Study On Inflammatory Response By The Nonstructural Protein 1 Of Porcine Parvovirus Through TLR2

Porcine parvovirus(PPV)mainly causes reproductive disorders in sows,resulting in abortion,fetal malformation,mummification and death,which has caused huge economic losses to the pig industry.PPV is a small,envelope-free DNA virus with a genome length of about 5 kb,and contains two open reading frames(ORFs).The 5’ terminal ORF encodes capsid proteins(VP1,VP2 and VP3),and the 3’ terminal ORF encodes non-structural proteins(NS1,NS2 and NS3).Among them,NS1 is the most important functional protein of PPV,which is involved in the replication and transcription,as well as a virulence protein of host cells.Nuclear transcription factor kappa-B(NF-κB)is an important factor in the regulation of innate and specific immune responses,which can regulate the expression of a variety of inflammatory cytokines and chemokines.Toll-like receptors(TLRs)are typical pattern recognition receptors,which can induce the activation of NF-κB and tumor necrosis factor receptor-related factor 6(TRAF6)by recognizing pathogen-associated molecular patterns(PAMPs).They play an important role in the immune response.Our previous studies have shown that PPV can activate NF-κB signaling pathway and induce inflammation through TLR2,but it is not clear whether PPV NS1 protein can also activate NF-κB signaling pathway through TLR2 to induce inflammation.Therefore,in this study,porcine kidney cells(PK-15)were used as an infection model in vitro.Firstly,the inflammatory response induced by PPV NS1 and the activation of NF-κB signaling pathway were researched.Secondly,the function of TLR2 in PPV NS1 activating NF-κB signaling pathway was studied,and finally the interaction between PPV NS1 and TLR2 was clarified.The specific research contents are as follows.1.PPV NS1 activates NF-κB signaling pathway and induces inflammation response in host cell.To investigate the effect of PPV NS1 on the host inflammatory response,PK-15 cells were first transfected with the PPV NS1 eukaryotic recombinant expression plasmid p CAGGS-HA-NS1,the expression of PPV NS1 and NF-κB signaling pathway-related proteins was detected by western blot(WB).The expression of the inflammatory cytokine interleukin-6(IL-6)and tumour necrosis factor-α(TNF-α)was measured by real-time quantitative PCR(q PCR).The results showed that PPV NS1 was successfully expressed in PK-15 cells,and its expression level was dose-dependent with the quality of recombinant plasmid transfection;PPV NS1 could significantly induce the degradation of IκBα and phosphorylation of p65(P<0.05).The expression of IL-6 and TNF-α were significantly increased after PPV NS1 transfection of PK-15 cells(P<0.01)and in a dose-dependent manner.The NF-κB inhibitor BAY11-7082 treatment of PK-15 cells significantly inhibited the ability of PPV NS1 to induce IL-6 and TNF-α expression(P<0.01).The above results suggest that PPV NS1 protein can activate NF-κB signaling pathway and induce inflammatory response in host cells.2.The role of TLR2 in the activation of the NF-κB signaling pathway by PPV NS1.To investigate the role of TLR2 in the inflammatory response induced by PPV NS1.Firstly,PK-15 cells were transfected with the recombinant plasmid of PPV NS1.q PCR and WB assays showed that the expression of TLR2 was significantly up-regulated(P<0.01).Furthermore,PK-15 cells were treated with TLR2 agonist Pam3CSK4 and inhibitor C29,to analyze the effect of TLR2 on PPV NS1 activating NF-κB signaling pathway.The results showed that the agonist of TLR2 significantly promoted the ability of PPV NS1 to induce the degradation of IκBα and phosphorylation of p65(P<0.05).The expression of IL-6 and TNF-α(P<0.01)was also up-regulated;while the inhibitor of TLR2 significantly inhibited the degradation of IκBα and phosphorylation of p65 by PPVNS1(P<0.05),and caused the down-regulation of the expression of IL-6 and TNF-α(P<0.01).These results suggest that activation of TLR2 is involved in the inflammatory response induced by PPV NS1 protein..3.Preliminary studies on the interaction between PPV NS1 and TLR2.To further investigate the interaction between PPV NS1 and TLR2,a TLR2 eukaryotic expression vector p CAGGS-FLAG-TLR2 with green fluorescent protein and FLAG tag protein was constructed and transfected into HEK-293 T cells.In addition,the expression of TLR2 in the cells was examined by fluorescence microscopy and WB.The results showed that the recombinant plasmid p CAGGS-FLAG-TLR2 was successfully expressed in HEK-293 T cells.Next,the recombinant plasmids p CAGGS-HA-NS1 and p CAGGS-FLAG-TLR2 were co-transfected into HEK-293 T cells,and the binding of PPV NS1 to TLR2 was analyzed by co-immunoprecipitation assay.The results showed that antibodies to both HA and FLAG tag proteins could cause immunoprecipitation of PPV NS1 with TLR2.The above results indicate that PPV NS1 can bind to TLR2.In summary,this experiment confirmed that PPV NS1 activates the NF-κB signaling pathway by binding to TLR2 and further induces the expression of inflammatory cytokines.The results of this study provide a theoretical basis for the elucidation of the immune mechanism and pathogenesis of PPV,as well as for the development of effective anti-PPV drugs and novel vaccines.