Functional Identification Of The Untranslated Region In The Newcastle Disease Virus Genome As A Regulator Of Foreign Gene Expression

Newcastle disease virus(NDV)provides a suitable option for the construction of live vaccine vectors due to its modular nature of transcription,minimal recombination frequency,and lack of DNA equivalence during replication,and vaccines developed using it as a vector have wide applicability.However,some NDV vector vaccines are not very immunogenic due to the low expression of the foreign gene(FG)and provide only partial protection after inoculation of the host.In addition,owing to the polar attenuation of transcription of the nonsegmented NDV genome,the capacity of the NDV vector is limited,and vaccines are mainly bivalent,with few studies of triplex or even multiplex vaccines being reported.In the development of vaccines using NDV as a vector,the use of different insertion strategies often results in various levels of gene expression.Currently,the most widely used insertion strategy is to insert the FG as an additional independent transcription unit,usually adding only the gene start(GS)and gene end(GE)sequences recognized by the viral transcription process to the 5’and 3’ ends of the FG.Studies have reported that certain viral genomic Untranslated regions(UTRs)can enhance the binding of messenger RNA(m RNA)to polyribosomes or enhance the stability of m RNA,thereby promoting protein expression.Whether the UTR of the NDV genome regulates gene expression and helps recombinant NDV express multiple FGs efficiently at the optimal FG insertion site is unclear.In this study,the UTRs on both sides of the NDV-encoded genes were used to identify and compare the regulatory functions of UTRs at the mini genome(MG)level and full-length genome level using reverse genetic manipulation.A method was developed to reduce the transcriptional attenuation of FGs,enhance the expression of downstream FGs,and efficiently express multiple FGs in a recombinant virus by chimerizing UTRs with FGs.The specific study and results include the following aspects:1.Probing the ability of the UTR to regulate FG expression at the MG level.In order to identify the key components affecting the efficiency of FG expression,MG vectors were constructed based on the UTR sequences of the NDV Spotted Dove strain genome with FG flanked by different UTRs,or deleted and truncated UTRs.After cotransfection of MG with auxiliary plasmids expressing NP,P,and L in BHK-21 cells,FG expression was examined by fluorescence microscopy,Western blot,and flow cytometry.The 5‘UTR of the NP gene was identified as an essential component for MG expression of FG,and the key sites(24–30 nt,30-36 nt,5–3’ direction)that significantly affected MG expression efficiency were located.it was confirmed that knockdown and replacement of 3’UTR had no significant effect on the expression of FG in MG.2.To investigate the regulatory role of UTRs on FG at the genome-wide level.In order to further determine the regulatory role of UTRs on gene expression in the NDV genome,the UTRs of different viral genes were constructed at the 5’ and 3’ ends of FG using reverse genetic techniques,and the corresponding recombinant viruses were rescued and tested for biological properties.The results showed that the rescued strains expressed FG correctly and had good genetic and replicative stability.The expression levels of FG were measured by fluorescence observation,WB,and flowmetry.The results confirmed that the UTR of NDV had no significant effect on the expression of foreign proteins when only one FG was expressed.3.The construction of a reverse genetic system for efficient expression of multiple FGs in NDV based on UTRs.Based on the identification of the necessary UTR elements for efficient expression of FGs in MG,FGs were inserted into MG by mimicking the NDV genomic structure by chimerizing FGs in the UTRs of NP5NP3,NP5P3,and NP5L3 in sequence,and the feasibility of MG to accommodate multiple FGs was verified after transfection.It was demonstrated that the chimerization of FGs with UTRs at the MG level could up-regulate the translation level of FGs in some cells when multiple FGs were inserted.Further,the FGs chimerized with UTRs were integrated into the infectious cloning vector as three independent transcription units,and four recombinant viruses capable of expressing two or three fluorescent proteins were successfully rescued.Growth kinetic assays showed that the recombinant viruses had similar proliferative capacities to the parental viruses;after in vitro infection of cells,a preliminary evaluation of the expression efficiency of FGs was performed.Fluorescence imaging,q PCR,flow,and WB results showed that the recombinant NDVs could accommodate the three inserted FGs and that chimerization of FGs with UTRs significantly up-regulated the expression efficiency of downstream FGs and reduced the transcriptional attenuation of FGs.In summary,the experiments compared the regulatory role of UTR at the MG level,validated the efficiency of UTR in regulating gene expression at the genome-wide level,and established a strategy for efficient expression of three FGs in the same NDV vector by chimerizing FG with UTR in combination with an ITU strategy.It has been strongly demonstrated that the NDV genomic UTR can be used as an important component to regulate the expression of multiple FGs and enhance the expression of downstream exogenous genes,but the specific mechanism of its involvement in regulation needs further study,and this study lays the foundation for the development of subsequent NDV multiplex vector vaccines.