The Molecular Mechanism Of Coronavirus N Proteins Inhibit IFN-? Production

Coronaviruses(CoVs)belong to Coronaviridae family in the order Nidovirales,which containing four genera based on phylogeny: alpha-CoV,beta-CoV,gamma-CoV and delta-CoV.The agents from alpha-CoV,beta-CoV,gamma-CoV and delta-CoV genera mainly infect mammals,or avains,or both.Coronaviruses are now becoming more a global concern since the worldwide emergence of severe acute respiratory syndrome CoV(SARS-CoV)in 2003.Recently,novel coronaviruses were successive identified,such Middle East respiratory syndrome CoV(MERS-CoV)in 2012 and porcine deltacoronavirus(PDCoV)in 2014,which spread though out America.In addition,coronavirus innovates quickly within the conditions of shifting surrondings and high immune pressures.Porcine epidemic diarrhea virus(PEDV)is a classic example,which variant pandemic and epidemic emerged in China in late 2010.The emergence and re-emergence of coronaviruses cause high morbidity and mortality in human and animal populations,and pose heavily public health and cause heavily economic losses.As structural protein,coronaviruse nucleocapsid(N)protein is the most abundant viral protein in both infected cells,and exhibits multiple functions throughout the viral life cycle.In addition to maintaining the viral RNA in an ordered conformation for replication and transcription,involving in the regulation of cellular transcription and apoptosis,N protein was also appointed to counteract the host innate immune defense.MHV and SARS-CoV N proteins have been reported to counteract IFN response or production with unclear mechanisms.The study reavealed the mechanisms of N proteins from PEDV(?-CoV),SARS-CoV and MHV(?-CoV),as well as PDCoV(?-CoV),inhibited IFN-? production.The study mainly includes:1.PEDV N protein antagonizes IFN-? productionN protein can express abundantly in early infected cells,which contributes N protein to regulate initial immune response.In this study,we constructed N expression plasmid and further study found that PEDV N protein inhibits Sendai-virus(SEV)-induced IFN-?production,IFN-stimulated gene expression,and activation of transcription factors IRF3 and NF-? B.We also found that N protein significantly impedes the activation of the IFN-? promoter stimulated by TBK1 or its upstream molecules(RIG-I,MDA5,IPS-1,and TRAF3),but does not counteract its activation by IRF3.A detailed analysis revealed that PEDV N protein targets TBK1 by direct interaction,and that this binding sequesters the association between TBK1 and IRF3,which in turn inhibits both IRF3 activation and type I IFN production.2.MHV and SARS-CoV N proteins share the same IFN-? antagonizing mechanismwe found that PEDV N protein blockades IFN-? signaling through targeting TBK1 to prevent its interaction with IRF3.We also investigated whether MHV and SARS-CoV N proteins utilized the same strategy to antagonize IFN-? production.However,results showed that N proteins of MHV and SARS-CoV do not interact with TBK1.Further study found that MHV N protein inhibits SEV-or poly(I:C)-induced IFN-? production.Meanwhile,it does not affect the activity of IFN-? promoter stimulated by RIG-I and MDA5 with no association,and similar observations were reported for SARS-CoV N protein with unidentified mechanism.Further studies showed that both MHV and SARS-CoV N proteins directly interact with PACT,a cellular dsRNA-binding protein which can bind to RIG-I and MDA5 to activate IFN-? production.The N-PACT interactions sequester PACT-RIG-I or MDA5 association,which in turn inhibits IFN-?production.In addition,we did not observe the association of PACT and N proteins from porcine epidemic diarrhea virus(PEDV)and porcine reproductive and respiratory syndrome virus(PRRSV),another coronavirus and an arterivirus,which is also classified in the order Nidovirales.3.PDCoV N protein involved diversity strategies to inhibit IFN-? production.PDCoV reported in United States swine in 2014 and subsequently was detected in Asia,which clinical cases in piglets were characterized with high mobidity and mortality.Unlike PEDV or MHV and SARS-CoV,PDCoV belongs to genera Deltacoronavirus,which during infection is little known about regulation of interferon(IFN)responses.We previously reported that PDCoV infection fails to induce,and even remarkably inhibits,SEV-or poly(I:C)-induced IFN-? production.In additon,coronavirus N proteins share conserved functions.To investigate the role of PDCoV N protein in IFN-? mediation,we first investigated whether PDCoV N protein could inhibit IFN-? production withluciferase reporter gene assay,quantitative real-time RT–PCR and ELISA,and found that PDCoV N prohibits SEV-or poly(I:C)-induced IFN-? sythesis,as well as SEV-or poly(I:C)-induced RIG-I activation for IFN-? production.Further study found that PDCoV N protein interactes with helicase and CTD domain of RIG-I,dsRNA and PACT,resulting in the agonists dsRNA and PACT failed to bind to RIG-I.The association of PDCoV N protein and PACT/RIG-I was RNA independent.Furthermore,we identified that NTD of PDCoV N protein harbores IFN-? antagonizing function,which is response for cytoplasm location and possess weak RNA binding ability.Taken togather,PDCoV N protein circumvents IFN-? production with a complicated mechanism: PDCoV N protein formed complexes with RIG-I,PACT and dsRNA,but N-RIG-I interaction contributes PACT and dsRNA failed to bind to RIG-I,leading to IFN-? inhibition.We revealed the distinct IFN-?-antagonizing mechanisms involved by PEDV,MHV,SARS-CoV and PDCoV N proteins,suggesting coronaviruses evolved quite complicated stragegies to counteract innate immunity.