Caspase Family Proteins Regulate The Replication Of Novel Coronavirus And Screening Of Antiviral Compound

Coronavirus Disease 2019(COVID-19),caused by Severe Acute Respiratory Syndrome Coronavirus-2(SARS-CoV-2),has circulated worldwide since the end of 2019.As of March 2,2022,COVID-19 has caused more than 450 million infections and 60.29 deaths,seriously endangering public health.Therefore,it is important to understand SARS-CoV-2 pathogenesis and develop antiviral drugs.The cysteine-containing aspartate protein hydrolase(Caspase)family,as cysteine proteases,is able to selectively cleave target proteins under specific physiological or pathological conditions to induce apoptosis.In this study,we first found that Z-VADFMK,a broad-spectrum caspase inhibitor,can significantly inhibit SARS-CoV-2 replication,suggesting that caspases may be involved in viral replication.By knocking down eight caspases using small interfering RNA(siRNA),we found that knockdown of caspase-2 or caspase-8,rather than other caspases,reproducibly inhibited SARSCoV-2 replication.Given that caspase-2 has a more potent function than caspase-8,we focused on caspase-2 in this study.We found that caspase-2 overexpression could promote SARS-CoV-2 replication,correlating with the pro-viral effect of caspase-2 discovered in knockdown experiments.Thus,caspase-2 could be a previously unreported regulator of SARS-CoV-2.To date,caspase-2’s role in apoptosis is not fully understood,and it was long thought to be an "orphan" caspase.To further investigate the mechanism of action of caspase-2,we constructed a series of plasmids expressing caspase-2 mutants.Overexpression of wild-type and nuclear localization sequence-mutated caspase-2 promoted SARS-CoV-2 replication,while mutations in enzyme activity site and caspase recruitment domain abrogated this effect,indicating that the enzymatic function caspase-2 is critical for its activity in SARS-CoV-2 replication.Recent studies showed that caspase-2 could inhibit cellular autophagy by suppressing the production of reactive oxygen species(ROS).Given that autophagy is involved in coronavirus replication,we further investigated whether caspase-2 regulates viral replication by modulating autophagy.Upon knockdown of caspase-2,we observed substantial LC3B conversion(LC3B-Ⅰ to LC3B-Ⅱ),a hallmark of cellular autophagy.Moreover,pharmacological inhibition of lysosomal proteases further promoted autophagy flux induced by caspase-2,indicating that knockdown of caspase2 triggered a complete autophagy process.Further analysis showed that SARS-CoV-2 replication,as indicated by viral nucleoprotein levels,was negatively correlated with the extent of LC3B-II conversion in caspase-2 knockdown cells,suggesting that caspase-2 may regulate viral replication through the autophagic pathway.To test this,we analyzed the effects of wortmannin,an autophagy inhibitor,and CCCP,a mitochondrial proton carrier uncoupling agent that promotes reactive oxygen species production,on viral replication.In contrast to the observation that caspase-2 substantially impeded SARS-CoV-2 replication,both drugs exerted marginal effects on viral replication,suggesting that caspase-2 may regulate viral replication independently of autophagy and reactive oxygen species-related mechanisms.The underlying mechanism needs to be further studied.With the progress of the COVID-19 epidemic,an increasing number of SARSCoV-2 variants with enhanced transmissibility,pathogenicity,and immune escape ability have emerged,posing challenges to the development of vaccines and therapeutic drugs.To rapidly identify safe and broad-spectrum antivirals,we conducted drug screening based on 20 U.S.Food and Drug Administration(FDA)-approved drugs that have been proved to inhibit the replication of the original SARS-CoV-2 strain in our prior research.By leveraging pseudo-typed SARS-CoV-2 viruses,we examine their efficacy on vial entry,a fundamental process of virus infection.The cytotoxicity of these drugs was examined concurrently.Six compounds were identified as safe and effective inhibitors against the pseudovirus derived from the original strain.Further,phenazine compounds trifluoperazine 2HCl and thioridazine HCl were found to inhibit the infection of B.1.617.2(Delta)and B.1.1.529(Omicron)pseudoviruses,two currently circulating strains.Finally,we discovered that these two compounds were highly effective against authentic SARS-CoV-2 viruses,including the original,Delta,and Omicron strains,suggesting that they are potential broad-spectrum SARS-CoV-2 inhibitorsIn summary,this study identified the pro-viral function of caspase-2 in SARSCoV-2 replication by siRNA-based screening of caspase family proteins.Further,enzymatic activity was found to be critical for caspase-2’s function.In addition,By using pseudotyped and authentic SARS-CoV-2 strains,this study discovered that the phenothiazine compounds trifluoperazine 2HCl and thioridazine HCl could potently inhibit SARS-CoV-2 entry with minimal cytotoxicity.These findings provide clues for understanding the mechanisms by which SARS-CoV-2 interacts with the host,and provide insights into the development of broad-spectrum therapeutics against SARSCoV-2.