Identification Of Key Virulence Sites Of Swine Influenza Virus And The Mechanism Of Host Proteins MATR3 And G3BP1 Inhibiting Virus Replication

Swine influenza virus(SIV)belongs to the genus of influenza A viruses and has been wid ely circulating in pig populations worldwide since its discovery during the Spanish influenza pandemic in 1918.SIV can cause respiratory diseases in pigs,causing great economic losses to the pig industry.Since pigs are susceptible to both human and avian influenza viruses,and can be used as a " mixing vessels" of influenza viruses to produce novel recombinant influenza viruses with pandemic potential,SIV also brings a continuous threat to human health.Therefore,research on SIV is of great public health significance.This study started from the epidemiological investigation of SIV,to understand the current epidemic trend of SIV in domestic pig populations,and to identify the key virulence sites and host proteins that inhibit virus replication,and to further analyze the pathogenic mechanism of SIV by studying their functions.In order to understand the prevalence of SIV in swine population in China,we conducted an epidemiological study on SIV in Hebei,Tianjin,Shandong,Jiangsu,Shanghai and Zhejian g from2018 to 2020.A total of 32 SIV strains were isolated and identified after inoculation with MDCK cells in 1006 nasal swab samples collected,with a virus isolation rate of 3.2%.The sequences of 8gene segments obtained by sequencing were analyzed for genetic evolution.The results showed that26 of the 32 SIV strains isolated belonged to Eurasian avian-like(EA)H1H1 subtype SIV,and 6belonged to H3N1 subtype SIV.The 26 EA H1N1 subtype SIV strains contained four different genotypes: G1,G2,G4 and G5.The G1 genotype strain was isolated only in 2018,and the G2 genotype strain was isolated only sporadically during 2018-2019.Since 2018,G4 and G5 genotypes have gradually become the main prevalent genotypes.The other 6 SIV strains of H3N1 subtype w ere a new type of recombinant virus,whose surface gene HA originated from the H3N2 lineage.The genetic evolution analysis showed that the emergence of the new recombinant SIV strain of H3N1 subtype was caused by the recombination of G4 genotype and H3N2 subtype SIV in pigs.This finding is the first report of a novel recombinant H3N1 subtype SIV in China.The findings of this study are of great significance for understanding the ecological distribution and genetic evolution trend of SIV in pig populations in China,and emphasize the importance of strengthening the monitoring of SIV in pig populations.In order to analyze the molecular pathogenic mechanism of SIV,this study first analyzed the evolutionary tree of H1N1 subtype SIV strains in China from 2012 to 2020.The results showed that the recombinant EA H1N1 subtype SIV strains with G5 and G4 genotypes were mainly prevalent in domestic pig populations.Further analysis of internal gene sources showed that Viral ribonucleoprotein originated from the 2009 pandemic(pdm/09)H1N1 lineage.The proportion of v RNP gene and NS gene derived from North American triple-reassortant(TR)lineage showed an increasing trend year by year.Pathogenicity experiments were conducted on mice and pigs between the recombinant strain and the pure G1 genotype strain.The results showed that NS gene derived from TR lineage had a key effect on virulence.Then,by mutating the different amino acid sites of the strong and weak virulence and evaluating the virulence,it was identified that the mutation of NS1 protein E153G played a key role in enhancing the virulence of the virus.The spatial structure analysis of NS1 protein showed that this mutation resulted in the reduction of the number of hydrogen bonds formed by amino acid 153 of NS1 protein.Further experimental results showed that the mutation of NS1 protein E153 G changed the stability of NS1 protein structure,resulting in enhanced inhibition of NS1 protein on IFN-β,and thus enhanced the replication ability of the virus and its pathogenicity in mice.NS1 protein,as a non-structural protein,plays an important role in the resistance to host innate immunity,while NP protein,as a structural protein,plays a central role in the process of influenza virus replication.In order to study the specific functional mechanism of how host restriction factors inhibit virus replication by influencing NS1/ NP protein,in this study,a variety of host proteins that may interact with NS1/NP proteins were identified by mass spectroscopic analysis.Through the effects on viral replication and Co-immunoprecipitation(Co-IP)experiments,Matrin 3(MATR3),which interacts with NS1 and inhibits virus replication,was identified.In addition,Ras-GAP SH3 domain binding protein 1(G3BP1),which can inhibit viral replication by interacting with and affecting NP proteins.As for the host protein MATR3,the laser confocal experiment showed that MATR3co-localized with NS1 protein in the nucleus and resulted in the accumulation of NS1 protein in the nucleus.NS1 protein can inhibit the expression of IFN-β by inhibiting the activation of RIG-I signaling pathway.In this study,the activation levels of TBK1 and IRF3,the key factors downstream of the RIG-I signaling pathway,were detected after overexpression of MATR3.The results show that the down-regulation of phosphorylated TBK1 and IRF3 caused by NS1 protein is restored to a certain extent,and the expression of IFN-β is up-regulated.Therefore,MATR3 is able to inhibit viral replication.For the host protein G3BP1,the results of laser confocal experiment showed that G3BP1 and NP protein co-localized in the cytoplasm,while the NP protein was not distributed in the cytoplasm.Further experimental results showed that G3BP1 could interfere with the nuclear i mport process of NP protein,and then lead to the residue of NP protein in the cytoplasm.To investigate how G3BP1 interferes with the nuclear import process of NP protein,Co-IP experiments in this study showed that G3BP1 could competitively bind nuclear import protein(Importin,IMP)α5 and inhibit the interaction between NP protein and IMP α5,thereby blocking the nuclear import process of NP protein.Since the nuclear import process of NP protein was inhibited by G3BP1,further experimental results verified that G3BP1 had a significant inhibitory effect on the polymerase activity of the virus.Therefore,the host protein G3BP1 could inhibit the virus replication.In summary,on the one hand,this study on the pathogenic mechanism of SIV,mainly through the epidemiological survey of SIV,understand the prevalence of SIV in pigs in our country in recent years,through the different strains isolated pathogenic experiments on mice and pigs found from TR lineage of NS gene has a critical influence on virulence,The E153 G mutation in NS1 protein was identified to enhance the virulence of the virus,and the main mechanism is that the mutation leads to the decrease of the number of hydrogen bonds,which changes the structural stability of NS1 protein,and then enhances the inhibition of NS1 protein on IFN-β.On the other hand,the host proteins that play a natural immune function in the process of resisting virus replication were identified and studied.Two host proteins,MATR3 and G3BP1,which can inhibit virus replication,were identified by mass spectrometry.Further studies found that Matriline3 interacts with NS1 protein and leads to its accumulation in the nucleus.The inhibition effect of NS1 protein on RIG-I signaling pathway was weakened.G3BP1 can interact with NP protein and inhibit the interaction between NP protein and IMP α5,resulting in the retention of NP protein in the cytoplasm,and then lead to the inhibition of the polymerase activity of the virus.Therefore,this study analyzed the pathogenic mechanism of SIV and the antagonistic mechanism of the host from the aspects of the virus and the host,which provided a certain theoretical basis for the prevention and control of SIV,and provided a direction and laid a certain theoretical basis for the in-depth study of the pathogenic mechanism of SIV.