National Institute of Diagnostics and Vaccine Development in Infectious Disease, School of Life Sciences, Xiamen University, Xiamen 361005, People's Republic of China.
Protein Sci. 2013 Mar;22(3):314-26. doi: 10.1002/pro.2213. Epub 2013 Jan 17.
The hepatitis E virus (HEV) capsid protein has been demonstrated to be able to assemble into particles in vitro. However, this process and the mechanism of protein-protein interactions during particle assembly remain unclear. In this study, we investigated the assembly mechanism of HEV structural protein subunits, the capsid protein p239 (aa368-606), using analytical ultracentrifugation. It was the first to observe that the p239 can form particles in 4M urea as a result of supplementation with salt, including ammonium sulfate [(NH₄)₂SO₄], sodium sulfate (Na₂SO₄), sodium chloride (NaCl), and ammonium chloride (NH₄Cl). Interestingly, it is the ionic strength that determines the efficiency of promoting particle assembly. The assembly rate was affected by temperature and salt concentration. When (NH₄)₂SO₄ was used, assembling intermediates of p239 with sedimentation coefficient values of approximately 5 S, which were mostly dodecamers, were identified for the first time. A highly conserved 28-aa region (aa368-395) of p239 was found to be critical for particle assembly, and the hydrophobic residues Leu³⁷², Leu³⁷⁵, and Leu³⁹⁵ of p239 was found to be critical for particle assembly, which was revealed by site-directed mutagenesis. This study provides new insights into the assembly mechanism of native HEV, and contributes a valuable basis for further investigations of protein assembly by hydrophobic interactions under denaturing conditions.
戊型肝炎病毒 (HEV) 衣壳蛋白已被证明能够在体外组装成颗粒。然而，这个过程以及在颗粒组装过程中蛋白质-蛋白质相互作用的机制尚不清楚。在这项研究中，我们使用分析超速离心法研究了 HEV 结构蛋白亚基，衣壳蛋白 p239（aa368-606）的组装机制。这是首次观察到 p239 在补充盐（包括硫酸铵 [(NH₄)₂SO₄]、硫酸钠 (Na₂SO₄)、氯化钠 (NaCl) 和氯化铵 (NH₄Cl)）的情况下，在 4M 尿素中能够形成颗粒。有趣的是，是离子强度决定了促进颗粒组装的效率。组装速率受温度和盐浓度的影响。当使用 (NH₄)₂SO₄ 时，首次鉴定出 p239 的组装中间体具有约 5 S 的沉降系数值，这些中间体主要是十二聚体。发现 p239 高度保守的 28-aa 区域（aa368-395）对于颗粒组装至关重要，并且 p239 的疏水性残基 Leu³⁷²、Leu³⁷⁵ 和 Leu³⁹⁵ 对于颗粒组装至关重要，这是通过定点突变揭示的。这项研究为天然 HEV 的组装机制提供了新的见解，并为进一步研究变性条件下疏水性相互作用的蛋白质组装提供了有价值的基础。
