Establishment Of SARS-CoV-2 Pseudovirus System And Its Application To The Study Of Mutant Strains And Screening Of Viral Entry Inhibitors

Objective:The pathogen 2019-n CoV（2019 Novel Coronavirus）of Corona Virus Disease 2019（COVID-19）is an enveloped,linear single-stranded positive-stranded RNA virus with a genome sequence length of approximately 29 kb,belonging to the genusβcoronavirus of the Coronaviridae family with severe acute Respiratory syndrome-associated coronavirus species^[1].It was officially named as Severe Acute Respiratory Syndrome Coronavirus 2（SARS-CoV-2）by the International Committee on Classification of Viruses on February 12,2020^[1].It belongs to Betacoronavirus genus including SARS-CoV and MERS-CoV,different branches of evolutionary tree,and is the seventh coronavirus that can infect humans^[2,3],with highly pathogenic and highly infectious characteristics.Among diverse human CoVs,HCoV-NL63,HCoV-229E,HCoV-OC43 and HCoV-HKU1 usually cause only mild upper respiratory tract disease and rare severe infections in infants,young children,and the elderly.But SARS-CoV,MERS-CoV,SARS-CoV-2 can infect the lower respiratory tract and cause severe respiratory disease in humans^[2].In particular,SARS-CoV-2brought about great political,economic,and life shocks and the spreading of a serious situation around the world^[4].Potential hosts of SARS-CoV-2 are a variety of mammals,which can invade the human body through the upper respiratory tract.Similar to SARS-CoV,SARS-CoV-2 infects multiple target organs,mainly in the lung,kidney and heart,through the human angiotensin-converting enzyme 2（ACE2）receptor^[5,6].The Receptor binding domain（RBD）of SARS-CoV-2 Spike（S）protein is responsible for recognizing and binding to the receptor ACE2 on the host cell surface and is critical for viral entry.The affinity of SARS-CoV-2 S protein for ACE2 is 10-20 times higher than that of SARS-CoV S protein^[7,8],which is the main reason for the high infectivity of this new coronavirus^[9,10].Thus,S proteins are important immunogens and key targets for vaccine design,therapeutic antibodies,and small molecule drugs that block viral entry.The cumulative number of confirmed SARS-CoV-2 infections worldwide has exceeded 510 million so far,and it is growing by millions every day.The isolation of the source of infection is the most important tool in the prevention and control of COVID-19 in the initial,however,vaccines,antibodies and drugs are also the ultimate weapons needed to defeat the epidemic.Several vaccines have been already in use,including recombinant RBD vaccines,inactivated vaccines,adenovirus vector vaccines,and m RNA vaccines^[11].Due to the highly variable nature of the SARS-CoV-2,multiple mutant strains resulting from mutations on its S protein have formed and spread around the world,from the original wild type strain,through D614G,Alpha,Beta,Gamma,Delta,and most recently the Omicron strain pandemic^[12-15].It is the amino acid site change on its S protein that causes antigenic drift,allowing the virus to evade the host immune response and affecting the immune protective effect of the vaccine.Therefore,the persistence of immune protection produced by vaccination and the cross-protect against mutant strains are still hot and difficult issues of concern^[16,17].With the emergence of mutant strains of SARS-CoV-2,the protective effect of neutralizing antibodies required further and deeper evaluation and systematically against current mutant strains.Prof.Aisun Jin’s team identified two RBD-specific monoclonal antibodies,58G6 and 510A5,which showed strong preventive effects in h ACE2 transgenic mice infected with SARS-CoV-2 and its mutant strain B.1.351（Beta）^[18].The joint therapy of BRII-196 and BRII-198 m Ab proposed by Linqi Zhang and his team received emergency marketing approval from the China Medical Products Administration（NMPA）for the treatment of mild and common types among adolescents,adults and those associated with high risk factors developed to severe COVID-19.However,in a global multicenter,double-blind,placebo-controlled randomized clinical trial,SARS-CoV-2 neutralizing monoclonal antibodies Sotrovimab,BRII-196 plus BRII-198 were not shown to be better than placebo in