Mechanism Of The Regulation Of Innate Immunity By The Nucleocapsid Protein Of Three Coronavirus

Coronaviruses(CoVs)refer to the highly diverse viruses of the order Nidovirales,family Coronaviridae,subfamily Orthocoronavirinae,which are further divided into four genera named α-,β-,γ-and δ-CoV.CoVs are enveloped viruses with a single-stranded,non-segmented and positive-oriented RNA genome and spreading widely in nature.Notably,α-,β-and δ-CoV can infect humans.Cross-species transmission is frequently reported for CoVs,which may lead to disease or epidemics of the new host.During the recent two decades,three worldwide epidemics caused by CoV originating from wide animals have emerged,seriously jeopardizing public health and global economic development.Unfortunately,the mechanism underlying cross-species transmission and pathogenesis of CoVs have not been fully understood yet,hindering the prevention and control of CoV.IFN-I(Type I interferon)is a critical factor of innate immune response against viral infection.However,a variety of pathogenic CoVs can inhibit the host IFN-I signaling pathway,though the molecular mechanism has not been fully revealed.N(nucleocapsid).an important structural protein of CoV,not only participates in virus packaging and replication,but also interacts with different host proteins to regulate gene expression and signal transduction of the host cell.In this study,we selected three typical pathogenic CoV with potential of cross-species transmission,including human β-CoV SARS-CoV-2,porcine α-CoV SADS-CoV and poultry δ-CoV HNU3-CoV,to investigate the roles of their N in modulating IFN-I pathway.To this end,we conducted the following three studies.Firstly,we explored the mechanism of SARS-CoV-2 N regulating IFN-I pathway in human cells.SARS-CoV-2 is a highly pathogenic coronavirus infecting humans which originated from certain bat CoV possibly.We found that SARS-CoV-2 N could inhibit SeV-mediated transcriptional activation of IFNβ,ISRE and NF-κB by luciferase-reporter assay and quantitative real-time PCR(q PCR).Further molecular studies showed that N protein could bind to several proteins of RLR(RIG-I-like receptor)signaling,including MAVS(mitochondrial antiviral signaling protein),G3BP(RAS GTPase-activating protein SH3 domain binding protein)and TRIM25(tripartite motif 25).These results revealed that SARS-CoV-2 N disrupted RLR activation,inhibited IRF3(Interferon regulatory factor 3)phosphorylation and thereby antagonized the production of IFN-I.Secondly,we explored the mechanism of SADSr-CoV N regulating IFN-I pathway of human cells.SADS-CoV is a newly identified porcine CoV belonging toα-CoV genus,which may originate from bats HKU2-related CoV.In order to reveal the function changes of the CoV N proteins from different species in regulating host innate immunity during the cross-species transmission,we carried out research from the following aspects.(1)We confirmed that the N proteins of porcine GDS04 and bat HKU2 could inhibit the activation of IFN-β,NF-κB and ISRE promoters mediated by SeV,and both N proteins could inhibit IFN and ISG transcription stimulated by SeV.ELISA results showed that both N proteins could inhibit the secretion of IFN-βactivated by SeV;VSV-GFP sensitivity experiment indicated that the N proteins reduced the sensitivity of the VSV to interferon.In addition,it was proved that the N proteins of GDS04 and HKU2 could not inhibit the ISRE promoter activity and ISG m RNA activated by IFN-β,indicating that the N proteins of GDS04 and HKU2 could inhibit the production of IFN-I but not the signal transduction of interferon.(2)Immunoblotting,immunofluorescence and nucleocytoplasmic separation experiments showed that both N proteins could inhibit the phosphorylation level and nuclear translocation of IRF3 and NF-κB induced by SeV.(3)the N proteins of GDS04 and HKU2 did not inhibit the activities of IFN-β,ISRE and NF-κB promoters activated by RIG-I,MAVS,TBK1,IKKε and IRF3 in RLR signaling pathway,suggesting that the potential targets of the N proteins of GDS04 and HKU2 are RIG-I or upstream of RIG-I.Co-IP found that both N proteins could not interact with G3BP1.The overall results showed that there was no significant difference between the N proteins of porcine GDS04 and bat HKU2 in inhibiting the IFN pathway and the specific mechanism.This suggests that N proteins inhibit the function of the new host’s innate immunity pathway,which may be used as an indicator to judge whether the CoV has the ability to spread across species and exhibit pathogenicity.Thirdly,we explored the mechanism of HNU3-CoV N regulating IFN-I pathway of human cells.HNU3-CoV belongs to the δ-CoV genus and is a new CoV identified by our research team from common magpies.δ-CoV mainly infect avian but have also reported to infect porcine and humans via cross-species transmission.In this study,we found that HNU3-CoV N inhibited the activation and the signal transduction of IFN-I but not NF-κB activition.Meanwhile,HNU3-CoV N could inhibit the activation of RLR signaling pathway,suggesting that its potential target was RIG-I or upstream of RIG-I.However,HNU3-CoV N protein could not interact with G3BP1 or TRIM25.In summary,our results have shown that N proteins of both porcine SADS-CoV and avian HNU3-CoV suppressed IFN-I pathway when infecting human cells,which was similar to SARS-CoV-2,indicating potential pathogenicity of these two animals CoV to humans.Therefore,the cross-species transmission of animal CoV should be well monitored and controlled.Nevertheless,the molecular mechanisms of inhibiting IFN-I pathway vary among N proteins of different CoVs.Especially,SADS-CoV cannot inhibit IFN-I signal transduction,and HNU3-CoV cannot inhibit the activity of NF-κB,while SARS-CoV-2 N protein can inhibit both pathways,indicating that animal SADS-CoV and HNU3-CoV had not established sophisticate strategy to antagonize human immunity.SARS-CoV-2 has evolved a complex antagonistic mechanism under the selective pressure of the human immunity.SARS-CoV-2 N protein inhibited more components upstream of the RLR signaling pathway than the other two CoVs.Such difference might explain the high pathogenicity of SARS-CoV-2 to humans.These findings provide new perspective about the mechanism employed by different CoV to suppress human innate immunity,which contributes to the understanding about the gene function,pathogenesis and cross-species transmission potential of CoV.