Liu Zheng, Guu Tom S Y, Cao Jianhao, Li Yinyin, Cheng Lingpeng, Tao Yizhi Jane, Zhang Jingqiang
Department of Biophysics, Health Science Centre, Peking University, Beijing, 100191 China.
Department of Biochemistry and Molecular Biophysics, Columbia University, New York, 10032 USA.
Biophys Rep. 2016;2(2):55-68. doi: 10.1007/s41048-016-0027-2. Epub 2016 Sep 2.
Virus 3D atomic structures provide insight into our understanding of viral life cycles and the development of antiviral drugs. X-ray crystallography and cryo-EM have been used to determine the atomic structure of viruses. However, limited availability of biological samples, biosafety issues due to virus infection, and sometimes inherent characteristics of viruses, pose difficulties on combining both methods in determining viral structures. These have made solving the high resolution structure of some medically important viruses very challenging. Here, we describe our recently employed protocols for determining the high-resolution structure of the virus-like particle of hepatitis E virus (HEV), a pathogen of viral hepatitis in human. These protocols include utilizing recombinant baculovirus system to generate sufficient amount of virus particles, single-particle cryo-EM to get an intermediate resolution structure as a phasing model, and X-ray crystallography for final atomic structure determination. Our protocols have solved the hepatitis E virus structure to the resolution of 3.5 Å. The combined methodology is generally applicable to other human infectious viruses.
病毒的三维原子结构有助于我们深入了解病毒生命周期以及抗病毒药物的研发。X射线晶体学和冷冻电镜已被用于确定病毒的原子结构。然而，生物样品的可获得性有限、病毒感染导致的生物安全问题以及病毒本身的一些固有特性，给结合这两种方法来确定病毒结构带来了困难。这些因素使得解析一些具有医学重要性的病毒的高分辨率结构极具挑战性。在此，我们描述了我们最近采用的用于确定戊型肝炎病毒（HEV）病毒样颗粒高分辨率结构的方案，HEV是人类病毒性肝炎的病原体。这些方案包括利用重组杆状病毒系统生成足够数量的病毒颗粒，采用单颗粒冷冻电镜获得中等分辨率结构作为相位模型，以及通过X射线晶体学确定最终的原子结构。我们的方案已将戊型肝炎病毒结构解析到3.5埃的分辨率。这种组合方法通常适用于其他人类感染性病毒。
