Human parainfluenza virus type 3 (HPIV3) causes the majority of childhood viral pneumonia around the world. Fusing the viral and target cell membranes is crucial for its entry into target cells, and the fusion process requires the concerted actions of two viral glycoproteins: hemagglutinin-neuraminidase (HN) and fusion (F) protein. After binding to the cell surface receptor, sialic acids, HN triggers F to undergo large conformational rearrangements to execute the fusion process. Although it has been reported that several domains of F had important impacts on regulating the membrane fusion activity, what role the DI-DII linker (residues 369-374, namely L1 linker) of the HPIV3 F protein plays in the fusion process still remains confused. We have obtained three chimeric mutant proteins (Ch-NDV-L1, Ch-MV-L1, Ch-HPIV1-L1) containing the full length of HPIV3 F protein but their corresponding DI-DII linker derived from the F protein of Newcastle disease virus (NDV), Measles virus (MV), and Human parainfluenza virus type 1 (HPIV1), respectively. One deletion mutant protein (De-L1), whose DI-DII linker was deleted, has been established simultaneously. Then vaccinia virus-T7 RNA polymerase transient expression system and standard plasmid system were utilized to express the mutant F proteins in BHK-21 cells. These four mutants were determined for membrane fusogenic activity, cell surface expression level, and total mutant F protein expression. All of them resulted in a significant reduction in fusogenic activity in all steps of cell-cell membrane fusion process. There was no significant difference in cell surface protein expression level for the mutants compared with wild-type F. The mutant proteins with inability in fusogenic activity were all at the form of precursor protein, F0, which were not hydrolyzed by intracellular protease furin. The results above suggest that the involvement of the DI-DII linker region is necessary for the complete fusion of the membranes.

中文翻译：

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人副流感病毒 3 型融合蛋白的 DI-DII 接头对病毒至关重要。

人类副流感病毒 3 型 (HPIV3) 导致了全世界大多数儿童病毒性肺炎。融合病毒和靶细胞膜对其进入靶细胞至关重要，融合过程需要两种病毒糖蛋白的协同作用：血凝素-神经氨酸酶 (HN) 和融合 (F) 蛋白。在与细胞表面受体唾液酸结合后，HN 触发 F 进行大的构象重排以执行融合过程。尽管有报道F的几个结构域对调节膜融合活性有重要影响，但HPIV3 F蛋白的DI-DII接头（残基369-374，即L1接头）在融合过程中所起的作用仍不清楚. 我们获得了三种嵌合突变蛋白（Ch-NDV-L1、Ch-MV-L1、Ch-HPIV1-L1) 含有全长 HPIV3 F 蛋白，但其相应的 DI-DII 接头来源于新城疫病毒 (NDV)、麻疹病毒 (MV) 和人类副流感病毒 1 型 (HPIV1) 的 F 蛋白，分别。同时建立了一种缺失突变蛋白（De-L1），其DI-DII接头被删除。然后利用牛痘病毒-T7 RNA聚合酶瞬时表达系统和标准质粒系统在BHK-21细胞中表达突变F蛋白。测定这四个突变体的膜融合活性、细胞表面表达水平和总突变体 F 蛋白表达。所有这些都导致细胞-细胞膜融合过程的所有步骤中的融合活性显着降低。与野生型F相比，突变体的细胞表面蛋白表达水平没有显着差异。无法融合活性的突变体蛋白均以前体蛋白F0的形式存在，不被细胞内蛋白酶弗林蛋白酶水解。上述结果表明，DI-DII 接头区域的参与对于膜的完全融合是必要的。