improving clinical outcome in adult hospitalized COVID-19patients^[19].In terms of antiviral drug development,there are still no drugs specifically targeting SARS-CoV-2 with significant efficacy,and most of the available antiviral drugs are ineffective.The anti-HIV drugs klonopinemide（lopinavir and ritonavir）have proved to be ineffective and have significant side effects in clinical trials for the treatment of COVID-19.Wang Manli et al from Wuhan Institute of Virology reported that chloroquine and raltegravir were able to inhibit SARS-CoV-2 virus in vitro with an IC₅₀ of 1.13 and 0.77μM,respectively^[20].Chloroquine was marketed as an anti-malarial drug to try to treat COVID-19,but follow-up studies found that chloroquine/hydroxychloroquine did not prevent viral infection and replication of lung adenocarcinoma cells Calu3^[21].In contrast,the phase III clinical trial of raltegravir conducted in China was terminated due to insufficient patient numbers,and the available data did not demonstrate a statistically significant difference^[22].Results from a phase II and III clinical trial of Pfizer’s COVID-19 agent PAXLOVID^TMin 1219 patients with COVID-19 showed an 89%reduction in patient hospitalization and mortality when being administered within 3 days of symptom onset compared to the placebo group;at 28 days of observation,there were 1%（6/607）admissions and no deaths in the treatment group and 6.7%（41/612）in the placebo group admissions and 10 deaths in the placebo group^[23].The NMPA has approved PAXLOVID^TMfor clinical use in February 2022,but the long-term antiviral efficacy needs to be further observed.Traditional virus neutralization assays require manipulation of authentic viruses with high biosafety risks,so there is an urgent need to establish a method that can be used in BSL-2 laboratories for SARS-CoV-2studies.In this study,a lentiviral system based on the HIV-1 backbone was used to package a SARS-CoV-2 pseudovirus in HEK293T cells by cotransfection of a recombinant plasmid lacking 19 amino acids of the C-terminal structural domain of the SARS-CoV-2 S protein with an HIV-1backbone plasmid encoding a single luciferase.We also established a 293T cell line stably expressing human 293T-ACE2 for assessing SARS-CoV-2pseudovirus infection efficiency.The luciferase activity of pseudoviruses and their mutant strains of infested cells was measured by chemiluminescence,and the plasma neutralizing ability and dynamics of COVID-19 patients during recovery,the neutralizing ability of monoclonal antibodies,the infection efficiency of SARS-CoV-2 mutant strains and the effect of cell entry inhibitors were quantitatively evaluated on the basis of luminescence values.In this study,we obtained a safe and efficient SARS-CoV-2pseudovirus system,and used it as a platform to facilitate the study of SARS-CoV-2 mutant strains and the screening of antiviral drugs.Methods:1.Establishment of SARS-CoV-2 S pseudovirus system:We established 293T-ACE2 cell line that can stably express ACE2.Recombinant plasmid p CMV3-SARS-CoV-2-S-FL（p S-FL）that can express the SARS-CoV-2 S gene was synthesized,and used as a template to construct the expression plasmid p CMV3-SARS-CoV-2-S-Mut（p S-Mut）with a C-terminal mutant of S protein and the expression plasmid p CMV3-SARS-CoV-2-S-C19del（p S-C19del）with a deletion of 19 amino acids at the C-terminal of S protein.Three S expression plasmids were cotransfected with the lentiviral vector p NL4-3.Luc.R-E-carrying the luciferase reporter gene,respectively,to package high-titer SARS-CoV-2 pseudovirus in HEK293T cells.The vesicular stomatitis virus（VSV）glycoprotein（G）expression plasmid p MD2.G was cotransfected with p NL4-3.Luc.R-E-in HEK293T cells to package VSV-G pseudovirus for control.The infection efficiency of three pseudoviruses of SARS-CoV-2 was assessed by assaying the luciferase activity of pseudoviruses after 72 hours of infection of 293T-ACE2 cells;subsequently,the infection efficiency of SARS-CoV-2 S protein and VSV-G protein-packed pseudoviruses on 293T and 293T-ACE2 cells was compared by assaying the luciferase activity at 72hours,and the effect of protease inhibitors Camostat and E-64d in inhibiting pseudovirus infection was evaluated.A SARS-CoV-2 