Dual R108K And G189D Mutations In The NS1 Protein Of A/H1N1 Influenza Virus Counteracted Host Innate Immunity

Influenza is an acute respiratory infectious disease caused by influenza virus infection.Influenza viruses belong to the family Orthomyxoviridae,including four types: type A,B,C and D.Influenza A and B viruses cause human seasonal influenza.Based on the antigenicity of hemagglutinin and neuraminidase surface glycoproteins,influenza A virus can be divided into 18 H subtypes(H1～H18)and 11 N subtypes(N1～N11).The World Health Organization(WHO)estimates that influenza outbreaks infect about 1 billion people,cause 3-5 million cases of serious illness and kill 300,000-500,000 people each year.The severity of pandemic influenza depends on a number of factors,including the virulence of pandemic influenza strains and the pre-existing immunity level of the population.The worst influenza pandemic in 1918 killed more than 40 million people worldwide.Influenza virus is a single-strand negative-chain multi-segment enveloped RNA virus.The genome contains eight genomic segments and the nucleotide length is between 890 bp and 2,341 bp.The non-structural protein(NS)fragment in the genome is the smallest RNA fragment,which encodes two kinds of viral proteins NS1 and NS2.NS1 plays a variety of important roles in viral infection,including regulating viral RNA synthesis,viral m RNA splicing and virion morphogenesis,and inhibiting host apoptosis by activating phosphatidylinositol 3-kinase(PI3K).As an interferon antagonist,it plays a role in the host,making the virus escape the antiviral effect of the host interferon and replicate efficiently.We analyzed 2,155 strains of H1N1 influenza virus from 2009 to2020 and found that the important functional sites such as I123 V and N205 S of one H1N1 influenza virus isolated from Xinjiang were mutated compared with the early strain A/California/07/2009,but 108 R and 189 g were highly conserved.In order to understand the effect of NS1 protein 108,189 on influenza virus,we made a preliminary exploration through this study.This study mainly includes three parts:Part Ⅰ: construction of reverse genetics system of influenza virus subtype H1N1Our laboratory isolated influenza A 2009H1N1 virus strain A/Urumqi/XJ49/2018(H1N1)from clinical throat swabs of patients with influenza in Urumqi in 2018.Sequence homology comparison showed that the homology with HA,NA,M,NS,NP,PA,PB1 and PB2 genes of A/CA/07/2009(CA07)strain was 96.0%,97.0%,98.0%,97.0%,98.0%,98.0%,97.0% and 98.0%,respectively.Through sequence alignment analysis between the rescued strain and the wild strain,it was found that there were a small amount of amino acid changes in the virus strain,most of which were nonsense mutations.Based on this strain,an infectious recombinant plasmid was constructed using influenza virus 8 plasmid system.There was no significant difference between rescue virus and wild virus in plaque and proliferation characteristics.Part Ⅱ: construction of NS1 mutant infectious clone plasmid and rescue of mutant virusXJ49 sequence analysis showed that there were mutations in E55 K,L90I,I123 V,V129I,N205 S and other important NS1 functional sites in the NS1 gene,but there was no mutation with R108 and G189,which affect the carboxyl terminal effect domain.Based on the reverse genetics system of H1N1 influenza XJ49 virus,we mutated the NS coding region of NS recombinant plasmid p HW2000-XJ49 NS at site 108,189 amino acids.The XJ49-NS1 mut mutant recombinant plasmid and other 7 wild type plasmids in the reverse genetics system were transfected into 293T/MDCK cells,and the R108K/G189 D double mutant virus XJ49-NS1 mut was successfully rescued.There was no significant difference between the mutant virus and the wild type in proliferation trend and plaque morphology in vitro.The pathogenicity of mutant virus in mice was slightly higher,although there was no significant difference in lung virus load,it caused more serious pathological damage in mouse lungs.Part Ⅲ: the effect of NS1 protein R108 K G189D mutation on antagonistic host innate immunityWe used double luciferase method to evaluate the effect of XJ49-NS1 mut mutant virus on the expression of IFN.The results showed that the mutant virus showed a higher level of inhibition of IFN than wild type virus.At the same time,after infection,the mutant virus showed a continuous inhibitory effect on the m RNA expression of IL-28/IL-29 and TRIM22.The results of infection experiment in C57BL/6N mice showed that R108K/G189 D double mutant virus significantly inhibited the expression of cytokines such as IL-6,GM-CSF,GRO-α/CXCL1,MCP-1/CCL2,MIP-1α,MIP-2,IFN-α and M-CSF in the early stage of infection,but there was no significant difference between wild type virus and wild type virus in the late stage of infection,which may be the reason why the mutant virus only slightly increased the pathogenicity.In summary,the virus encoding NS1 protein with dual R108 K and G189 D mutations was rescued based on the seasonal 2009 p H1N1 A/Urumqi/XJ49 virus backbone.The dual mutations in NS1 protein exhibited little effects on viral replication but inhibited m RNA expression levels of IL-28,IL-29 and TRIM22.The NS1 mutated virus showed significant efficient at suppression of early cytokine induction in mouse lung and cause slightly increased mortality double mutations of R108 K and G189 D could enhance the inhibition of host gene expression by NS1 protein.