Effects Of The Diversity Of Amino Acid Sequence At The Cleavage Site And Other Regions Of Newcastle Disease Virus F Protein On Fusogenic Activity

Newcastle disease virus(NDV)is an enveloped virus with a single-stranded,non-segmented,negative-sense RNA genome.It can cause Newcastle disease(ND)in avian species,which leads to substantial economic losses in the poultry industry worldwide.The amino acid sequence at the F protein cleavage site is a major determinant of NDV virulence.In general,the F protein cleavage site(Fcs)sequence of velogenic and mesogenic strains contains multi-basic amino acids with the motif “(R/K)-R-(Q/R/K)-(R/K)-R?F”.Lentogenic Fcs contains a single or two basic amino acids with the motif “(G/E)-(K/R)-Q-(G/E)-R?L”.The type,property and position of amino acids at the Fcs present diversity due to the strains.Although a large number of mutations in single or multiple amino acids at Fcs have been reported to affect the ability to induce cell fusion and viral virulence,the role of the diversity of amino acid sequences at the Fcs remains unknown.In addition,some studies have found that although the strains have same amino acid sequence in the Fcs,their ability to induce cell fusion and cause pathogenicity are quite different.This indicated that in addition to Fcs,the amino acids in other regions of F protein also plays an important roles in the fusogenic activity and viral virulence.In this study,we analyzed the diversity of amino acid sequence of NDV Fcs.The Fcs mutants were constructed on the base of the F genes of virulent and avirulent strains,respectively.Using in vitro assay,we explore the effect of the amino acid sequence diversity of the Fcs and other regions of F protein under specific Fcs motif on fusogenic activity.The contents and results of our studies are following:1.Comprehensive analysis of the diversity of Fcs in natural isolatesThe 1572 F gene sequences of NDV natural isolates were collected from the GenBank database and previous reports,and the diversity of Fcs was analyzed by software Lasergene7.1.Based on the pathogenicity of these natural isolates,the Fcs was classified into eight types of virulent Fcs(VFcs)with 1073 isolates,which belonged to Class II.Ten types of avirulent Fcs(AFcs)with 499 isolates,which mostly belonged to Class I,partly belonged to the genotype I,II and X of Class II.By analyzing the epidemiology of the strains,we foundthat there was a significant correlation between Fcs and bird species,temporal and spatial distribution.Some of them were species-dependent and region-specific characteristics.2.Effect of the Fcs amino acid sequence diversity on cell membrane fusionThe F gene of the virulent F48E9 strain and avirulent LaSota strain were used as a backbone to contruct 10 types of AFcs mutants and 8 types of VFcs mutants,respectively.The effect of inducing cell fusion efficiency was observed by co-transfected BHK-21 cells with F mutants and HN plasmids.The result showed that the neutral residue Q at the P3 position of the VFcs played an enhancing role compared to the basic residues R and K.The single residue K is more efficient of fusogenic activity at VFcs with five basic residues.The types VFcs-1and VFcs-2 are the most efficient for cell-cell membrane fusion activity.The contribution of these VFcs to fusion efficacy occurs in the order VFcs-1 > VFcs-3 and VFcs-4;VFcs-2 >VFcs-4 > VFcs-5,VFcs-6,VFcs-7 and VFcs-8.The AFcs mutants couldn't induce syncytium formation in the absence of trypsin,except for the type AFcs-10,and could induce syncytium formation in the presence of trypsin,except for the type AFcs-9,which has the amino acid Q at the P4 position.3.Effect of other region of F protein with a cleavage motif “RRQRR?L” on ability of inducing fusogenic activityThe Fcs motif “RRQRR?L” was found to be an AFcs in above study.In generally,both virulent and avirulent F proteins with AFcs are unable to induce cell fusion.However,we found that the F protein of virulent strain F48E9 with this motif had a high ability of inducing fusogenic activity.Inversely,the F of avirulent strain La Sota with this motif was unable to induce cell fusion.The results suggested that other region of the F protein are involved in fusogenic activity in addition to the Fcs.To identify which region of the F proteins is involved in cell fusion,we divided the F protein into 9 segments based on the functional domains and exchanged them between that of the strains F48E9 and La Sota.Using cell transfection experiments,we analyzed the effect of these F protein mutants on the efficiency of cell fusion.Furthermore,the identifiacton of the F regions were narrowed by replacement or mutation.Finally,we identified that two amino acids(D479,S486)of the virulent strain F protein with this unique motif were critical for fusogenic activity under the condition of Fcs was“RRQRR?L”.4.Establishment of a new approach for NDV induced cell fusionNDV infection causes syncytia formation in host cells.The extent of cell fusion can be used as a measure of viral pathogenicity.At present,the average number of nuclei in the syncytium is widely used to evaluate the cell fusion.Although this method is more accurate than the earlier determination of syncytia area,time-consuming and strong subjective are easyto be error.In order to explore a more convenient and effectively quantitative detection of the cell fusion,we successfully selected two fluorescent cell lines BHK-21/GFP and BHK-21/tRFP in this study.The two kinds of cells were mixed and then infected with virus,the cell fusion was determined by the number of syncytium(double fluorescence positive cells)through flow cytometry.The result showed that the proportion of double fluorescent positive cells reflected the linear relationship between the dose of virus infection and the number of syncytium after infected with NDV.This study provides a new technical method for the development of convenient and effectively quantitative detection of the cell fusion.In conclusion,our studies comprehensively elucidated the effect of amino acid sequence diversity of NDV Fcs on cell membrane fusion.It would provide the theoretical basis for the Fcs motifs in viral pathogenesis.Next,other regions of F protein also play an important role for inducing fusogenic activity in addition to Fcs.It provides new insights into the cell fusion triggered by F protein and design of antiviral peptides.