pseudovirus neutralization assay was established to verify the neutralization effect of COVID-19 recovery plasma with recombinant angiotensin-converting enzyme 2-Immunoglobulin Ig G1（ACE2-Ig）against pseudovirus.Finally,44 SARS-CoV-2 monoclonal antibodies targeting RBD were screened for neutralizing ability using neutralization assays.2.SARS-CoV-2 S-G614 pseudovirus construction and infectivity study:The amino acid sequence of SARS-CoV-2 S protein from the GISAID database was analyzed using bioinformatics.The p S-G614 recombinant plasmid was constructed by point mutation using pS-D614 recombinant plasmid and cotransfected with the lentiviral vector p NL4-3.Luc.R-E-carrying the luciferase reporter gene in a 1:1 system to package high titer SARS-CoV-2 S-G614 pseudovirus in HEK293T cells.The cleavage of S protein was analyzed by Western Blot after exogenous transfection of pS-D614 with p S-G614 in HEK293T cells for 48 hours.By detecting the luciferase activity of SARS-CoV-2 S-D614 and SARS-CoV-2S-G614 pseudoviruses 72 hours after infection of 293T-ACE2 cells,we investigated the effect of S-D614 and S-G614 pseudoviruses on cell entry efficiency,ACE2-Ig neutralization ability,elastase-2 and svelestat on pseudovirus entry at 24,48 and 72 hours.We performed serial gradient dilutions of Camostat and E-64d by measuring luciferase activity in 293T-ACE2 cells 72 hours after pseudovirus infection to compare their differences in their ability to counteract VSV-G,S-D614,and S-G614 pseudovirus entry.Finally,the ID₅₀ of plasma against S-D614 and S-G614 pseudoviruses during the recovery period of a total of 70 COVID-19 patients from Yongchuan Hospital of Chongqing Medical University,Chongqing Public Health Rescue Center,and Wanzhou Three Gorges Central Hospital of Chongqing,respectively,was analyzed using neutralization tests.3.Identification of bibenzyltetrahydroisoquinoline alkaloids as SARS-CoV-2 S-G614 pseudovirus entry inhibitors:We constructed mutant expression plasmids p S-N501Y.V1 and p S-N501Y.V2 for the N501Y amino acid locus variant of SARS-CoV-2 S gene,SARS S gene expression plasmid p S-SARS,and MERS S gene expression plasmid p S-MERS by genetic recombination using the pS-D614recombinant plasmid as a template.pS-G614,p S-N501Y.V1,p S-N501Y.V2,p S-SARS,p S-MERS and p NL4-3.Luc.R-E-were cotransfected with HEK293T cells by a 1:1 ratio and packaged with S-G614,S-N501Y.V1,S-N501Y.V2,S-SARS,S MERS pseudoviruses.We collected a drug library containing 188 compounds and analyzed their ability to resist S-G614 pseudovirus entry in 293T-ACE2 cells at a concentration of 20μM.The stages of action of these nine compounds against S-G614 pseudovirus entry were determined by different combinations（pre-pseudovirus entry treatment,virus entry phase,and post-virus entry）,and subsequently their EC₅₀ and CC₅₀ against S-G614pseudovirus entry in Calu3,A549-ACE2,and 293T-ACE2 cells,respectively.We determined their EC₅₀ against S-G614,S-N501Y.V1,S-N501Y.V2,S-SARS,S-MERS,VSV-G pseudovirus entry cells by means of multiplicative dilution using the five compounds with the best EC₅₀ in different cell lines.We investigated the mechanism of cell entry of these five compounds against SARS-CoV-2 using cell membrane fusion assay,competitive ELISA assay,differential scanning fluorometry（DSF）assay,calcium depletion assay,and cellular cholesterol assay.Finally,we infected VERO E6 cells with authentic SARS-CoV-2 virus and examined the effect of our compounds against SARS-CoV-2 infection by observing the cytopathic effect and real-time fluorescence quantitative PCR.Results:1.293T-ACE2 stably transfected cell lines were successfully constructed.Three SARS-CoV-2 S gene expression plasmids,p S-FL,p S-Mut and p S-C19del,all detected S protein expression in HEK293T cells.The recombinant plasmid p S-C19del,which lacks the C-terminal 19 amino acids of the S protein structural domain,also detected the complete band of S protein and the cleaved S1 subunit.Meanwhile,laser confocal microscopy results showed that S-FL was mainly localized to the endoplasmic reticulum and S-Mut and S-C19del facilitated the transport of S proteins to the cell membrane surface.We successfully packaged SARS-CoV-2 S pseudovirus and VSV-G pseudovirus