Discovery And Research On SARS-CoV-2 Entry Pathway And Natural Small Molecule Inhibitor

Objective:Since the outbreak of Severe Acute Respiratory Syndrome Coronavirus 2(SARS-COV-2)for more than one year,it brought irreparable losses to the lives and health of human beings,and has also caused a huge impact on the economy of all countries in the world.Understanding the process of SARS-CoV-2 infecting host cells is of great significance for the development of vaccines and antiviral drugs.Thus,this work intends to construct pseudovirus system of SARS-CoV-2 and its epidemic variant strains,study the entry pathway of SARS-CoV-2,screen the natural small molecule inhibitors against SARS-CoV-2 infection.At the same time,using the pseudovirus system,a mouse infection model of SARS-CoV-2 pseudovirus is about to be established.Methods:1.Construction of dual-reporter pseudovirus:(1)Construction and identification of spike protein expression vectors:The target fragments were obtained through molecular biological means such as sequence optimization synthesis and point mutation,and then connected to the eukaryotic expression vectors to obtain the target plasmids containing genes of spike protein from SARS-CoV-2 and its variants,SARS-CoV and MERS-CoV.The correctness of the plasmids was confirmed by agarose gel electrophoresis and sequencing;(2)Pseudovirus packaging and identification:Dual-reporter pseudoviruses of SARS-CoV-2 and its variants and SARS-CoV,MERS-CoV were packaged using lentivirus three-plasmid packaging system and concentrated by ultrafiltration.The spike proteins of pseudoviruses were detected by western blot.The infection abilities and titers of pseudoviruses were detected by infection experiment.The function of pseudovirus spike protein was verified by using recombinant proteins Angiotensin Converting Enzyme 2(ACE2)-Fc and ACE2-His.2.Studies on the entry pathway of SARS-CoV-2:(1)The localization of SARS-CoV-2 pseudovirus was observed by using confocal imaging technique.(2)Chemical inhibitors against various entry pathways were used to detect that by which entry pathway SARS-CoV-2 entered host cells through infection and inhibition experiments;(3)Co-transfection of human angiotensin converting enzyme 2(hACE2)plasmid and the dominant negative or active mutants was carried out to study the related proteins involved in the entry process of SARS-CoV-2.3.Screening of natural small molecule inhibitors against SARS-CoV-2 infection:(1)Primary screening:A library containing 89 kinds of plant-derived natural small molecule compounds was screened by SARS-CoV-2 pseudovirus infection and inhibition experiments;(2)Evaluation of anti-SARS-CoV-2 infection activity of the candidate molecules:CCK-8 method was used to detect the cytotoxicity and safe concentration of candidate molecules.The anti-SARS-CoV-2 infection activity of candidate molecules was analyzed by observing the number of green fluorescent cells and detecting luciferase activity;(3)Mechanism study on the candidate molecules against SARS-CoV-2 infection:The specific stage of the candidate molecules worked was studied through time-of-treatment experiments.The influence of the candidate molecules on intracellular localization of SARS-CoV-2 was observed by confocal imaging technology.The influences of candidate molecules on pseudovirus binding and uptake were detected by western blot and real-time quantitative PCR(q RT-PCR).The influence of candidate molecules on membrane fusion was analyzed by cell-cell fusion experiments.Effect of Ca²⁺on the inhibitory activity of candidate molecules against SARS2pp infection was determined by extracellular and intracellular Ca²⁺depletion assays.4.Establishment of SARS-CoV-2 infected mouse model:A mouse model with high expression of hACE2 in the lungs was established by thoracic injection of recombinant adeno-associated virus 9(rAAV9)carrying hACE2 gene.The feasibility of this mouse model was verified by infecting with SARS-CoV-2 pseudovirus.Results:1.The pseudoviruses of SARS-CoV-2 original strain and its variants,as well as SARS-CoV and MERS-CoV were successfully constructed through the lentivirus three-plasmid packaging system:(1)Western blot results showed that the spike proteins were successfully packaged into pseudoviruses;(2)The infection of SARS-CoV-2pseudovirus was inhibited by recombinant proteins ACE2-Fc and ACE2-His,verifying the binding ability of the pseudovirus to ACE2.The results above indicated that these pseudoviruses could be used to simulate the process of viral infection mediated by spike proteins.2.Results of studies on the entry pathway of SARS-CoV-2:(1)Confocal imaging results verified that SARS-CoV-2 pseudovirus could enter the host cells through the endosome-dependent pathway and locate in the endosome;(2)The use of chemical inhibitors and dominant negative/active mutants of entry-related proteins verified the presence of clathrin-dependent pathway and p21-activated kinase 1(Pak1)and Rac1-dependent macropinocytosis pathway,both of which were also regulated by ADP-ribosylation factor 6(Arf6).3.Screening results for natural small molecule inhibitors against SARS-CoV-2infection:(1)based on the pseudovirus system,neferine showed good broad-spectrum anti-coronavirus activity,including inhibitory activities against SARS-CoV-2 original strain and its variants(including D614G variant,N501Y/D614G variant,UK variant501Y.V1,SouthAfrican variant 501Y.V2 and Brazil variant 501Y.V3),SARS-CoV and MERS-CoV.(2)In terms of anti-SARS-CoV-2 pseudovirus infection,the effect of neferine was better than that of its analogs liensinine and isoliensinine.(3)Studies on the mechanism showed that like SARS-CoV and MERS-CoV,the membrane fusion of SARS-CoV-2 was Ca²⁺-dependent.Neferine could inhibit Ca²⁺-dependent membrane fusion by blocking host calcium channels,thus suppressing SARS2pp infection.4.Research results on the establishment of SARS-CoV-2 infected mouse model:A mouse model susceptible to SARS-CoV-2 was successfully established by thoracic injection of rAAV9-hACE2.After SARS-CoV-2 pseudovirus infection,the luciferase expression of the model gradually improved from the 7th day,reached stable level on the 14th day,and could last for at least 5 weeks.Immunohistochemical results confirmed the high expression of hACE2 in the lungs of this mouse model.Conclusion:In this study,SARS-CoV-2 was found to infect host cells through the clathrin-dependent pathway and Pak1 and Rac1-dependent macropinocytosis pathway,both of which were regulated by Arf6 based on the pseudovirus system.At the same time,a bisbenzylisoquinoline alkaloid,neferine,with broad-spectrum inhibition to coronavirus infection was screened from a plant-derived natural small molecule library.Neferine could inhibit the Ca²⁺-dependent fusion of viral envelope and endosomal membrane.These results provided abundant clues and theoretical proofs for the development of broad-spectrum anti-coronavirus drugs.In addition,the rAAV9-hACE2mouse model was successfully constructed,which made it possible to evaluate the protective effect of SARS-CoV-2 entry inhibitors(such as small molecules and antibodies)and vaccines in vivo.