Screening And Validation Of Anti-EV-A71 Virus Targets And Drug Candidates

Objective:Identification of host factors involved in EV-A71 infection based on the CRISPR/Cas9system.It provides a novel host target for the discovery of anti-EV-A71 virus drugs.Besides,the traditional Chinese medicine with anti-EV-A71 activity was screened and evaluated to provide candidate drugs for the research and development of anti-EV-A71 drugs.Method:1.Identification of host factors involved in EV-A71 infection based on the CRISPR/Cas9 systemThe CRISPR/Cas9 library was used to screen host factors associated with EV-A71 virus replication.First,a negative control virus expressing green fluorescent protein was used to determine the optimal MOI of lentivirus-infected cells by flow cytometry-based on the abundance of fluorescent protein expression.Then,the optimal screening concentration of puromycin was determined.Finally,library cells infected with lentivirus were screened successfully.After determining the optimal conditions for lentivirus infection,the CRISPR/Cas9 library virus was used for mass infection.After the library cells were successfully constructed,the last survived cells were collected after two rounds of lethal doses of EV-A71 virus infection to extract genomic DNA for sequencing.The abundance of sg RNA in the EV-A71 infected group and the uninfected group was compared.The genes corresponding to the increased abundance of sg RNA were host genes associated with EV-A71 virus infection.Combined with gene function query,new candidate genes were identified,and a gene silencing technique was used to verify the effect of knockdown of candidate host gene proteins on EV-A71 virus infection.RT-q PCR and Westen blot were used to detect the copy number of EV-A71 virus RNA and the expression of EV-A71 virus protein in cells with candidate gene protein knockdown.The effect of the host factor on EV-A71 virus replication was preliminarily verified.2.Screening and evaluation of traditional Chinese medicine monomer with anti-EV-A71 virus activity.The traditional Chinese medicine monomer with anti-EV-A71 virus activity was screened.The anti-EV-A71 activity of 14 traditional Chinese medicine monomers was detected by using the EV-A71 virus-infected cell model.The compounds with anti-EV-A71activity were screened preliminarily by detecting the inhibitory effect of each compound on the cytopathy caused by the EV-A71 virus.The anti-EV-A71 virus activity of the screened Mulberroside C was evaluated in vitro and in vivo.CCK-8 assay was used to detect the inhibitory effect of the compounds on EV-A71 virus-induced cytophysiopathy.The effects of the compounds on the RNA and protein synthesis of the EV-A71 virus were detected by RT-q PCR,Westen blot and immunofluorescence.The effect of the compound against the EV-A71 virus was preliminarily discussed by time-of-addition assay.Besides,to evaluate the effect of Mulberroside C on body weight,survival rate,and clinical score of neonatal ICR mice infected with EV-A71 virus at lethal dose.Viral load in different tissues and pathological observation were used to evaluate the effect of Mulberroside C on virus replication in vivo.Viral capsid protein VP1 is a recognized antiviral target,the virus protein VP1 was identified as a potential target by Pharm Mapper Server prediction.The interaction between viral protein VP1 and Mulberroside C was simulated by molecular docking.The interaction between Mulberroside C and viral protein was verified by Biolayer interferometrvy technolgy using Fortebio Octet Red96e.Results:1.Identification of host factors involved in EV-A71 infection based on the CRISPR/Cas9 system.To determine the optimal conditions for lentivirus infection,a negative control virus expressing green fluorescence was used for the study.When MOI=7,the infection efficiency of lentivirus is about 20%.The optimal concentration of puromycin showed that wild RD cells were all killed at 1?g/m L within 48 hours.Therefore,the optimal MOI of the CRISPR/Cas9 library was 7,and the optimal screening concentration of puromycin was 1?g/m L.Based on the above screening criteria,the CRISPR/Cas9 library lentivirus was used to infect RD cells on a large scale.After the library cells were successfully constructed,the surviving cells