人副流感病毒3型(hPIV-3)通过膜融合进入和宿主内传播是由两种包膜糖蛋白启动的,血凝素-神经氨酸酶(HN)和融合(F)蛋白。HN蛋白通过其受体结合位点与细胞受体的结合引发F蛋白的构象变化,导致病毒-细胞融合。然而,关于包含受体结合位点的单个氨基酸在融合过程中的作用知之甚少。这里,位于受体结合位点Ⅰ内的残基R192、D216、E409、R424、R502、Y530和E549,在推定位点Ⅱ处的残基N551和H552被丙氨酸取代,并进行定点突变。除N551A外,所有突变体均显示出统计学上较低的血液吸附活性,范围为野生型(wt)水平的16.4%至80.2%。随着结合红细胞数量的标准化,同样,除了N551A,所有突变体在三个连续阶段显示降低的融合活性:脂质混合(半融合),内容混合(全融合)和合胞体发育。半融合过程的动力学测量表明,R192A的初始半融合程度,D216A,E409A,R424A,R502A,Y530A,E549A和H552A降至69.9%,80.6%,71.3%,67.3%,50.6%,87.4%,84.9%和25.1%,分别,相对于wt,而E409A的初始半输注率,R424A,R502A和H552A突变体减少到69.0%,35.4%,62.3%,37.0%,分别。此外,四个初始半融合率降低的突变体也显示F蛋白切割百分比从wt的43.4%降低到56.3%。一起来看,突变体R192A,D216A,E409A,R424A,R502A,Y530A,E549A和H552A可能导致半融合初期的融合活性受损,其中降低的程度和速率可能与受损的受体结合活性有关,导致F蛋白的活化屏障增加及其裂解,分别。
Human parainfluenza virus type 3 (hPIV-3) entry and intrahost spread through membrane fusion are initiated by two envelope glycoproteins, hemagglutinin-neuraminidase (HN) and fusion (F) protein. Binding of HN protein to the cellular receptor via its receptor-binding sites triggers conformational changes in the F protein leading to virus-cell fusion. However, little is known about the roles of individual amino acids that comprise the receptor-binding sites in the fusion process. Here, residues R192, D216, E409, R424, R502, Y530 and E549 located within the receptor-binding site Ⅰ, and residues N551 and H552 at the putative site Ⅱ were replaced by alanine with site-directed mutagenesis. All mutants except N551A displayed statistically lower hemadsorption activities ranging from 16.4% to 80.2% of the wild-type (wt) level. With standardization of the number of bound erythrocytes, similarly, other than N551A, all mutants showed reduced fusogenic activity at three successive stages: lipid mixing (hemifusion), content mixing (full fusion) and syncytium development. Kinetic measurements of the hemifusion process showed that the initial hemifusion extent for R192A, D216A, E409A, R424A, R502A, Y530A, E549A and H552A was decreased to 69.9%, 80.6%, 71.3%, 67.3%, 50.6%, 87.4%, 84.9% and 25.1%, respectively, relative to the wt, while the initial rate of hemifusion for the E409A, R424A, R502A and H552A mutants was reduced to 69.0%, 35.4%, 62.3%, 37.0%, respectively. In addition, four mutants with reduced initial hemifusion rates also showed decreased percentages of F protein cleavage from 43.4% to 56.3% of the wt. Taken together, Mutants R192A, D216A, E409A, R424A, R502A, Y530A, E549A and H552A may lead to damage on the fusion activity at initial stage of hemifusion, of which decreased extent and rate may be associated with impaired receptor binding activity resulting in the increased activation barrier of F protein and the cleavage of it, respectively.