The Roles And Mechanisms Of SARS-CoV-2 Proteins Antagonizing Host Interferon Antiviral Response

Coronavirus disease 2019(COVID-19),caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),has spread worldwide and infected more than 490 million people.It seriously affects human health and causes great losses to the global economy.SARS-CoV-2 is a positive-strand RNA virus that belongs to the enveloped coronaviridae β-coronavirus.The viral genome encodes 16 nonstructural proteins(nsp 1-16),four structural proteins(spike[S],envelope[E],membrane[M],nucleocapsid[N]),and seven accessory proteins(ORF3a,ORF3b,ORF6,ORF7a,ORF7b,ORF8,and ORF10).A typical feature of COVID-19 is the lack of type Ⅰ interferon(IFN-Ⅰ)-mediated antiviral immunity in patients.Nevertheless,the specific molecular mechanisms by which SARS-CoV-2 proteins evade the IFN-Ⅰ-mediated antiviral responses are not completely clear.Therefore,we constructed the expression plasmids for 22 SARS-CoV-2 viral proteins based on the genomic structure,and screened a set of SARS-CoV-2 proteins that antaginize IFN-Ⅰ signaling pathway by using interferon P(IFN-β)promoter-based luciferase reporter assays.Initially,we found that the nsp7,nsp 15,M,3CLpro(domain of nsp5),Helicse(nsp 13),and N proteins inhibit IFN production,whereas the nsp7,Helicse,nsp9,ORF7a,ORF8,E,and S proteins inhibit the activation of the IFN-stimulated response element(ISRE).We further used the Real Time Quantitative PCR(RT-qPCR),western blotting,and immunofluorescence experiments to explore the mechanisms.We found that Helicase protein significantly inhibited the phosphorylation levels of the TANK Binding Kinase 1(TBK1)and Interferon Regulatory Factor 3(IRF3),and N protein inhibited the phosphorylation levels of IRF3 but had little effect on the phosphorylation levels of TBK1.Although M protein did not affect the activation of IRF3,it clearly inhibited IRF3 imported into the nucleus.To further explore the specific mechanisms of above SARS-CoV-2 virus proteins,we performed immunoprecipitation and protein mass spectrometry(IP-MS)experiments.We found that M protein interacts with many importin karyopherin subunits.These interactions were subsequently validated by immunoprecipitation(IP)experiments,immunofluorescence,and nucleocytoplasmic fractionation assays.We further confirmed that M protein inhibited the IFN-Ⅰproduction by blocking IRF3 binding to importin karyopherin subunit alpha-6(KPNA6).In IFN-Ⅰ downstream signaling pathway,we found that p-STAT1 and p-STAT2 were significantly inhibited by Helicase and envelope protein(E).Spike(S)not only inhibited the phosphorylation of Signal Transduction and Activation of Transcription 1(STAT1)and Signal Transduction and Activation of Transcription 2(STAT2),but also inhibited the nuclear translocation of p-STAT1.To further explore the specific mechanisms of these proteins,we also performed IP-MS,and immunofluorescence experiments.We found that S protein interacted with STAT1.Further studysuggested that S protein antagonizes the IFN-Ⅰ signaling pathway by inhibiting the binding of Janus Kinase 1(JAK1)and STAT1 through its S1 subunit.In this study,we screened for SARS-CoV-2 proteins that antagonizing the IFN-Ⅰproduction pathway,and elucidated the specific mechanism of M impairing the IFN-Ⅰproduction.We also screened for SARS-CoV-2 virus proteins antagonizing IFN-Ⅰdownstream signaling pathway(JAK-STAT)and preliminarily elucidated the specific mechanism for S protein inhibiting IFN-Ⅰ signaling.These studies will provide essential targets and theoretical references for vaccine and antiviral drug development.