Study On Function And Mechanism Of Notch Signaling Pathway In Host Anti-hantaanviral Innate Immune Responses

With the outbreak of Ebola hemorrhagic fever in Africa in 2014,viral hemorrhagic fever has attracted great attention worldwide.Hemorrhagic fever caused by the hantavirus is the most endemic and most common hemorrhagic fever virus in the world and poses a serious threat to public health.Hantavirus belongs to the Bunyavirales family(Hantaviridae)orthohantavirus genus,carried and transmitted by rodents,causing hantavirus lung syndrome(HPS)and renal syndrome hemorrhagic fever(HFRS).HPS is predominantly endemic in the Americas,while HFRS is predominantly endemic in Eurasia.Hantan virus(HTNV),the main pathogen of severe HFRS in China,is the prototype virus of Hantavirus.The incidence of HFRS in China accounts for more than 80%of the world’s,and the mortality fluctuate between 0.1%and 15%.At present,treatments of HFRS are mainly based on supportive and symptomatic treatment,and there are no effective antiviral drugs.Therefore,further exploration on molecular mechanisms will facilitate clinical transformation of HFRS.The Notch signaling pathway has high conservatism of evolution and activated by interactions of adjacent cells,which plays a significant role in cell differentiation,proliferation,apoptosis,tumorigenesis,and cancer delivery.Recent studies indicated the key regulatory role of Notch signaling pathway in the host innate immune response to anti-infection.Studies in our group also demonstrated that Notch signaling facilitate the progression of HFRS by influencing macrophage activation patterns.However,it is still not clear how HTNV regulates Notch signal activation during the interaction between the virus and target cells(blood vessel endothelial cells and other tissue cells),and whether Notch signal itself can directly affect the process of virus infection and replication.First,we detected that HTNV can block the nucleation of NICD and inhibit the activation of Notch signal using q PCR,immunofluorescence,western blotting and other molecular biological techniques.Secondly,on the one hand,theγsecretase inhibitor DAPT was used to block notch signal activation,and cell infection model was constructed by q PCR and western blotting.Then,the replication of HTNV was determined at the nucleic acid and protein levels,and the level of ISG molecules,the downstream of interferon(IFN),was detected.The results suggested that inhibition of Notch signal could promote HTNV replication,but has no influence on the expression of ISG.on the other hand,Notch signal was activated by using overexpressed plasmids upregulating NICD and a cell infection model was built.Then,we detected the viral replication and the level of ISG,and the results indicated that overexpression of NICD can significantly inhibit HTNV replication but not production,and Notch signal exerted antiviral effect through interferon-independent ways.Finally,we overexpressed NICD by western blotting,q PCR and other methods,and detected the influence of Notch signal on autophagy,N-6 methylation modification(m6A)and other biological processes.The results demonstrated that Notch signal has no influence on the expression of the key proteins in autophagy such as Beclin1 and p62,however,it significantly facilitate the production of METTL14,a key"writer"of m~6A,suggesting that Notch signal may exert antiviral effects by regulating the N-6 methylation modification of viral nucleic acids.However,its specific molecular mechanisms still need further studied.In summary,with the help of immunofluorescence,western blotting,q PCR,molecular cloning and other experimental means,this study confirmed the antiviral effect of Notch signal in the process of HTNV-target cells interaction,and preliminarily revealed that the antiviral effect does not rely on interferons or autophagy pathways,but is related to m~6A modification,which providing a new way of antiviral therapy in HFRS.