Construction Of Newcastle Disease Virus Minigenome And Research On M,V And W Proteins On Virus Replication And Transcriptional Regulation

NDV is a non-segmented single-stranded negative RNA virus.Due to its large genome about 15 kb,it is technically difficult to manipulate directly in the full-length genome that hinders the study of mechanism of viral replication,transcription and protein functions.Therefore,a minigenome technology,which uses reporter gene to replace the viral coding region,and retains simplest regulatory sequences of viral genome.The viral replication and transcription depend upon the NP,P and L proteins of the RNP complex.Other viral proteins may involve in regulation of the RNP complex activity.However,it is complete unknown.In this study,four models of the minigenome were designed.After experimental examination,the sequences of the correct model of minigenome were identified.The rescue efficiency of two promoters driving minigenome were compared.In addition,a replication-defective minigenome was established.The effects of M,V and W proteins on viral replication and transcription process were preliminarily investigated.The main results are following:1.Construction of four minigenomes of NDVDesign scheme:(1)According to mimic viral genome,the EGFP reporter gene was reversely inserted between sequences of 5' Trailer and 3' Leader,named Mini.(2)Based on the Mini,the corresponding GE,GS and UTR sequences were deleted,named Mini2.(3)According to the approach of constructing reverse genetics,the EGFP was positively inserted between sequences of 5' Leader and 3' Trailer,named MG.(4)Based on MG,the corresponding GE,GS and UTR sequences were deleted,named as MG2.Four migenomes cloned into pUC19 vector under T7 promoter were co-transfected with NP,P,L and T7 RNA polymerase expression plasmid to analyze EGFP expression in BHK-21 cells.Only the Mini was found to be functional.The results demonstrated that the reporter gene could only be inserted reversely,and the GE-5' UTR and 3' UTR-GS sequences were essential gene elements for the minigenome construction.2.Effects of two promoters on the efficiency of minigenome rescue.The Mini minigenome was cloned into pUC19 vector under chicken ?-actin promoter,pCAGGS-Mini-EGFP.Compared to pUC19-Mini-EGFP,analysis of the rescue efficiency showed that the T7 promoter was much higher than the chicken ?-actin promoter.3.Construction of a replication-defective minigenome.To study the two independent processes of viral replication and transcription,the 5'Trailer sequence of the minigenome was deleted that results in the replication of the viral genome(vRNA)to produce the deleted 3' Trailer(?T)cRNA,named pUC19-Mini-?T.The?T cRNA is unable to promote the replication to produce vRNA that can distinguish between gene replication and transcription.4.Effects of M,V and W proteins on the regulation of viral replication and transcription.For quantitative analysis expression of reporter gene,in normal(replication-sensitive)and replication-defective minigenome,firefly luciferase was used for instead of EGFP,respectively named pUC19-Mini-Luc and pUC19-Mini-?T-Luc.The expression plasmids of M,V and W were co-transfected with the two minigenoms to analyze the firefly luciferase activity.The results showed that the only V protein inhibited transcription of the viral genome,while M and W protein had no effect.In conclusion,the construction mode of minigenome was demonstrated,and the rescue efficiency of the minigenome was optimized.Using the normal and replication-defective minigenome systems,it was suggested that the V protein had an effect on processes of viral transcription.This study provides a fundamental basis for the roles of viral replication,transcription and related proteins in NDV.