Plum Island Animal Disease Center, ARS, USDA, Greenport, New York, USA.
Biofisika Institute (CSIC-UPV/EHU), University of the Basque Country, Bilbao, Spain.
J Virol. 2018 Nov 12;92(23). doi: 10.1128/JVI.01360-18. Print 2018 Dec 1.
Nonstructural protein 2B of foot-and-mouth disease (FMD) virus (FMDV) is comprised of a small, hydrophobic, 154-amino-acid protein. Structure-function analyses demonstrated that FMDV 2B is an ion channel-forming protein. Infrared spectroscopy measurements using partially overlapping peptides that spanned regions between amino acids 28 and 147 demonstrated the adoption of helical conformations in two putative transmembrane regions between residues 60 and 78 and between residues 119 and 147 and a third transmembrane region between residues 79 and 106, adopting a mainly extended structure. Using synthetic peptides, ion channel activity measurements in planar lipid bilayers and imaging of single giant unilamellar vesicles (GUVs) revealed the existence of two sequences endowed with membrane-porating activity: one spanning FMDV 2B residues 55 to 82 and the other spanning the C-terminal region of 2B from residues 99 to 147. Mapping the latter sequence identified residues 119 to 147 as being responsible for the activity. Experiments to assess the degree of insertion of the synthetic peptides in bilayers and the inclination angle adopted by each peptide regarding the membrane plane normal confirm that residues 55 to 82 and 119 to 147 of 2B actively insert as transmembrane helices. Using reverse genetics, a panel of 13 FMD recombinant mutant viruses was designed, which harbored nonconservative as well as alanine substitutions in critical amino acid residues in the area between amino acid residues 28 and 147. Alterations to any of these structures interfered with pore channel activity and the capacity of the protein to permeabilize the endoplasmic reticulum (ER) to calcium and were lethal for virus replication. Thus, FMDV 2B emerges as the first member of the viroporin family containing two distinct pore domains. FMDV nonstructural protein 2B is able to insert itself into cellular membranes to form a pore. This pore allows the passage of ions and small molecules through the membrane. In this study, we were able to show that both current and small molecules are able to pass though the pore made by 2B. We also discovered for the first time a virus with a pore-forming protein that contains two independent functional pores. By making mutations in our infectious clone of FMDV, we determined that mutations in either pore resulted in nonviable virus. This suggests that both pore-forming functions are independently required during FMDV infection.
口蹄疫病毒（FMDV）的非结构蛋白 2B 由一个小的、疏水性的 154 个氨基酸组成。结构-功能分析表明，FMDV 2B 是一种离子通道形成蛋白。使用跨越氨基酸 28 至 147 之间区域的部分重叠肽的红外光谱测量表明，在残基 60 至 78 之间和残基 119 至 147 之间的两个假定跨膜区域以及残基 79 至 106 之间的第三个跨膜区域中采用螺旋构象，主要采用延伸结构。使用合成肽，在平面脂质双层中进行离子通道活性测量并对单个巨大的单分子层囊泡（GUV）进行成像，揭示了存在两个赋予膜穿孔活性的序列：一个跨越 FMDV 2B 残基 55 至 82，另一个跨越 2B 的 C 末端区域从残基 99 到 147。对后者序列的作图确定了残基 119 到 147 负责该活性。评估合成肽在双层中插入程度以及每个肽相对于膜平面法线的倾斜角度的实验证实，2B 的 55 至 82 残基和 119 至 147 残基作为跨膜螺旋积极插入。使用反向遗传学，设计了一组 13 种 FMD 重组突变病毒，这些病毒在氨基酸残基 28 至 147 之间的关键氨基酸残基中具有非保守和丙氨酸取代。这些结构的任何改变都会干扰孔道活性以及该蛋白使内质网（ER）透钙的能力，并且对病毒复制是致命的。因此，FMDV 2B 成为包含两个不同孔域的第一个 viroporin 家族成员。口蹄疫病毒非结构蛋白 2B 能够将自身插入细胞膜以形成孔道。该孔道允许离子和小分子通过膜。在这项研究中，我们能够表明电流和小分子都能够通过 2B 形成的孔道。我们还首次发现了一种具有形成孔道的蛋白的病毒，该蛋白包含两个独立的功能孔道。通过对我们的 FMDV 感染性克隆进行突变，我们确定了两个孔中的任何一个突变都会导致无活力的病毒。这表明在 FMDV 感染过程中，两个孔形成功能都是独立需要的。
