Studies On The Molecular Mechanism That The Leader And 3C Proteinase Of FMDV Regulated Antiviral Innate Immunity

Innate immunity is not only the first defensive line to pathogen (including viruses and bacteria etc.) but also the basis to activate acquired immunity. Act as an important anti-virus factor, interferon plays a fundamental role in innate immunity research. And many viruses have the capability to escape or inhibit the effect of interferon in their evolution. It is a hot point in present virological and immunological research to deeply study for virus how to modulate the production of interferon.At present, the innate immunity of human and mice is relatively clear in researches, while little is known about the porcine innate immunity. A large amount of porcine signal molecules and effective molecules in innate immunity haven't been cloned and identified, and the lack of luciferase reporting system for effective molecule promoter partly impedes deeply study at the interaction between virus and porcine innate immunity. Since this project took the signal pathway of porcine innate immunity as entry point to clone 16 important molecules related to signal pathway in porcine innate immunity and constructed Iuciferase reporting system for effective molecule promoter in porcine innate immunity, preliminarily build up technological platform to study signal pathway of porcine innate immunity. On the basis, to systematically analyze the mechanism and molecular details in the production of interferon inhibited by non-structure protein Lpro and 3Cpro in FMDV. The main studies were as following:1. Construction and identification of luciferase reporter gene vectors directed by porcine IFN-βpromoter and its NF-kB binding siteTo establish a method for detection of porcine proteins and genes related to IFN-βsignal transduction, we analyzed the regulatory elements that regulate the transcrip tion of porcine IFN-βgene. The promoter region of porcine IFN-βgene and its four copies NF-kB (nuclear factor kB) binding site regions were amplified from porcine genomic DNA by PCR and were cloned into promoter-free plasmid pGL3-basic. Then these constructs were transiently transfected into PK-15 cells and luciferase activities were measured with or without the transfection of poly(I:C) or poly(dA:dT). Higher expression of luciferase was obviously detected in PK-15 cells transfected with poly(I:C) or poly(dA:dT). These reporter constructs are important tools for investigation of porcine IFN-βsignaling transduction pathways.2. Molecular cloning of the porcine RANTES promoter:functional characterization of dsDNA/dsRNA response elements in PK-15 cellsThe chemokine RANTES (regulated upon activation, normal T-cells expressed and secreted) plays an essential role in inflammation and immune response. In this study, we cloned the nucleotide sequence of the 5'-flanking region of the porcine RANTES (poRANTES) gene and characterized the regulatory elements that activate transcription. Analyses of a series of 5'deletion constructs demonstrated that a 266 bp region (-220/+46) that spanned the potential transcription start site of the poRANTES gene was sufficient to activate transcription in PK-15 cells. Furthermore, our results indicated that dsDNA/dsRNA significantly induced poRANTES promoter activity and expression of mRNA levels in a time-and dose-dependent manner. Promoter deletions and mutagenesis experiments indicated that an interferon-stimulated responsive element (ISRE) was critical for dsDNA/dsRNA-induced poRANTES transcription. In addition, porcine interferon regulatory factor 3 (IRF-3) and IRF-7 play important roles in dsDNA/dsRNA-induced poRANTES expression.3. Molecular cloning and functional characterization of porcine IFN-βpromoter stimulator 1 (IPS-1)The IFN-βpromoter stimulator 1 (IPS-1), also known as MAVS/VISA/Cardif, is an adaptor molecule for retinoic-acid-inducible protein I (RIG-I) or melanoma-differentiation-associated gene 5 (MDA5) that senses intracellular double-stranded RNA (dsRNA) and triggers a signal for producing type I IFN. In the present study, the porcine IPS-1 cDNA was cloned using RT-PCR coupled with rapid amplification of cDNA ends (RACE)-PCR from porcine peripheral blood monouclear cells. The open reading frame of porcine IPS-1 consists of 1575 bp encoding 524 amino acids. The putative porcine IPS-1 protein contains an N-terminal CARD-like domain, a proline-rich domain in the middle, a C-terminal transmembrane domain, and exhibits similarity to mouse, rat, monkey, human and cattle counterparts ranging from 59 to 79%. Semi-quantitative RT-PCR showed that porcine IPS-1 mRNA was widely expressed in different tissues. Porcine kidney (PK-15) cells transfected with a DNA construct encoding porcine IPS-1 could produce type I IFN, and activate IRF-3/7 (interferon regulatory factor 3/7) and NF-kB. Deletion mutant analyses further revealed that both the CARD-like domain and transmembrane domain are essential for these functions. In addition, poly(I:C)-induced porcine IFN-βpromoter activation in PK-15 cells could be significantly reduced by siRNA targeting IPS-1, indicating that IPS-1 is an important immunoregulator in porcine innate immune system. The availability of porcine IPS-1 and establishment of its function in type I IFN signaling pathway provides a useful molecule for defining its role during the course of pig infectious diseases.Besides porcine IPS-1 gene, this research in succession cloned RIG-I,MDA5,TRAF6 etc 15 important molecules in signal pathway of porcine innate immunity. It laid basis on the research in porcine innate immunity in future and provided convenient tool to study the interaction between porcine pathogen and host.4. Foot-and-mouth disease virus leader proteinase inhibits dsRNA-induced type I interferon transcription by decreasing interferon regulatory factor 3/7 in protein levelsThe leader proteinase (Lpro) of FMDV has been identified as an interferon-β(IFN-β) antagonist that disrupts the integrity of transcription