Inhibition Mechanism Of Type ? Interferon Response By Viral Proteins Of SARS-CoV-2

Coronavirus disease 2019 is also known as COVID-19,which is caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2).Typical clinical symptoms of SARS-CoV-2 infection range from mild-to-severe respiratory illness,including fever,dry cough,breathing difficulties,and acute respiratory distress.Today,the number of SARS-CoV-2 infected people and the spatial scope of COVID-19 epidemic area have spread all over the world,which has formed a serious threat to human survival and public health,as a result,WHO declared COVID-19 as a pandemic by WHO at March 11 th,2020.SARS-CoV-2 is a member of ?-coronavirus,which emerged after two other highly pathogenic coronaviruses in the past two decades [Severe Acute Respiratory Syndrome Coronavirus(SARS-CoV,emerged in 2002)and Middle East Respiratory Syndrome Coronavirus(MERS-CoV,emerged in 2012)].SARS-CoV-2,together with SARS-CoV and MERS-CoV,causing fatal respiratory disease,makes coronavirus one of the major obstacles that seriously delay the development of globe health.In addition,four endemic human coronaviruses(i.e.,OC43,229 E,NL63,and HKU1)cause common cold respiratory diseases.SARS-CoV-2 genome is a positive-sense,single-stranded RNA that encodes 16 nonstructural proteins(NSP1–16),4 structural proteins(S [spike],E [envelop],M [membrane],and N[nucleocapsid]),and several accessory proteins(such as ORF3 a,ORF3b,ORF6,ORF7 a,ORF7b,ORF8,and ORF10).The nonstructural proteins make up the replicase,the structural proteins form the virion,and the accessory proteins modulate the host response to facilitate infection and pathogenesis.Understanding the molecular mechanisms of the virus and its host interactions is the key to studying COVID-19 pathogenesis and transmission,as well as to develop diagnosis method and make the countermeasures against these coronaviruses.The innate interferon(IFN)response constitutes the first line of host defense against viral infections.IFN is a class of cytokines with powerful functions,which can directly induce an immune response against pathogenic microorganisms by controlling the inflammatory response and coordinating the immune response,resulting in resisting the infection of foreign pathogens.Upon infection,the induced IFN will eventually lead to the transcription and expression of hundreds of IFN-stimulated genes(ISGs)through different signal transduction pathways,and further play a role in resisting viral infections.To figure out the role of individual SARS-CoV-2 proteins in IFN-I production and signaling,and to understand the molecular mechanisms,we cloned individual viral proteins of SARS-CoV-2 into a mammalian expression plasmid p XJ.Individual viral proteins were expressed with FLAG tag in C-terminal to facilitate the detection of their expressions.Then we screened the role of individual viral proteins in antagonism of IFN-I production and signaling.Individual suppressors were mapped to their inhibitory steps in IFN-I production/signaling pathway.Our results indicate that SARS-CoV-2 proteins antagonize distinct steps in IFN-I production and signaling:(1)Among the 26 tested viral proteins,3nonstructural proteins(NSP1,NSP6,and NSP13)and 1 accessory protein(ORF6)significantly inhibited IFN-I production.NSP6 suppresses IRF3 phosphorylation by binding TBK1,while NSP13 interacts with TBK1,the interaction between NSP13 and TBK1 results in the inhibition of TBK1 and IRF3 phosphorylation;in addition,OFR6 blocks IRF3 nuclear translocation,resulting in antagonism of IFN-I production.(2)Ten proteins(NSP1,NSP6,NSP7,NSP13,NSP14,ORF3 a,M,ORF6,ORF7 a,and ORF7b)act as suppressors of IFN-I signaling,NSP1,NSP6,NSP13,ORF3 a,M,and ORF7 b block STAT1 phosphorylation;NSP6,NSP13,ORF7 a,and ORF7 b suppress STAT2 phosphorylation;and ORF6 inhibits nuclear translocation of STAT1 through interacting with KPNA2.As an important host factor,ubiquitin is involved in regulation of innate immunity.Ubiquitination is a post-translational modification process,which plays an important role in many biological processes.Based on viral proteins of SARS-CoV-2 which antagnize IFN-I response,we screened them for ubiquitination level,the result shows that 3 viral proteins(NSP13,ORF3 a,ORF7a)undergo high level of ubiquitination.Subsequently,ORF7 a was selected to study the relationship between its ubiquitination and antagonism of IFN-I signaling.And then,we identified that ORF7 a could be poly K63-ubiquitinated at residue K119,followed by a mutant(K-to-A)at K119 was induced to generate ubiquitination-defective ORF7a(ORF7a-K119A).We also compared antagonism level of IFN-I signaling between ORF7a-K119 A with wild type ORF7a(ORF7a-WT).Finally,we concluded that ubiquitination of ORF7 a promotes antagonism of IFN-I signaling by blocking STAT2 phosphorylation.Importantly,we have developed a reporter replicon of SARS-CoV-2 suitable for studying viral replication at BSL-2 laboratories,by replacing nucleotides 21,563 – 28,259)with Rluc/FDMV 2A/Neo reporter cassette.The deleted region includes S,E,M,and ORF3 a,ORF3b,ORF6,ORF7 a,ORF7b,and ORF8.We demonstrate the utility of the replicon using remdesivir,a well-known inhibitors of SARS-CoV-2 in cell culture.Finally,the replicon was used in antagonism comparison in IFN-I signaling of NSP1/NSP6 from three highly pathogenic coronaviruses,followed by evaluation of their roles in viral replication.As a result,NSP1 and NSP6 from three coronaviruses inhibit IFN-I signaling at different efficiency.In SARS-CoV-2,the potent inhibition of IFN-I signaling by NSP1 and NSP6 leads to higher level of viral replication than those of SARS-CoV or MERS-CoV,which may result in stronger replication kinetics of SARS-CoV-2.Briefly,we screened inhibitory abilities of IFN-I response by all viral proteins of SARS-CoV-2,and mapped their antagonism targets in IFN-I pathway.This study provides a theoretical basis for the vaccine development and antiviral drugs discovery of coronavirus.In addition,we also developed a reporter replicon of SARS-CoV-2,which allows experiments at biosafety level 2,providing a useful tool for research about coronaviruses.