Key Tim, Duncan Roy
Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia, Canada.
Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia, Canada; Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada; Department of Pediatrics, Dalhousie University, Halifax, Nova Scotia, Canada.
PLoS Pathog. 2014 Mar 20;10(3):e1004023. doi: 10.1371/journal.ppat.1004023. eCollection 2014 Mar.
The homologous p10 fusion-associated small transmembrane (FAST) proteins of the avian (ARV) and Nelson Bay (NBV) reoviruses are the smallest known viral membrane fusion proteins, and are virulence determinants of the fusogenic reoviruses. The small size of FAST proteins is incompatible with the paradigmatic membrane fusion pathway proposed for enveloped viral fusion proteins. Understanding how these diminutive viral fusogens mediate the complex process of membrane fusion is therefore of considerable interest, from both the pathogenesis and mechanism-of-action perspectives. Using chimeric ARV/NBV p10 constructs, the 36-40-residue ectodomain was identified as the major determinant of the differing fusion efficiencies of these homologous p10 proteins. Extensive mutagenic analysis determined the ectodomain comprises two distinct, essential functional motifs. Syncytiogenesis assays, thiol-specific surface biotinylation, and liposome lipid mixing assays identified an ∼25-residue, N-terminal motif that dictates formation of a cystine loop fusion peptide in both ARV and NBV p10. Surface immunofluorescence staining, FRET analysis and cholesterol depletion/repletion studies determined the cystine loop motif is connected through a two-residue linker to a 13-residue membrane-proximal ectodomain region (MPER). The MPER constitutes a second, independent motif governing reversible, cholesterol-dependent assembly of p10 multimers in the plasma membrane. Results further indicate that: (1) ARV and NBV homomultimers segregate to distinct, cholesterol-dependent microdomains in the plasma membrane; (2) p10 homomultimerization and cholesterol-dependent microdomain localization are co-dependent; and (3) the four juxtamembrane MPER residues present in the multimerization motif dictate species-specific microdomain association and homomultimerization. The p10 ectodomain therefore constitutes a remarkably compact, multifunctional fusion module that directs syncytiogenic efficiency and species-specific assembly of p10 homomultimers into cholesterol-dependent fusion platforms in the plasma membrane.
禽呼肠孤病毒（ARV）和纳尔逊湾呼肠孤病毒（NBV）的同源p10融合相关小跨膜（FAST）蛋白是已知最小的病毒膜融合蛋白，也是融合性呼肠孤病毒的毒力决定因素。FAST蛋白的小尺寸与为包膜病毒融合蛋白提出的典型膜融合途径不相符。因此，从发病机制和作用机制的角度来看，了解这些微小的病毒融合原如何介导复杂的膜融合过程具有相当大的意义。使用嵌合ARV/NBV p10构建体，确定36-40个残基的胞外域是这些同源p10蛋白不同融合效率的主要决定因素。广泛的诱变分析确定胞外域包含两个不同的、必不可少的功能基序。细胞融合试验、硫醇特异性表面生物素化和脂质体脂质混合试验确定了一个约25个残基的N端基序，该基序决定了ARV和NBV p10中胱氨酸环融合肽的形成。表面免疫荧光染色、荧光共振能量转移（FRET）分析和胆固醇消耗/补充研究确定胱氨酸环基序通过一个两残基的连接子与一个13个残基的膜近端胞外域区域（MPER）相连。MPER构成了第二个独立的基序，控制p10多聚体在质膜中可逆的、胆固醇依赖性的组装。结果进一步表明：（1）ARV和NBV同型多聚体在质膜中分离到不同的、胆固醇依赖性的微结构域；（2）p10同型多聚化和胆固醇依赖性微结构域定位是相互依赖的；（3）多聚化基序中存在的四个近膜MPER残基决定了物种特异性微结构域关联和同型多聚化。因此，p10胞外域构成了一个非常紧凑的多功能融合模块，它指导细胞融合效率以及p10同型多聚体在质膜中组装成胆固醇依赖性融合平台的物种特异性过程。