factor nuclear factor kB (NF-kB). In this study, we showed that the reduction of double stranded RNA (dsRNA)-induced IFN-αl/βexpression caused by Lpro was also associated with a decrease of IRF-3/7 in protein levels, two critical transcription factors for activation of IFN-a/β. Furthermore, overexpression of Lpro significantly reduced the transcription ofro multiple IRF-responsive genes including 2',5'-OAS, ISG54, IP-10, and RANTES. Screening Lpro mutants indicated that the ability to process eIF-4G of Lpro is not required for suppressing dsRNA-induced activation of the IFN-al/βpromoter and decreasing IRF-3/7 expression. Taken together, our results demonstrate that, in addition to disrupting NF-kB, Lpro also decreases IRF-3/7 expression to suppress dsRNA-induced type I IFN production, suggesting multiple strategies used by FMDV to counteract the immune response to viral infection.5. The leader proteinase of foot-and-mouth disease virus negatively regulates type I interferon pathway by acting as a viral deubiquitinaseHere, we demonstrate that FMDV Lpro has deubiquitinating activity. Sequence alignment and structural bioinformatics analyses revealed that the catalytic residues (Cys51 and His 148) are highly conserved in FMDV Lpro of all seven serotypes and the topology of FMDV Lpro is remarkably similar to that of ubiquitin-specific protease 14 (USP14), a cellular deubiquitylation enzyme (DUB), and that of severe acute respiratory syndrome coronavirus (SARS-CoV) papain-like protease (PLpro), a coronaviral deubiquitylation enzyme (DUB). Both purified Lpro protein and in vivo ectopically expressed Lpro removed ubiquitin (Ub) moieties from cellular substrates, acting on both lysine-48-and lysine-63-linked polyubiquitin chains. Furthermore, Lpro significantly inhibited ubiquitination of RIG-I TANK-binding kinase 1 (TBK1), TNF receptor-associated factor 6 (TRAF6) and TRAF3, key signaling molecules in activation of type I IFN response. Mutations in Lpro that ablate the catalytic activity (C51A or D163N/D164N) or disrupt the SAP (for SAF-A/B, Acinus, and PIAS) domain (I83A/L86A) abrogated the DUB activity of Lpro as well as its ability to block signaling to the IFN-βpromoter. Collectively, these results demonstrate that FMDV Lpro possesses DUB activity in addition to serving as a viral proteinase and describe a novel mechanism evolved by FMDV to counteract host innate antiviral responses.6. FMDV could be inhibited by interferon-Iambda 1 and had mechanisms to block this actionFMDV causes an economically important disease in swine-producing area, and interferon lambda 1 (IFN-λ1), a newly identified type III interferon, has antiviral activity against a broad spectrum of viruses. In this study, we found that replication of FMDV in IBRS-2 cells was significantly reduced following treatment with the purified recombinant porcine IFN-λ1 in a dose-dependent manner. However, FMDV could not activate IFN-λ1 promoter in IBRS-2 cells, and the activity of IFN-λ1 promoter was much lower than that triggered by poly(I:C). Furthermore, we found that the leader proteinase (Lpro) of FMDV is involved in IFN-λ1 regulation. The obtained results showed that FMDV Lpro inhibited poly(I:C)-induced IFN-λ1 promoter activity. Screening Lpro mutants indicated that the catalytic activity and a SAP (for SAF-A/B, Acinus, and PIAS) domain of Lpro were required for suppressing dsRNA-induced IFN-λ1 production. In conclusion, our results suggested that FMDV could be inhibited by IFN-λ1 and had mechanisms to inhibit this action.7. Foot-and-mouth disease virus leader proteinase inhibits dsRNA-induced RANTES transcription in PK-15 cellsThe chemokine RANTES plays an essential role in inflammation and immune response. Previous study has been demonstrated that infection with wild-type (WT) foot-and-mouth disease virus inhibits the expression of RANTES compared to infection with a genetically engineered mutant lacking the leader protein (Lpro) coding region in PK-15 cells. However, a complete analysis of the promoter cis-regulatory elements and nuclear factors involved in this inhibition of RANTES gene transcription has not been fully elucidated. In this study, we showed that transfection of PK-15 cells with Lpro of FMDV expression plasmid, in the absence of other FMDV proteins, inhibited dsRNA-induced RANTES in luciferase activity and mRNA transcription. Promoter mutagenesis experiments indicate that interferon-stimulated responsive element (ISRE) was important for the ability of Lpro to inhibit dsRNA-induced the RANTES promoter. Furthermore, the overexpression of Lpro also inhibited IRF-3/7-mediated activation of the RANTES promoter. Screening Lpr0 mutants indicated that the catalytic activity and a SAP (for SAF-A/B, Acinus, and PIAS) domain of Lpro were required for suppressing dsRNA-induced RANTES production. These findings reveal one of the important mechanisms underlying the innate immune evasion by FMDV during infection.8. Foot-and-mouth disease virus 3C proteinase inhibits type IIFN transcription The 3C proteinase (Lpro) of FMDV has proteinase activity and is involved in processing the viral polyprotein. In this study, our results show that 3Cpro is involved in the inhibition of type I IFN response, suggesting multiple proteins used by FMDV to counteract the immune response to viral infection. FMDV 3Cpro negatively regulates IFN-al/βexpression by disrupting activation of IRF-3/7. Furthermore, overexpression of 3Cpro significantly reduced the transcription of multiple IRF-responsive genes including 2',5'-OAS, ISG54, IP-10, RANTES, and IFN-λ1.In a word, this project preliminarily build up technological platform to study signal pathway of porcine innate immunity and systematically analyzed the mechanism and molecular details in the production of interferon inhibited by non-structure protein Lpro and 3Cpro in FMDV, proclaimed the immune inhibitory characteristic and immune escaping strategy, which established theoretical basis to exploit safer and more effective vaccine of FMD from now on.