Reovirus FAST Proteins Drive Pore Formation and Syncytiogenesis Using a Novel Helix-Loop-Helix Fusion-Inducing Lipid Packing Sensor

Pore formation is the most energy-demanding step during virus-induced membrane fusion, where high curvature of the fusion pore rim increases the spacing between lipid headgroups, exposing the hydrophobic interior of the membrane to water. How protein fusogens breach this thermodynamic barrier to pore formation is unclear. We identified a novel fusion-inducing lipid packing sensor (FLiPS) in the cytosolic endodomain of the baboon reovirus p15 fusion-associated small transmembrane (FAST) protein that is essential for pore formation during cell-cell fusion and syncytiogenesis. NMR spectroscopy and mutational studies indicate the dependence of this FLiPS on a hydrophobic helix-loop-helix structure. Biochemical and biophysical assays reveal the p15 FLiPS preferentially partitions into membranes with high positive curvature, and this partitioning is impeded by bis-ANS, a small molecule that inserts into hydrophobic defects in membranes. Most notably, the p15 FLiPS can be functionally replaced by heterologous amphipathic lipid packing sensors (ALPS) but not by other membrane-interactive amphipathic helices. Furthermore, a previously unrecognized amphipathic helix in the cytosolic domain of the reptilian reovirus p14 FAST protein can functionally replace the p15 FLiPS, and is itself replaceable by a heterologous ALPS motif. Anchored near the cytoplasmic leaflet by the FAST protein transmembrane domain, the FLiPS is perfectly positioned to insert into hydrophobic defects that begin to appear in the highly curved rim of nascent fusion pores, thereby lowering the energy barrier to stable pore formation.

病毒诱导的膜融合过程中，孔道形成是耗能最多的步骤：融合孔缘的高曲率会增大脂质头部基团之间的间距，使膜内部的疏水核心暴露于水环境中。目前仍不清楚蛋白质融合因子如何突破孔道形成所需的热力学障碍。我们在狒狒呼肠孤病毒p15融合相关小跨膜(FAST)蛋白的胞质端结构域中，发现了一种新型诱导融合的脂质包装传感器(FLiPS)，该结构对于细胞间融合和合胞体形成过程中的孔道形成至关重要。核磁共振(NMR)光谱学与突变研究表明，该FLiPS依赖于疏水螺旋-环-螺旋结构。生化与生物物理实验显示，p15 FLiPS优先富集于具有高正曲率的膜中，而双苯胺萘磺酸盐（bis-ANS）——一种可插入膜内疏水缺陷的小分子——会阻碍这一富集过程。尤为关键的是，p15 FLiPS的功能可被异源两亲性脂质包装传感器(ALPS)替代，但无法被其他膜相互作用性两亲螺旋所替代。此外，在爬行动物呼肠孤病毒p14 FAST蛋白的胞质结构域中，此前未被发现的两亲螺旋可在功能上替代p15 FLiPS，而该两亲螺旋自身也可被异源ALPS基序替代。FAST蛋白的跨膜结构域将FLiPS锚定在胞质侧膜小叶附近，使其处于理想位置，可插入新生融合孔高弯曲度边缘处逐渐出现的疏水缺陷中，从而降低稳定孔道形成所需的能量障碍。