with p S-FL,p S-Mut,p S-C19del,p MD2.G and p NL4-3.Luc.R-E-co-transfected HEK293T cells and infected HEK293T and 293T-ACE2 cells with them,and the results showed that VSV-G pseudovirus were effective in invading both cell lines,and the measured luciferase activities were comparable.However,the luciferase activity of SARS-CoV-2 S pseudovirus in 293T-ACE2 cells was 1200 times higher than that of HEK293T（P<0.001）.The highest viral infectivity of（8.02±0.11）×10⁴ RLU was observed in S-C19del pseudovirus during infection of293T-ACE2 cells by SARS-CoV-2 S pseudovirus,with the highest infection efficiency（（5.77±0.28）×10⁴ RLU）reached at 72 hours.We detected the inhibitory activity of two protease inhibitors,Camostat and E-64d,against SARS-CoV-2 S pseudovirus entry in 293T-ACE2 cells,with Camostat failing to inhibit infection and E-64d having a blocking efficiency of 95%（RLU=2909±986.2）at 2μM concentration.We used the pseudovirus system to establish methods for testing plasma-neutralizing antibodies as well as monoclonal antibodies during the recovery period of COVID-19.The results of three COVID-19 recovery plasma showed that their half neutralization potencies(NT₅₀)were 412,745 and 1930,respectively.8^#,15^#,16^#,20^#,25^#and 30^#monoclonal antibodies to the S protein receptor binding structural domain（RBD）were determined to have the best neutralization effect of SARS-CoV-2-S pseudovirus.2.We analyzed the amino acid sequence of the SARS-CoV-2 S protein from the viral genome sequence of the GISAID database and identified a globally distributed S protein mutant strain,D614G,which accounted for 64.6%of the sequence analysis.The PROSPER software was used to predict the potential protease cleavage sites in the S protein mutant strain,and a new serine protease（elastase-2）cleavage site was identified at residues 615-616of the S1-S2 linkage of the S-G614 protein.We found that the luciferase activity of 293T-ACE2 cells increased approximately 250-fold（P<0.001）and 530-fold（P<0.001）when infected with S-D614 and S-G614 pseudoviruses,respectively,compared with HEK293T cells,and both S-D614 and S-G614 pseudoviruses were effectively inhibited by ACE2-Ig,with their IC₅₀ values of 0.13μg/m L and0.15μg/m L,respectively.For S-G614 pseudovirus,the entry efficiency at48 h increased 2.2-fold compared to S-D614（P<0.01）,and the highest entry efficiency（（2.50±0.41）×10⁴ RLU）was observed after 72 h of S-G614pseudovirus infection at a level approximately 2.4-fold higher than that of S-D614 pseudovirus（P<0.01）.When 293T-ACE2 cells were treated with 100μg/m L elastase prior to infection with pseudovirus,the RLU value of S-G614 pseudovirus-infected cells（6.9×10⁴）was 3.1-fold higher than that of S-D614（2.2×10⁴）;in the presence of 100μg/m L elastase,the serine protease inhibitor sivelestat dose-dependent blockade of S-G614 entry.These results demonstrate that the infectivity of S-G614 pseudoviruses containing additional elastase-2cleavage sites is enhanced by exogenous elastase,and thus S-G614pseudoviruses are more sensitive to svelestat than S-D614 pseudoviruses.In293T-ACE2 cells lacking TMPRSS2 expression,the serine protease inhibitor Camostat failed to inhibit S-D614 or S-G614 pseudovirus infection,whereas the cysteine protease inhibitor E-64d significantly blocked the entry of both pseudoviruses with IC₅₀ of 0.37μM and 0.24μM,respectively.In a neutralization assay of 70 plasma from patients recovering from COVID-19 against S-D614 and S-G614,we found that 65 plasma showed comparable neutralizing activity against both S-D614 and S-G614pseudoviruses,while 5 plasma samples（1^#,7^#,40^#,42^#,and 52^#）showed decreased inhibition against S-G614 pseudovirus（P<0.05）.1^#,7^#,40^#,42^#and 52^#plasma showed high neutralizing activity against S-D614pseudovirus with ID₅₀ ranging from 729 to 1524,but decreased neutralizing activity against S-G614 pseudovirus with ID₅₀ ranging from 216 to 367 and2.5 to 5.9-fold decrease in neutralizing activity（P<0.05）.These data suggest that the majority（93%）of anti-sera from patients infected with the early SARS-CoV-2 variant cross-neutralize the S-G614 variant,but the D614G variant reduces the neutralization susceptibility to 7%of recovered sera.3.We screened 188 compounds for cell