Identification Of Pathogen Associated Molecular Patterns And Recognition Mechanism Of Porcine Reproductive And Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome(PRRS)is the most common viral disease in pork industry.It is caused by porcine reproductive and respiratory syndrome virus(PRRSV).At present,PRRS is widely spread in pig industry around the world,causing huge economic losses.In 2006,there was a sudden outbreak of swine fever in China,which was characteristic of persistent high fever,rapid death,highly contagious,and the incidence rate was close to 100%.At the same time,it caused serious miscarriage and other breeding disorders in pregnant sows.The pathogen of the disease was subsequently identified as highly pathogenic PRRSV(HP-PRRSV),which is a variant PRRSV strain.Until now,there have been no better methods for the prevention and control of PRRSV.Innate immunity plays an important role in the antiviral response and it is necessary to further study the immunologic mechanism of PRRSV.Upon virus infection,the host pattern recognition receptors(PRRs)quickly recognize the pathogen-associated molecular patterns(PAMPs)to activate downstream signals,inducing large production of interferon(IFN)and downstream interferon-stimulated genes(ISGs)to restrict virus replication.Previous reports have placed on how PRRSV evades host immunity due to the immunosuppressive characteristic of PRRSV.However,there are few studies on the specific mechanism of how PRRSV is recognized and induces IFN production.Thus,my thesis focuses on this and the main results obtained are as follows:1.In this study,we analyze the nucleotide sequences recognized by retinoic acid-inducible gene I(RIG-I)and melanoma differentiation associated gene 5(MDA5)via RNA-binding protein immunoprecipitation sequence(RIP-seq)in PRRSV infected porcine alveolar macrophages(PAM).The results demonstrated that the regions of RIG-I and MDA5 binding center on the 3' untranslated regions(3' UTR)region of the PRRSV positive-strand RNA genome and the binding sequences show U/C and U/G specificity.2.PRRSV belongs to the Arteriviridae family,order Nidovirales and the genome and subgenome share common 5' and 3' UTR regions which are essential for viral replication.The RIP-seq results also indicate that the PRRSV 3' UTR region is important for immune recognition.Therefore,we further studied the relationship between PRRSV genome 5' or 3' UTR and immune recognition.Here,we reported that 5' and 3' UTR of PRRSV genome could induce IFNs.Moreover,the pseudoknot region residing in the 3' UTR of PRRSV genome sensed as a new PAMP by RIG-I and Toll like receptor(TLR3)and strongly induced type I IFNs and ISGs in PAMs.The pseudoknot structure is a tertiary structure formed by the interaction of two stem loops at the end of RNA virus which can be used as a key molecule to regulate viral RNA synthesis and virus replication.In this study,we showed that it acted as PAMP to induce antiviral response.The interaction between the two stem-loops inside the pseudoknot structure was sufficient for IFN induction,since disruption of the pseudoknot interaction powerfully dampened the IFN induction.The results of RNA-pulldown indicated that RIG-I regulation domain(RD)and TLR3 ectodomain(ECD)bound to the pseudoknot.Furthermore,transfection of the 3' UTR pseudoknot transcripts in PAMs inhibited PRRSV replication in vitro.The sequence alignment and RNA secondary structure prediction revealed that the pseudoknot sequences were conserved in different arteriviruses.Importantly,the predicted similar structures of other arterivirus members also displayed strong IFN induction activities.3.pRIG-I-RD protein was successfully expressed and purified,and its interaction with the PRRSV 3' UTR pseudoknot RNA was identified by gel filtration chromatography.Moreover,Small-angle X ray scattering(SAXS)was used to analyze the status of the complex of pRIG-I-RD and PRRSV 3' UTR pseudoknot in solution,the results showed that the complex was elongated macromolecules and may have multiple lobes and at least two large centers.Since the PRRSV 3' UTR pseudoknot RNA was capped on the 5' end,the recognition mechanism is different from the previously reported recognition mechanism.This study provides novel insights into innate immune recognition during virus infection.Together,in this work we identified an innate recognition mechanism by which PRRSV 3' UTR pseudoknot region served as PAMPs of arteriviruses and activated innate immune signaling to produce IFNs that inhibit virus replication.Combining with previous research,we proposed a model on the balance between the IFN stimulatory effect of 3' UTR pseudoknot structure and the IFN inhibitory effect by the viral proteins.At the early endosome,following the virus uncoating and disassembly the virus genome is exposed to cytoplasm.The genomic RNA serves as the mRNA for immediate translation of the large replicase polyproteins,pp1 a and pp1 ab.After cleavage,these proteins assemble into a replication and transcription complex(RTC).The RTC immediately binds 3' UTR to initiate minus-strand RNA synthesis.During infection,the pseudoknot of the genome is recognized by RNA sensors RIG-I through its RD domain and TLR3 through its ECD domain resulting in IFN regulatory factor 3(IRF3)or nuclear factor-?B(NF-?B)activation and triggering IFN genes expression.The IFNs activate ISGF3 rapidly inducing ISG products through the JAK/STAT pathway to restrict infection.The generated viral proteins such as nonstructural proteins 1/2/4/11(NSP1/2/4/11)and N protein interfere the host antiviral response by targeting signaling pathways,such as IRF3 or NF-?B.During this process,the conformational change of the 3' end which acts as a molecular switch to regulate the timing of viral synthesis might affect virus recognition and subsequent IFN response.All these results provide novel insights into innate immune recognition during virus infection.