Institut National de la Recherche Agronomique, Unité de Virologie et Immunologie Moléculaires, UR892, Domaine de Vilvert, F-78350 Centre de Jouy-en-Josas, France.
J Biol Chem. 2010 Apr 23;285(17):13233-43. doi: 10.1074/jbc.M109.067710. Epub 2010 Feb 19.
The influenza A virus PB1-F2 protein, encoded by an alternative reading frame in the PB1 polymerase gene, displays a high sequence polymorphism and is reported to contribute to viral pathogenesis in a sequence-specific manner. To gain insights into the functions of PB1-F2, the molecular structure of several PB1-F2 variants produced in Escherichia coli was investigated in different environments. Circular dichroism spectroscopy shows that all variants have a random coil secondary structure in aqueous solution. When incubated in trifluoroethanol polar solvent, all PB1-F2 variants adopt an alpha-helix-rich structure, whereas incubated in acetonitrile, a solvent of medium polarity mimicking the membrane environment, they display beta-sheet secondary structures. Incubated with asolectin liposomes and SDS micelles, PB1-F2 variants also acquire a beta-sheet structure. Dynamic light scattering revealed that the presence of beta-sheets is correlated with an oligomerization/aggregation of PB1-F2. Electron microscopy showed that PB1-F2 forms amorphous aggregates in acetonitrile. In contrast, at low concentrations of SDS, PB1-F2 variants exhibited various abilities to form fibers that were evidenced as amyloid fibers in a thioflavin T assay. Using a recombinant virus and its PB1-F2 knock-out mutant, we show that PB1-F2 also forms amyloid structures in infected cells. Functional membrane permeabilization assays revealed that the PB1-F2 variants can perforate membranes at nanomolar concentrations but with activities found to be sequence-dependent and not obviously correlated with their differential ability to form amyloid fibers. All of these observations suggest that PB1-F2 could be involved in physiological processes through different pathways, permeabilization of cellular membranes, and amyloid fiber formation.
甲型流感病毒 PB1-F2 蛋白由 PB1 聚合酶基因中的一个备用读码框编码，其显示出高度的序列多态性，并被报道以序列特异性的方式促进病毒发病机制。为了深入了解 PB1-F2 的功能，研究了在不同环境中在大肠杆菌中产生的几种 PB1-F2 变体的分子结构。圆二色性光谱显示，所有变体在水溶液中均具有无规卷曲的二级结构。在三氟乙醇极性溶剂中孵育时，所有 PB1-F2 变体均采用富含α-螺旋的结构，而在模拟膜环境的中等极性溶剂乙腈中孵育时，它们则显示出β-折叠二级结构。与大豆卵磷酯脂质体和 SDS 胶束孵育时，PB1-F2 变体也获得了β-折叠结构。动态光散射表明β-折叠的存在与 PB1-F2 的寡聚/聚集有关。电子显微镜显示 PB1-F2 在乙腈中形成无定形聚集体。相比之下，在低浓度 SDS 存在下，PB1-F2 变体表现出形成纤维的各种能力，在硫代黄素 T 测定中，这些纤维被证明为淀粉样纤维。使用重组病毒及其 PB1-F2 敲除突变体，我们表明 PB1-F2 也在感染细胞中形成淀粉样结构。功能膜透化测定表明，PB1-F2 变体可以在纳摩尔浓度下穿透膜，但活性发现是序列依赖性的，与它们形成淀粉样纤维的不同能力没有明显相关。所有这些观察结果表明，PB1-F2 可以通过不同途径，即细胞内膜的通透性和淀粉样纤维的形成，参与生理过程。