entry inhibition activity at 20μM using the SARS-CoV-2 S-G614 pseudovirus system and identified 41compounds with S-G614 pseudovirus relative infection rates<30%.Using VSV-G pseudovirus control,we identified nine compounds（SC9,SC161,SC171,SC182,SC183,SC184,SC185,SC186 and SC187）that specifically inhibited SARS-CoV-2 S-G614 pseudovirus entry,all of which had half effective concentrations(EC₅₀)less than 10μM against SARS-CoV-2 S-G614 pseudovirus.Among these compounds,all of them were bisbenzylisoquinoline alkaloids（calcium channel antagonists）,except SC171.We are assured that all nine compounds could block S-G614 pseudovirus infection by targeting host factors at the initial stage by adding compounds at three stages:before,during,and after pseudovirus infection,respectively.In the S-G614 pseudovirus experiments in three cell lines,293T-ACE2,A549-ACE2,and Calu3,we found that the EC₅₀ of five compounds,SC9,SC161,SC171,SC182,and SC185,were all below 5μM and could foresee good application prospects.Cepharanthine（SC9）,Hernandezine（SC161）,3,3’-（1,3-phenylenebis（oxy））bis（1-（4-methylpiperidin-1-yl）propan-2-ol）（SC171）,Tetrandrine（SC182）,and Neferine（SC185）significantly inhibited coronavirus entry.They effectively protected three cell lines（293T,Calu3,and A549）from infection by different coronaviruses（SARS-CoV,MERS-CoV,SARS-CoV-2[S-D614,S-G614,N501Y.V1 and N501Y.V2 variants]）in vitro with EC₅₀values of 0.1-10μM.We then ruled out that SC9,SC161,SC171,SC182 and SC185 acted by interfering with RBD-ACE2 interaction by competitive ELISA assay,differential scanning fluorometry（DSF）assay.These compounds were further found to be effective in inhibiting S protein-mediated membrane fusion by cell membrane fusion assays.In HEK293T cells,depletion of intracellular Ca²⁺by 20μM calcium chelator BAPTA-AM significantly reduced S-G614 pseudovirus entry by approximately 70%（P<0.001）.More importantly,when cells were treated with 20μM BAPTA-AM in combination with any of the four compounds,the entry blocking effects of these compounds on anti-S-G614 pseudoviruses were reduced by more than 10-fold（P<0.001）.Results consistent with those in the 293T-ACE2 cells were also observed in the Calu3 cells.We confirmed the anti-SARS-CoV-2 activity of compounds SC9,SC161,SC171,and SC185 in Vero E6 cells.Structural and morphological changes of Vero E6 cells induced by viral infection were observed after 48-h infection of SARS-CoV-2 authentic viruses.With the treatment of 10μM SC9,SC161,SC171,or SC185,the viral RNA levels were 0.08%（P<0.001）,70.27%（P<0.01）,43.55%（P<0.001）,and 76.98%（P<0.05）relative to those of DMSO control group,respectively.These results indicated that SC9,SC161,SC171,and SC185 inhibited SARS-CoV-2 authentic virus to different degrees.Conclusions:We have successfully constructed a high-titer SARS-CoV-2 S protein-mediated pseudovirus system using an HIV-1 lentiviral vector with a luciferase reporter gene that can be used in the BSL-2 laboratory for the study of SARS-CoV-2,a highly infectious and important emerging pathogen.It was clarified that S-G614 pseudoviruses have a greater ability to enter cells than S-D614 pseudoviruses,and the D614G mutation was found to have an additional elastase 2 cleavage site in the S protein,thus facilitating the cleavage of its S protein leading to easier virus entry into cells and causing SARS-CoV-2 to become more infectious,as well as in 7%of patients recovering from COVID-19,the D614G mutation reduced the susceptibility of the virus to plasma-neutralizing antibodies.A group of bibenzyltetrahydroisoquinoline alkaloids were identified as coronavirus entry inhibitors.These inhibitors effectively protected different cell lines（293T,Calu3 and A549）in vitro from the infection of different coronaviruses（SARS-CoV,MERS-CoV,SARS-CoV-2 S-D614,SARS-CoV-2 S-G614,SARS-CoV-2 S-N501Y.V1,SARS-CoV-2 S-N501Y.V2）.These compounds block host calcium channels,thereby inhibiting calcium-mediated membrane fusion and suppressing coronavirus invasion.Our study provides new types of lead compounds for anti-SARS-CoV-2 drug discovery.And the pseudoviral system will contribute to the understanding of SARS-CoV-2mutant strains and the design of vaccines and therapeutic interventions against COVID-19.