The Underlying Mechanism Of The Antagonism Between SARS-CoV-2 And Host Innate Immunity

Coronavirus disease 2019(COVID-19)has now developed a pandemic.According to data of WHO,as of 4:28pm CEST,19 April 2021,there have been 141,057,106 confirmed cases of COVID-19,including 3,015,043 deaths.Its pathogen is identified as a new type of coronavirus(SARS-CoV-2).Compared with some other respiratory RNA viruses,SARS-CoV-2 is a poor inducer of type I interferon(IFN).Therefore,understanding the antagonism between SARS-CoV-2 and the host at the molecular level is crucial to further clarify the pathogenic mechanism of the virus.In order to understand the specific mechanism of SARS-CoV-2 antagonizing the host’s natural immunity from a viral perspective,we first constructed the expression plasmids of 23 proteins encoded by SARS-CoV-2 virus.After verifying the expression of each protein on HEK293T cells,we used the dual luciferase reporter assay to detect the effects of various viral proteins on the activation of IFN-β promoter induced by RIG-IN,MDA5 and TRIF.The results showed that multiple viral proteins including nsp1,nsp3,nspl2,nspl3,nsp14,ORF3,ORF6 and M play an inhibitory role in the RLR or TLR signaling pathway.In this study,we focused on the RNA-dependent RNA polymerase(RdRp)of SARS-CoV-2 to explore the mechanism of its inhibition of type I IFN signaling.We first confirmed that nsp12 could inhibit the expression of endogenous IFN-β mRNA induced by SeV or high molecular weight(HMW)poly(I:C),and then confirmed that nsp12 could inhibit the activation of IFN-β promoter induced by SeV or HMW poly(I:C)in a dose-dependent manner through dual luciferase reporter system.By detecting the activity of ISRE promoter downstream of IFN-β,it was found that nsp12 had no significant effect on the activation of ISRE promoter stimulated by exogenous IFN-β.These results suggested that nsp 12 inhibited the upstream IFN-β signaling pathway but not the downstream.Next,we tested the activation of the IFN-β promoter induced by RIG-IN\MDA5\MAVS and IRF3-5D and found that nsp 12 inhibited the activation of the downstream signals of the above four molecules,indicating that the target of nsp 12 is downstream of IRF3.Then we tested the effect of nsp 12 on SeV-stimulated IRF3 activation,and found that nsp 12 did not affect the phosphorylation of IRF3.The co-IP experiment showed that there is no direct interaction between nsp 12 and IRF3.Nuclear and cytoplasmic separation and immunofluorescence experiments indicated that nsp 12 could suppress the nuclear translocation of IRF3 stimulated by SeV or RIG-IN.These results indicated that nsp12 antagonized IFN-β production by inhibiting IRF3 translocating to the nucleus.In order to verify whether nsp12’s antagonism of IFN-β production is related to its RdRp activity,we mutated the key sites of polymerase activity and found that the mutated nsp12 variants could still inhibit the activation of IFN-β promoter and the nuclear translocation of IRF3 induced by SeV.In the presence of nsp12 inhibitor Remdesivir,nspl2 still inhibited the activation of downstream signal of IRF3-5D.Meanwhile,the ability of nspl2 to antagonize IFN-β was not affected by its cofactors nsp7 and nsp8.The above results showed that nsp12 antagonized IFN-β production was polymerase activity independent.In addition,we used interferon induced gene(ISG)knockout cells to screen the key host factors that can inhibit the replication of SARS-CoV-2 virus.Through previous screening,we found that the replication of SARS-CoV-2 virus in Galectin-9(Gal-9)knock out A549-ACE2 or THP-1 cells was significantly enhanced,while the replication of SARS-CoV-2 virus in cells with Gal-9 overexpression was significantly inhibited.We found that Gal-9 could inhibit the activity of SARS-CoV-2 replicon.We also found that Gal-9 could directly interact with nsp2,nsp8,nsp12 and nsp16,which are the key proteins involved in viral genome replication.These results indicated that Gal-9 could inhibit the replication of SARS-CoV-2.In addition,we also examined the effect of Gal-9 on other viruses,and found that the replication of enterovirus,another important single stranded positive RNA virus,was also significantly enhanced in Gal-9 knock out cells.In Gal-9 overexpression cells,enterovirus replication was also significantly reduced.We found that Gal-9 may affect the early stage of viral infection by comparing RNA and protein at the early stage of viral infection.When the viral genome was directly transfected into cells,the replication of viral RNA in Gal-9 deficient cells was obviously enhanced.These results indicated that Gal-9 could also inhibit the replication of enterovirus.In conclusion,we found that a variety of viral proteins of SARS-CoV-2 could directly inhibit the type Ⅰ IFN signal.Among them,viral polymerase nsp12 could inhibit the production of type Ⅰ IFN by suppressing the nuclear translocation of IRF3,which is polymerase activity independent.In addition,we found that Gal-9 could inhibit the replication of SARS-CoV-2 and bind to nsp12 and other viral proteins directly.Gal-9 could also inhibit the replication of enterovirus.