Inhibition Of Porcine Rotavirus NSP4 Protein On Type Ⅰ Interferon Signaling Pathway And Screening Of Interaction Proteins

Porcine rotavirus disease is one of the main causes of diarrhea in piglet.At present,there is no specific treatment for rotavirus,and the prevention by attenuated vaccine is often used in practice.Porcine rotavirus（PoRV）is the pathogen of the disease.The genome of PoRV consists of 11 double-stranded ribonucleic acids,which encode 6 structural proteins and 6 non-structural proteins,respectively.The NSP4 protein is a non-structural protein encoded by the 10th segment of the gene.It is also the first identified viral enterotoxin that causes diarrhea in sick piglets.It promotes virus replication by regulating calcium ions and can also be used as an adjuvant to stimulate the immune response.However,the regulatory effect of NSP4 protein on type Ⅰ interferon signaling pathway and the mechanism by which it works have not been elucidated.In this study,we investigated the effect of NSP4 protein on the type Ⅰ interferon signaling pathway,and further screened the intestinal epithelial cell proteins that interact with NSP4 by GST pull-down combined with mass spectrometry analysis,so as to provide a basis for studying the mechanism of interaction between NSP4 and host molecules and revealing the function of NSP4in regulating innate immune response.The main research content is divided into four parts:（1）The viral stock with 3.1623×10⁵ TCID₅₀/m L was obtained by amplifying PoRV DN30209 strain,and its replication and cytopathic effect in target cell IPEC-J2 and rhesus monkey embryonic kidney cell（MA104）were determined.IPEC cells became round,exfoliated and died 12 hours after PoRV infection.The replication kinetics curve of PoRV in IPEC-J2 cells showed that the virus replicated rapidly within 12 hours of infection,and then the amount of virus remained at a relatively stable level.The replication of PoRV in MA104 cells led to vacuolar and reticular lesions,and the amount of virus reached the peak at 48 h.The NSP4 gene of PoRV was obtained by RT-PCR technology.The prokaryotic plasmid pGEX-4T-1-NSP4（47-175）was constructed and GST-NSP4（47-175）protein was purified for subsequent GST pull-down assay to detect NSP4 host target protein.The eukaryotic expression plasmid pRK-Flag-NSP4,pRK-HA-NSP4 were constructed and the expression of NSP4 protein in HEK293T was proved by Western blot.These eukaryotic expression plasmids were used for co-transfection with different labeled signal molecules to prepare for the follow-up study of the regulation of NSP4 protein on IFN-βsignal pathway.（2）The mRNA expression of interferon and downstream antiviral factors in the presence of NSP4 were detected by fluorescence quantitative PCR,including IFNB1,ISG56,CXCL10,IFIT2,and ISG15.The results showed that NSP4 significantly inhibited the expression of cytokines stimulated by RIG-IN（domain of RIG-I-mediated signal transduction）and MDA5-N（domain of MDA5-mediated signal transduction）.By detecting the phosphorylation level of IRF3 in HEK293T induced by SeV,it was found that NSP4 inhibited the phosphorylation of IRF3.Through the dual luciferase reporter gene assay,it was found that NSP4 protein inhibits the IFN-βand ISRE promoter activities stimulated by RIG-IN and MDA5-N,which proves that NSP4 protein has an inhibitory effect on the IFN-βsignaling pathway,and the action site was upstream of MAVS.However,through immunoprecipitation experiments,it was found that NSP4 has no interaction with RIG-I,MAVS,TBK1,IKKi and IRF3,and the target of NSP4needs to be further explored.（3）The host proteins that interact with NSP4 under conditions of viral infection were identified by GST pull-down combined with mass spectrometry.A total of 30 specific proteins that interact with NSP4 were detected.The GO function enrichment and KEGG signal pathway enrichment analysis of differential proteins are mainly involved in biological processes such as cytoskeleton organization,extracellular secretion,messenger RNA processing and neurotransmitter receptor transport,etc.The molecular function is mainly to RNA binding,GTP binding,GTPase acticity,mRNA bingding,etc.The signal pathways involved mainly include endocrine and other factors regulating calcium reuptake,vasopressin regulating water reuptake,JAK/STAT signaling pathway,RNA transport,phospholipase D signaling pathway,autophagy,endocytosis,and Fc R-mediated Phagocytosis.In this study,prokaryotic plasmids pGEX-4T-1-NSP4（47-175）expressing NSP4 protein and eukaryotic expression plasmids pRK-Flag-NSP4 and pRK-HA-NSP4,were constructed to evaluate the effect of NSP4 on type Ⅰ interferon pathway by detecting antiviral factor transcription level,ISRE and IFN-βactivity and IRF3 phosphorylation level.It was proved that NSP4 could inhibit type Ⅰ interferon pathway.Through mass spectrometry identification and bioinformatics analysis,it is speculated that NSP4 may play a role through IFNLR1-mediated JAK/STAT pathway and IFITM3 through autophagy to degrade IRF3,thus inhibiting type Ⅰ interferon pathway.