were collected for NGS sequencing after two rounds of lethal doses of EV-A71 virus infection,Mageck-RRA statistical analysis.A total of 150 candidate genes that may affect EV-A71 virus infection were identified in this study(P<0.05).These include host genes that have been reported to be associated with EV-A71 virus replication,such as SCARB2,ATF4,ANKRD17,and SETD3.Through gene function queries and literature research,PSMA6 was selected as the key host factor for EV-A71 virus infection.PSMA6si RNA was further designed and synthesized.After transfection with RD cells,RT-q PCR results showed that PSMA6 knockdown significantly reduced the production of progeny virus compared with wild-type RD cells,and Westen blot results showed that PSMA6knockdown significantly reduced the expression of viral protein VP1.We screened out the PSMA6 gene as an important gene in EV-A71 virus infection.2.Screening and evaluation of traditional Chinese medicine monomer with anti-EV-A71 virus activity.The anti-EV-A71 activity of 14 traditional Chinese medicine monomers was screened by using the model of EV-A71 infected RD cells.The results showed that Mulberroside C showed good anti-EV-A71 activity.Furthermore,the toxicity and anti-EV-A71 activity of Mulberroside C were evaluated systematically by in vitro experiments.Toxicity assay showed that the CC₅₀of Mulberroside C in RD cells was 94.82?M and that in Vero cells was 257.1?M.The results of the antiviral activity test showed that Mulberroside C could inhibit the cytopathic effect of EV-A71 virus infection in a dose-dependent manner.The IC50of Mulberroside C against RD cytopathic effect was 2.09?M,and the IC₅₀ of Mulberroside C against Vero cytopathic effect was 35.48?M.RT-q PCR,Westen blot,and immunofluorescence assay showed that Mulberroside C could significantly inhibit the synthesis of EV-A71 virus RNA and viral protein.Time-of-addition assay showed that Mulberroside C mainly inhibited the early stage of EV-A71 virus replication.Mulberroside C could significantly inhibit the proliferation of the EV-A71 virus in vivo.reduce the mortality of virus-infected mice and alleviate the clinical symptoms of virus-infected mice.Compared with model mice,Mulberroside C significantly reduced viral load in the heart,brain,lung,small intestine,and hind limb muscle of virus-infected mice,and alleviated inflammatory infiltrates and tissue lesions caused by viral infection.Besides,Pharm Mapper Server prediction results showed that viral protein VP1 is one of the important targets of Mulberroside C.Molecular docking results also showed that Mulberroside C was similar to the natural molecule sphingosine,which could occupy the space in the viral pocket more fully,thus stabilizing the viral capsid and preventing the virus from uncoating and releasing RNA into cells.The results of biomolecular interaction analysis further showed that Mulberroside C could bind to EV-A71 VP1 protein molecule,and the affinity(KD)between virus-VP1 protein and Mulberroside C was 1.289×10^-3 M,and the binding showed a significant concentration-dependent relationship.Conclusions:1.In this study,CRISPR/Cas9 library screening technology was used to screen host factors related to EV-A71 virus replication,which is of great significance for revealing the complex interaction between EV-A71 virus and host cells.We screened and found that PSMA6 is an important host factor associated with EV-A71 virus infection.Knock-down of PSMA6 protein can significantly reduce the RNA replication of EV-A71 virus and the expression of viral protein VP1,suggesting that PMSA6 may be developed as a candidate host target against EV-A71 virus.2.We screened and found that Mulberroside C from the traditional Chinese medicine Morus alba L had a strong anti-EV-A71 effect in vivo and in vitro.Mulberroside C significantly reduced the RNA copy number and inhibited the synthesis of viral protein in vitro.Mulberroside C can effectively reduce the survival rate and clinical score of mice caused by an EV-A71 virus infection,reduce viral load in a variety of tissues,and improve tissue lesions caused by EV-A71 virus infection in vivo.The mechanism study showed that Mulberroside C mainly targeted EV-A71 VP1 protein.This study provides a candidate drug for anti-EV-A71 drug development.