Characterization Of SARS-CoV-2 Variants And Its In-host Function Study Of Non-structural Protein 1(NSP1)

SARS-CoV-2(severe acute respiratory distress syndrome coronavirus 2),homologous to SARS-CoVs severe acute respiratory distress syndrome coronavirus),both belong to the coronavirus family,and have certain structural and functional characteristics.similarity.The diameter is about 50-200,and the total length is about 30kb.The 5-terminal includes four structural proteins and several accessory proteins.Like other coronaviruses,SARS-CoV-2 converts ORF1a and ORF1ab into the polyproteins pp1a and pplb inside the host cell.Proteolytic cleavage of these polyproteins by NSP3(i.e.papain-like protease)and NSP5(i.e.major protease)yields 16 nonstructural proteins(NSPs).The 3-terminal of SARS-CoV-2 has four structural proteins,namely S(spike),E(envelope),M(membrane)and N(nucleocapsid)proteins.Since the outbreak of SARS-CoV-2 in December 2019,as of April 10,2021,more than 134 million confirmed cases have been reported in 192 countries and regions around the world,of which more than 6 million have died and 76.537 million have been cured,the number of infections and deaths is still rising rapidly.However,due to the high mutagenicity of the virus,the circulating strains of the virus are constantly changing.The mutations that affect the characteristics of virus infection and transmission are mainly concentrated in the S protein.These mutations affect the binding ability of the virus S protein to the host ACE2 receptor.At the same time,many mutations on the S protein also promote the immune escape ability of the virus.There are also some differences in the infection and replication ability of the strains.Current studies have shown that SARS-CoV-2 NSP1 can inhibit host translation by binding to the 40S ribosomal subunit,while the special stem-loop structure at the 5 end of the virus’s own RNA can escape the inhibition,but other transcriptome data have shown that NSP1 It may also play some other important roles in the host.First,we characterized the differences in antigenicity and tertiary structure of the S protein of different strains of SARS-CoV-2 through an online program.The mutation of the S protein of each strain has a certain impact on the antigenicity.Except for Omi cron,other viruses The strain difference does not seem to be particularly pronounced,and the D614G mutation in the mutant strain makes the RBD region of the S protein more exposed,which may be the reason for the more rapid spread of the mutant strain.Then,to verify the binding ability of S protein and ACE2 of different mutant strains,we carried out pseudovirus infection experiments and in vitro binding ability experiments of S protein and ACE2 protein of different mutant strains.Low binding capacity of S protein to ACE2.To further verify these results,we used different strains to infect Vero cells,and detected the viral load at different time points.It was found that the Omicron virus genome load was the lowest at 2h after infection,and the Prototype was the highest at 24 hours after infection.Delta is the highest,Omicron second.This shows that Omicron’s ACE2 binding ability and cell infection ability are not strong enough,but its immune evasion ability is strong,and its cytotoxicity is weaker than other strains.Then,to study other functions and molecular mechanisms of SARS-CoV-2 NSP1 in the host,and to search for NSP1-interacting proteins and their regulated signaling pathways,we expressed the full-length NSP1 in prokaryotic and eukaryotic cells,respectively.The total protein of H1299 cells was used for GST-pull down experiment,silver staining analysis,and mass spectrometry was used to search for interacting proteins.The eukaryotic expression vector was transfected into H1299 cells and sent to omics analysis.Combined with mass spectrometry and omics data,the signaling pathway regulated by NSP1 and its possible mechanism were preliminarily analyzed.We found many host proteins that bind to NSP1 by mass spectrometry,including many ribosomal proteins,and finally verified the hnRNPK and FXR1 proteins that bind to NSP1.In the proteome,a highly expressed interferon signal inhibitor GPATCH3 protein was found and verified.Finally,the inhibitory effect of NSP1 and GPATCH3 on interferon was proved by the interferon reporter gene detection system,but no difference in GPATCH3 expression was detected by knockdown or overexpression of FXR1.Viral infection experiments found that NSP1 promotes viral infection while GPATCH3 appears to have the opposite effect.In short,we found through a series of experiments that the inhibitory effect of NSP1 on interferon may be caused by the interaction between NSP1 and a certain protein in the host to promote the expression of GPATCH3.At the same time,NSP1 creates favorable conditions for virus infection,but the mechanism still needs further study.Our research on different SARS-CoV-2 mutants provides a certain reference for further analysis and evaluation of viral infection transmission and prevention.In addition,our research on non-structural proteins revealed the promoting effect of non-structural proteins on virus infection,which provided a certain reference for virus prevention and target development.