Molecular Mechanism For The Inhibition Of Host Gene Expression By Alphacoronavirus Nsp1

Alphacoronavirus(?-CoV)consists of several members including Human coronavirus 229E(HCoV-229E),Human coronavirus NL63(HCoV-NL63),Transmissible gastroenteritis virus(TGEV),Porcine epidemic diarrhea virus(PEDV),Feline infectious peritonitis(FIPV),Porcine respiratory coronavirus(PRCV)and Swine acute diarrhea syndrome coronavirus(SADS-CoV).They can cause respiratory,digestive and neuron systems diseases in both human kinds and animals,which have great risks to human safety and properties.Nonstructural protein 1(nsp1)is the N-terminal part of the polyprotein encoded by coronaviruses,which is only expressed by ?-CoV and betacoronaviruse(?-CoV).Studies focused on the ?-CoV nsp1 had been thoroughly delved these days.?-CoV nsp1 could suppress the expression of cytokines and antigen representing processes by reducing its host protein synthesis,and then reduced the cytotoxic impacts on the infected cells,so it was a crucial pathogenic factor.At present,the effect of ?-CoV nsp1 on viral virulence and the detailed mechanism of inhibiting host protein synthesis are unclear.This study chose ?-CoV nsp1 as the research subjects,exploring the structural basis and molecular mechanisims of how nsp1 protein will inhibit hosts gene expression.Specifically as follows:1.Through molecular cloning,protein expression and purification,crytal primary filtration and optimization as well as data collection and processing,we first figured out 4 types of ?-CoVs(PEDV,TGEV,FIPV and SADS-CoV)nsp1,full-length crystal structures.This work reveals that ?-CoV nsp1 displayed a conservative secondary structure which is composed by two ?-helices and six ?-stands.Furthermore,?-CoV can be divided into 2 types based in the foundation of nsp1 amino acid(aa)sequences and crystal structures.The first type includes SADS-CoV,PEDV,HCoV-NL63 and HCoV-229 E,the other type includes FIPV,TGEV and PRCV.2.Although the degrees of amino acid sequences homology between ?-CoV and ?-CoV nsp1 are low,the degrees of homology between their tertiary structures are quite high,which indicate that nsp1 may undertake a similar biological function between ?-CoV and ?-CoV nsp1.Using Ribopuromycylation(RP)and Renilla luciferase(Rluc)reporter assays,we corroborated that ?-CoV nsp1 could significantly inhibit the expression of Rluc gene.Furthermore,they can generally induce a time and concentration dependent inhibition of host gene expression in a broad spectrum.Based on the crystal structures of PEDV nsp1,we constructed a series of deletions and mutations.The results of Rluc demonstrated that PEDV nsp1 tended to showing three loops(67-71 aa,78-85 aa,103-110 aa)forming the enssential functional region.Unfortunately this discovery could not be conjectured with other ?-CoV nsp1.Additionally we elucidated that the motif(91-95 aa)was the key region to promote its function by using TGEV nsp1 as the model.The results of its crystal structure and biochemistry experiments were conductive to our hypothesis as well,which appeared to trigger similar reactions among ?-CoV nsp1.3.To test how this key region impacted on the virus,we constructed TGEV and FIPV mutants through CRISPR-Cas9 technology.According to results of recombinant virus on the level of cells and animals,we found that nsp1 was an important virulence factor in ?-CoV,as it did not affect their replicant activity in cells,it would reduce the pathogenecity on the level of animals.Integrating the results of RNA-Sequence and biochemistry experoiments,we confirmed that ?-CoV nsp1 made effects on hosts immune evasion and cell cycle to enhance its virulence.This article revealed ?-CoVs nsp1 crystal structures,and confirmed the key domain and amino acid regions of nsp1 that regulate hosts translation.We also clarified the detailed molecular mechanism of how nsp1 worked in the interaction between virus replication and host factors,providing insight into the development of a new attenuated vaccine with nsp1 modifications.