Identification Of Porcine Torovirus 3C-like Protease And Screening For Small Molecule Inhibitors

Torovirus(ToV)belongs to the order Nidovirales and infects a wide range of hosts,including mammals,reptiles,and fish.Based on the tenth report ICTV,toroviruses were formally separated from coronaviruses and formed a separate suborder.As a member of the subfamily torovirus,porcine torovirus(PToV)is a potential pathogen causing diarrhea in piglets,and its genome is highly variable,with high recombination risk and potential for cross-species infection.Its unique taxonomic position(between the Arteriviridae and Coronaviridae)is an important link in the study of the evolution of the set viruses,and the analysis of the structure and function of its key genes is important for understanding the evolution of the nidoviruses.The 3C-like protease(3CL^pro),also known as the main protease,cleaves the virus-encoded polyproteins pp1a and pp1ab to generate most of the mature nonstructural proteins of the virus,which play an essential role in the replication process and pathogenesis of the virus and is one of the important targets for antiviral drug design.The analysis of the structure and function of 3CL^pro of the nidovirus will provide a theoretical basis for the study of broad-spectrum inhibitors of 3CL^pro.At present,coronavirus and arteritis virus3CL^pro have been relatively well studied,but little is known about torovirus 3CL^pro,which to a certain extent hinders the in-depth study of the relationship between the structure and evolution of 3CL^pro in the set of viruses.Therefore,in this study,the specific amino acid composition of PToV 3CL^pro and its substrate recognition properties were analyzed,an enzyme activity assay was established,and small molecule inhibitors targeting PToV3CL^pro were screened.The details of the study are as follows:1.Whole-genome sequence determination of porcine torovirus(PToV)strain HB1To better understand the extent of the recent prevalence of PToV variation,we compared the sequences of the published viruses of the family torovirus on the Genbank,selected conserved nucleotide sequence regions for primer design,and designed a total of22 pairs of primers that could cover the whole genome.A positive piglet fecal samples was selected for epidemiological investigation,and the whole genome was sequenced and named as PToV HB1.All 22 fragments were sequenced by reverse transcription,PCR amplification,cloning and sequencing.The whole genome sequence of PToV was obtained by splicing the sequencing results,and the sequence analysis showed that the total length of PToV HB1 strain was 28276 bp.The analysis of its molecular variation and evolutionary characteristics revealed that the sequence had only 92%nucleotide homology with the previously reported PToV,suggesting that the PToV virus was highly variable.Further analysis of the genome composition revealed that this virus is similar to other torovirus in genome composition,mainly consisting of six open reading frames(ORF1a,ORF1b,S,M,HE and N).2.Identification of the specific amino acid composition of porcine torovirus 3C-like protease and its substrate specificityTo identify the specific amino acid composition of PToV 3CL^pro,the predicted PToV3CL^pro and its two flanking sequences were firstly cloned using PToV HB1 strain as a template,and the self-cleaved bands were sequenced by prokaryotic expression as well as N-terminal sequencing by Edman degradation method,and the specific region of mature PToV 3CL^pro was found to be ORF1a 3096-Subsequent site-directed mutagenesis experiments showed that PToV 3CL^pro is a serine protease with a catalytic center composed of His53 and Ser160 duplexes.Further,using a cyclized luciferase-based biosensor,the3CL^pro cleavage site of PToV polyprotein was systematically scanned and it was found that the most typical feature of PToV 3CL^pro substrate recognition was the recognition of glutamine(Gln,Q)at the substrate P1 position.In addition,the substrate P4 position is also relatively conserved for phenylalanine(Phe,F).Interestingly,unlike most sets of viral3CL^pro,the P1-Gln residue is not a sufficient determinant for PToV 3CL^pro to undergo N-terminal self-cleavage,but is influenced by the P1 and P4 residues in concert.Subsequent homology modeling and biological experiments showed that PToV 3CL^pro forms distinct S1 and S4 pockets with the substrate,and mutation of key amino acids in the S1(D159A,H174A)or S4(P62G/L185G)pockets resulted in a complete loss of the ability of PToV3CL^pro to cleave the substrate,confirming that the S1 and S4 pockets play important role in protease cleavage.3.Establishment of a cell-based assay for PToV 3CL^pro enzymatic activity and screening of inhibitorsIn order to establish an enzymatic activity assay for PToV 3CL^pro,the cleavage substrate short peptide sequence(nsp3/nsp4)of PToV 3CL^pro was cloned into a cyclized fluorophore enzyme vector,and a method was established to detect the protease activity of PToV 3CL^pro at the cellular level,and dose experiments and enzymatic mutation experiments confirmed that the method could effectively reflect the cleavage specificity of PToV 3CL^pro cleavage specificity and protease activity.The established enzyme activity assay was used to screen the natural product library for drugs that specifically target PToV3CL^pro and four natural products(Doxorubicin HCl,Daunorubicin HCl,Lycorine and3,4',5-Trimethoxy-trans-stilbene)were found to significantly inhibit PToV 3CL^pro enzyme activity.Further studies revealed that the resveratrol derivative 3,4',5-trimethoxy-trans-stilbene(3,4',5-Trimethoxy-trans-stilbene,MR-3)inhibited the activity of coronavirus PEDV and arteritis virus PRRSV 3CL^pro in addition to PToV 3CL^pro enzymatic activity in a dose-dependent,suggesting that MR-3 is most likely a broad-spectrum inhibitor targeting the 3CL^pro of the nidovirus.4.Inhibition of nidovirus roliferation by resveratrol and its derivativesTo investigate the anti-nidovirus potential of resveratrol components,this study investigated the anti-nidovirus capacity and mechanisms of of resveratrol derivatives with strong anti-PEDV ability,using various isolated and cultured coronaviruses(coronavirus:PEDV,PDCoV,MHV;arteritis virus:PRRSV)as models.Firstly,in vitro biochemical assays confirmed that MR-3 and piceatannol among the six drugs significantly inhibited PEDV 3CL^pro enzymatic activity,with piceatannol showing better inhibitory activity.Subsequent antiviral assays also confirmed that piceatannol significantly inhibited PEDV proliferation and had the strongest inhibitory ability among the six drugs.Replication cycle assays revealed that piceatannol inhibited both the adsorption and replication phases of PEDV and had an inactivating effect on PEDV.However,compared with the ability to inhibit PEDV proliferation,piceatannol inhibited the proliferation of PDCoV and MHV and had almost no inhibitory ability against PRRSV and had no inhibitory ability against PRRSV 3CL^pro activity,confirming that the antiviral ability of piceatannol was species-specific.In conclusion,this study found that P.albicans significantly inhibited the proliferation of several coronaviruses(PEDV,PDCoV and MHV)and may be associated with the inhibition of their 3CL^pro enzymatic activity,providing useful information for the rational development of anti-coronaviral drugs.In conclusion,whole-genome sequencing of porcine torovirus(PToV)strain HB1was performed,and identified the self-cleavage activity and substrate specificity of PToV 3CL^pro,and based on this,we constructed a cell-based assay for PToV 3CL^proenzymatic activity using PToV 3CL^pro cleavage substrate properties,and used the assay to screen natural product libraries for inhibitors specifically targeting PToV3CL^pro,and subsequently,confirmed at the viral level that the screening drug piceatannol has broad-spectrum anti-coronavirus activity,laying a theoretical foundation for the development of safer and more effective anti-coronavirus drugs in the future.