Mutational Analysis of Conserved Amino Acids on the Active Domain of Newcastle Disease Virus Fusion Protein
To make sure the effects of conserved amino acids on the active domain that interacts with homologous hemagglutinin-neuraminidase(HN) on fusion protein(F) of Newcastle disease virus and to know the molecular mechanism of cell fusion,the site-directed mutagenesis was used to obtain mutants by creating a new enzymatic site,the mutants were sequenced and expressed in eukaryocytes.Some of the mutants were assayed by Giemsa staining and reporter gene assay for fusion activities and the cell surface expression efficiency by fluorescence-activated cell sorter(FACS) analysis.The results showed that NDV F mutant F117L had the same fusion activity as wild-type NDV F.Mutant of R112G-K115G had 44% of activities as wild type,but cell surface expression efficiency revealed no change.Mutant of N147K had only 15% activity as wild type,but expression efficiency had no change.Mutant of L154K had no fusion activity at all and expression efficiency was 94% of the wild type.D462 was highly conserved and when replaced by N,neither fusion activity nor expression efficiency on cell surface was observed.While mutated to R or E,there was no change in fusion activity,but expression efficiency decreased to 63% and 44%,respectively.These data proved that some conserved amino acids on the specific domain of NDV F for fusion play an important role in the cell fusion process.If they are mutated,the fusion activity of F will decrease for most of them.They play different roles in folding,processing and transportation of NDV F,and thus affect fusion activity and/or expression quantity on cell surface.