation-associatedgene5(MDA-5) and retinoid acid-inducible gene I (RIG-I) or by restrictingIFN-secretion through repressing the cellular secretory pathway. Recent studiesrevealed that the3C protease of EV71associated with RIG-I and cleaved TRIF andIRF7; moreover, EV71inhibited IFN-I-induced ISG activation by reducing IFNAR1levels in host cells. However, additional work is required to understand themechanisms for EV71to escape from innate antiviral responses.IFN-I, as the first line of host immune response, is critical in mediating antiviraldefense. The host senses viral and bacterial pathogen invasion by recognition ofpathogen-associated molecular patterns with pattern recognition receptors, includingmembrane-bound TLRs and cytosolic sensory molecules, such as the multi-domaincontaining NOD proteins, RIG-Ⅰ, and MDA-5helicases. Both RIG-I and MDA-5contain caspase recruitment domains (CARDs) that interact with the CARDdomain-containing protein mitochondrial antiviral signaling (MAVS) upon binding touncapped RNA, resulting in MAVS association with I B kinase (IKK) proteins.While MAVS association with IKK/activates NF-B, its association with TBK1as well as IKKε leads to the activation of IFN regulatory factor (IRF)-3; coordinatedactivation of NF-B and IRF-3pathways further results in the assembly of amulti-protein enhancer complex that drives the expression of IFN-and theIFN-mediated antiviral immunity.RIG-I signaling is negatively regulated at multiple levels and involvesdeubiquitination of specific proteins. Previous reports showed that the ubiquitination status of RIG-I is controlled by CYLD, a tumour suppressor which was originallyidentified as a genetic defect in familial cylindromatosis. Indeed, CYLD was shown tointeract with the CARDs of RIG-I and to remove K63-linked polyubiquitin chainsfrom RIG-I, which inhibits downstream signaling. DC lacking CYLD constitutivelypolyubiquitinate RIG-I and show enhanced activity of TBK1and IKKε, suggestingthat CYLD regulates basal RIG-I activity by modulating its K63-polyubiquitin status.CYLD also acts as a negative regulator of NF-B and Jun N-terminal kinase signalingpathways by removing Lys63-linked polyubiquitin from NEMO/IKK, TRAF2andBCL3. These findings thus establish CYLD as a critical regulator of antiviral innateimmune response.miRNAs, an abundant class of highly conserved noncoding RNAoligonucleotides (22nt long in average), suppress gene expression by binding to the3’-untranslational region (UTR) of target mRNAs. miRNAs play key roles in theregulation of diverse biological processes. Recently, a role for miRNAs has beenproposed in the regulation of innate immune responses in monocytes andmacrophages. Direct roles of miRNAs in innate immune response were discovered ina report that identified miR-146a as a negative feedback regulator in RLR signalingby targeting IL-1R associated kinase (IRAK)1and TRAF6. Further reports showedthat both miR-155and miR-132were induced in monocyte cell line treated withlipopolysaccharide (LPS). Given the important roles of the RIG-I signaling pathwayin innate antiviral immune response, identifying more miRNAs that can regulateRIG-I-dependent IFN-I production is of vital importance. However, to date, there isfew report about the regulation of RIG-I signaling pathway by miRNAs, especiallyduring EV71infection. Here we show:1. Viral infection upregulates miR-526a expression through the IRF Pathway: Byquantitative PCR(q-PCR) we found miR-526a is upregulated after VSV infection.;InIRF3/7knockdown cells VSV-triggered miR-526a expression is remarkably inhibited.These data indicate miR-526a is upregulated in an IRF-dependent manner in responseto viral infection.2. miR-526a positively regulates VSV-triggered IFN-I production:miR-526amimics could enhance RIG-I signaling pathway by detecting IFN--luc, NF-B-lucand IRF3-luc activity. However miR-526a inhibitor blocked this promotion; Byq-PCR and AlphaLISA we found that miR-526a can promote virus-triggered IFN-production; Also q-PCR analysis revealed that miR-526a can induce the expression of ISG56, ISG54, IL-8and CXCL-2. These data show that miR-526a is a positiveregulator of RIG-I signaling pathway.3. miR-526a suppresses VSV and NDV virus replication: By immunoblotting wefound that miR-526a mimics could suppresse VSV-G expression; By quantification ofNDV-GFP, immunofluorescent and flow cytometry we observed that miR-526amimics can inhibit GFP expression. These data show that miR-526a can suppressRNA virus replication.4. miR-526a targets CYLD, triggering RIG-I signaling pathway: By prediction ofnew targets of miR-526a via bioinformatics methods including TargetScan software,we screened that CYLD is a potent target;3’UTR luciferase assay, q-PCR andimmunoblotting analysis revealed that miR-526a mimics can downregulate CYLDexpression; Immunoblotting analysis revealed that miR-526a can enhance thephosphorylation of IKK/, I B and IRF3. These data show that miR-526a canenhance RIG-I (K63) ubiquitination by downregulating CYLD, resulting in theactivation of RIG-I signaling pathway.5. Downregulating of miR-526a by EV713C inhibits RIG-I signaling pathway:By q-PCR and immnuoblotting we found that EV713C protease can inhibit theexpression of miR-526a, IRF7and IFN-; Ectopically-expressed miR-526a or siRNAto CYLD could suppress EV71VP1expression. These data show that EV713Cprotease targets miR-526a to inhibit RIG-I signaling pathway.In the present study, we found that miR-526a is significantly upregulated inmacrophages upon viral infection in an IRF-dependent manner. Then wedemonstrated that miR-526a feedback positively regulates VSV-triggered IRF3andNF-B activation by suppressing CYLD expression and subsequent RIG-Ⅰ(K63)ubiquitination. Furthermore, we found that miR-526a upregulation is blocked byEV713C protease, whereas ectopic miR-526a expression inhibited the replication ofEV71. Thus the present study has for the first time demonstrated that miR-526a is apositive feedback regulator of the RIG-I signaling and that EV71targets miR-526a tosuppress RIG-Ⅰ-dependent IFN-I production.