Infection Characteristics Of SARS-CoV-2 Variants And Screening And Mechanism Of Antiviral Drugs

The coronavirus disease 2019(COVID-19)pandemic is threatening human health worldwide.As of March 23,2022,SARS-coronavirus 2(SARS-CoV-2),pathogen of COVID-19,has infected over 256 million people and has caused more than 5 million deaths(https://covid19.who.int).SARS-CoV-2 uses its spike(S)protein to bind host receptor angiotensin-converting enzyme 2(ACE2)and mediate virus entry into host cells.Virus entry into target cells requires activation of the S protein by cellular proteases,which triggers cleavage at the S1/S2 and S2’ sites,and then mediates fusion of the virus with the cell membrane.SARS-CoV-2 entry can be disrupted by protease inhibitors or endocytosis inhibitors.The therapeutic applicability of these drugs for COVID-19 treatment is being evaluated within clinical trials.Although the genome of SARS-CoV-2 remains relatively stable,it still has a high mutation rate during virus transmission.With the extension of the COVID-19 pandemic,a wide variety of variants have been produced in the host.Recently,several new SARS-CoV-2 variants have appeared more infectious in the population.The variants make their antigenicity different from the original strain,which can reduce or even invalidate the protective efficacy of current antibodies and vaccines.Among the variants of concern(VOC),the B.1.351 and P.1 strains have attracted wide attention because of their extensive mutations and the ability to escape from neutralizing antibodies.In addition,B.1.617.2 and B.1.1.529 have spread widely around the world,raising concerns about the infectivity of the virus and the effectiveness of antibodies and vaccines against the virus.As widely vaccinated SARS-CoV-2 vaccines in china,Corona Vac and BBIBP-CorV need to be tested for their protection ability against concerned variants.The SARS-CoV-2 spike protein is a key molecule for entry into host cells and a major target for therapeutic monoclonal antibodies(mAbs)or vaccination-elicited polyclonal antibodies.To investigate the effect of S protein mutation on viral transmission,we used a pseudoviral system of vesicular stomatitis virus(VSV)to evaluate the infection and immune escape characteristics of SARS-CoV-2 virus.First,we used site-directed mutagenesis and gene recombination techniques to construct the S protein expression plasmids of the major circulating SARS-CoV-2 variants,and used pseudovirus packaging technology to produce VSV pseudoviruses carrying the S protein of different variants.We detected the expression and cleavage of S protein in cells and viruses by western blot.Next,we detected the infectivity of SARS-CoV-2 variants in different cells.We also tested the neutralizing capacity of mAbs against pseudovirus infection in Vero cells,as well as the neutralizing activity of vaccinated sera against variant pseudoviruses.Finally,we tested the inhibitory effect of some protease inhibitors and endocytosis inhibitors on different SARS-CoV-2 variants.We found that some SARS-CoV-2 variants have changed significantly in viral infectivity.Pseudovirus carrying the B.1.351 S protein had higher infection efficiency than wild type and D614G.B.1.617.2 is more likely to infect less susceptible cells than D614G,and the virus infection process can be completed in a shorter time.In addition,neutralizing mAbs and vaccinated sera partially or completely failed to inhibit entry mediated by the S protein of certain SARS-CoV-2 variants.However,SARS-CoV-2 variants S protein mediated viral infection can be blocked by protease inhibitors and endocytosis inhibitors.In the screening of SARS-CoV-2 small molecule inhibitors,we screened 48 FDA-approved drugs using the VSV pseudovirus system.We tested the cytotoxicity of small molecule inhibitors by CCK-8 and determined the safe effect concentration of the candidate drugs.We used TMPRSS2-overexpressing cells to examine whether the drug candidate Tetrandrine affects viral entry into target cells by proteolysis.The specific stage of the candidate drugs worked was studied through time-of-treatment experiments.The influence of the candidate drugs on intracellular localization of SARS-CoV-2 pseudovirus was observed by confocal imaging technology.The calcium channel TPC2 knockout cells were constructed by gene editing CRISPRCas9 technology,and the ability of the virus to infect the TPC2 knockout cells was tested.We found that Chloroquine and Tetrandrine could significantly inhibit virus infection in 293T-hACE2 cells.Unlike neutralizing mAbs,Tetrandrine has a significant inhibitory effect on all SARS-CoV-2 variants infection.Further research found that Tetrandrine mainly plays an inhibitory role in the process of virus infecting cells through the endocytic pathway.Disruption of TPC2 function by gene knockout blocks viral trafficking and prevents SARS-CoV-2 infection.In summary,we demonstrate that the infection efficiency of pseudovirus with SARS-CoV-2 variants S protein has changed in some target cells.This may be the main reason for the widespread of SARS-CoV-2 variants.In addition,our data suggest that SARS-CoV-2 variants may compromise the therapeutic effect of neutralizing antibodies or reduce the protective effect of vaccines.Our findings highlight the need to strengthen virus surveillance and assess the effectiveness of current antibodies and authorized vaccines against emerging SARS-CoV-2 variants.Meanwhile,increasing the proportion of people vaccinated with effective SARS-CoV2 vaccines is the key strategy for reducing the emergence of new variants and ending the pandemic of COVID-19.