The Regulatory Functions And Mechanisms Of Prion Protein In H7N7-IAV Infection

Influenza A virus(IAV)is an enveloped,negative sense,single-stranded RNA virus,which often causes seasonal epidemics,posing a potential risk for a new influenza pandemic.IAV first invades respiratory tract or lung epithelial cells via binding virus surface protein hemagglutinin(HA)to sialic acid receptor on cell surface.The result of infection depends on the balance between the virus replication and host immune response.Overactivation of inflammatory response caused by IAV will lead to over-production of pro-inflammatory cytokines,as well as"cytokine storm".Several lines of evidence indicate that inflammatory response cytokines induced by IAV play a pivotal role in virus infection-induced lung injury and inflammation.The normal cellular prion protein,designated PrP~c,is a membrane glycoprotein expressed abundantly in brains and to a lesser extent in lungs and other tissues.PrP~c participates in neuroprotection,immune response and plays a complex regulatory role in various virus infections.A recent study indicated that PrP~c has a protective role against lethal infection with IAV.It was believed that PrP~c functions in combining the copper ions,and regulates the reactive oxygen species producing enzyme(SOD1)through the N-terminal OR region,thereby reducing excessive reactive oxygen species(ROS)in IAV-infected lungs,inhibiting cell apoptosis,eventually protecting them from lethal infection with IAV.However,whether the non-OR region(other regions of PrP~c)has the function of anti-virus infection remains to be studied.In addition,PrP~c is expressed in T lymphocytes,natural killer cells,macrophages and alveolar type?,type?epithelial cells and bronchiolar Clara cells.Therefore,PrP~c may play an important role in the immune system of the lung.However,the function of PrP~c in the inflammatory response induced by IAV remains largely unknown.Based on this,this thesis intended to explore the role and the mechanism of PrP~c in in pathogenicity and inflammation of IAV infection in vitro and in vivo by two types of PrP~c knockout mice and PrP~c overexpression plasmid,and the molecular mechanism was further discussed by RNA-Seq,differential genes expression analysis and verification.The content of the thesis could be briefly summarized as follows.The first part,to explore the effect of PrP~c in the pathogenicity of mice post IAV infection in vivo.We constructed two types of PrP~c-deficient mice by CRISPR/Cas9(119 KO mice with prion protein knocked out and 120 KO mice with N-terminal-OR region of PrP~c deleted).We then investigated the differences in clinical symptoms and lung histopathology between wild-type(WT)mice and 119KO/120KO mice at day 3,5,8 and 11 post H7N7-IAV infection.Compared with WT mice,120 KO mice were more susceptible to H7N7,showed lower survival rate and more wight loss,accompined with more severe lung damage.In contrast,119 KO mice were less susceptible to H7N7 compared with WT mice.These results suggested that the OR region could play an important role for PrP~c to protect against lethal infection with H7N7-IAV in mice,while non-OR-domains of PrP~c might promote virus infection significantly.Therefore,we speculated that PrP~c could have double regulatory efffects in the process of IAV infection.The second part of this thesis was RNA-seq sequencing,differential gene expression profile analysis and qPCR were performed of 120KO,119KO and WT mice lungs at at day 3,5,8 and 11 post infection of H7N7 and equal saline.Analysis and qPCR results showed that cytokines TLRs pathways(TLR4/7,MyD88,TAK1,IRF3),interferons(IFN?/?/?),IL6,TNF?,Nrf2,CSF3 and chemokines CCL2/3/11 and receptors CCR2/3,CXCL2/3/10/11 and receptors CXCR3/4/6 and CX3CR1 were significantly up-regulated post infection of H7N7.The expression level of these cytokines and chemokines in 120KO mice were significantly higher than WT mice;while these genes in 119KO mice were significantly lower than in WT mice.Therefore,we believe that OR region could inhibit the up-regulation of cytokines and chemokines after H7N7 infection and played a protective role in inflammatory storms induced by IAV infection.However,the non-OR region of PrPc might act in promoting up-regulated of chemokines,as well as in enhancing the inflammatory response post H7N7infection.That is,the prion protein might have a dual-regulatory effects on lung injury and inflammatory response in H7N7-IAV infection.The third part of this thesis was transfection of overexpression PrP~c plasmid on mouse lung epithelial cells(MLE-12)in vitro,to further verify the role of PrP~c in influenza virus H7N7 infection on MLE-12.The qPCR results showed that at different stages after H7N7 infected cells,the interactions between virus particles and PrP~c were different.During the early stage,within 12h post H7N7 infection,the virus induced up-regulation of PrP~c on the basis of transfectio PrP~c overexpression plasmid.After 24h of virus infection,the expression of PrP~c decresed significantly and continuously down-regulated M gene.48h post-infection,PrP~c recovered to its original expression level.Moreover,24h post transfection of mo-PrP~c-pcDNA3.1,IFN-?and TLR3 were significantly up-regulated,and IFN-?expression level was significantly reduced but TLR3 expression level was obviously up-regulated after IAV infection.It was speculated that overexpressed PrP~c might have different regulatory effects on different cytokines during IAV infection.Overall,it came a conclusion that PrP~c was involved in lung injury and inflammatory responses induced by H7N7-IAV in mice and MLE-12 cells for the first time.These data could be helpful for further understanding the the role of PrP~c in pathogenesis of RNA virus infection and suggest that PrP~c might be a novel target molecule for anti-influenza A virus therapeutics.