新城疫病毒F蛋白细胞融合活性位点中保守氨基酸基因突变分析

Mutational Analysis of Conserved Amino Acids on the Active Domain of Newcastle Disease Virus Fusion Protein

为了确定新城疫病毒融合蛋白(F)分子上活性位点中保守氨基酸在F蛋白的细胞融合作用,弄清F细胞融合的分子机理,采用基因定点突变法,创造一个酶切位点,用酶切反应初步筛选突变株,然后用DNA序列分析进一步确定,并于真核细胞内进行表达,Giemsa染色定性和指示基因法定量检测细胞融合功能,荧光强度分析(FACS)检测表达效率情况。结果表明,NDV F第117位苯丙氨酸(F)突变成亮氨酸(L)时对细胞融合作用没有显著影响。R112和K115同为保守序列,分别突变为G时,细胞融合活性只有原来的44%,下降了56%。细胞表面表达效率没有明显的改变。N147突变为K时,细胞融合活性明显下降,只有原来的15%,而细胞表面表达效率没有明显的改变。L154为保守序列,突变为K时,细胞融合活性消失,说明L154是一个非常关键的氨基酸,对维持F蛋白的细胞融合活性非常重要。细胞表面表达效率也有所下降(为原来的94%)。D462属于高度保守氨基酸,当突变为N时,细胞融合活性消失,但经细胞表面表达效率分析证明,此突变蛋白未表达于细胞表面,证明在细胞浆转运至细胞表面的过程中发生了问题。当突变为R和E时,细胞融合活性未发生改变,但细胞表面表达效率有所下降,分别为野毒株的63%和44%。说明NDV F分子上与HN相互作用的特异性区域中的某些保守氨基酸在细胞融合中发挥着重要作用,对F蛋白的折叠、加工、转运等,发挥着不同作用,从而影响F蛋白的细胞融合作用和/或在细胞表面的表达量。

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.