检测PRRSV-NSP4酶活性的FlipNLuc报告系统的构建与应用OA

To establish a FlipNLuc reporter system for detecting the enzymatic activity of non-structural protein 4(NSP4)of porcine reproductive and respiratory syndrome virus(PRRSV),a nanoluciferase-based reporter plasmid,pCMV-NLuc-Flag,was constructed in this study.The optimal cleavage sequence for the FlipNLuc reporter system was screened,and the system was subsequently characterized and optimized with respect to its operating conditions and compatibility.Furthermore,the established system was used to screen compounds targeting NSP4 protease activity and to evaluate their antiviral effects.The results showed that among the NSP4 cleavage sequences derived from the PRRSV polyprotein,GSLLE/GAFR was identified as the optimal cleavage sequence.The optimal detection performance was achieved when the pCMV-NLuc-Flag reporter plasmid and the pCMV-NSP4-HA eukaryotic expression plasmid were co-transfected at a ratio of 1:1 for 24-36 h.The system efficiently accommodated NSP4 proteases from both PRRSV-1 and PRRSV-2 strains.Using this system,one NSP4 protease inhibitor,tanshinone IIA,was successfully identified.Treatment of porcine alveolar macrophages(PAMs)with tanshinone IIA at concentrations below 50 μmol/L for 24 h showed no obvious cytotoxicity(P>0.05).In contrast,treatment with 50 μmol/L tanshinone IIA for 24 h significantly reduced the mRNA expression level of the PRRSV-2 open reading frame 7(ORF7)gene,N protein expression level,and viral titer(P<0.01),indicating that the compound possessed anti-PRRSV-2 activity.In conclusion,this study successfully established a FlipNLuc reporter system for detecting NSP4 enzymatic activity.The system can be used not only for high-throughput screening of anti-PRRSV compounds targeting NSP4 protease at the cellular level,but also as a methodological reference for activity detection of other viral proteases.