Kim Jonghwa, Zhang Xing, Centonze Victoria E, Bowman Valorie D, Noble Simon, Baker Timothy S, Nibert Max L
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.
J Virol. 2002 Dec;76(23):12211-22. doi: 10.1128/jvi.76.23.12211-12222.2002.
The reovirus core particle is a molecular machine that mediates synthesis, capping, and export of the viral plus strand RNA transcripts. Its assembly and structure-function relationships remain to be well understood. Following the lead of previous studies with other Reoviridae family members, most notably orbiviruses and rotaviruses, we used recombinant baculoviruses to coexpress reovirus core proteins lambda1, lambda2, and sigma2 in insect cells. The resulting core-like particles (CLPs) were purified and characterized. They were found to be similar to cores with regard to their sizes, morphologies, and protein compositions. Like cores, they could also be coated in vitro with the two major outer-capsid proteins, micro 1 and sigma3, to produce virion-like particles. Coexpression of core shell protein lambda1 and core nodule protein sigma2 was sufficient to yield CLPs that could withstand purification, whereas expression of lambda1 alone was not, indicating a required role for sigma2 as a previous study also suggested. In addition, CLPs that lacked lambda2 (formed from lambda1 and sigma2 only) could not be coated with micro 1 and sigma3, indicating a required role for lambda2 in the assembly of these outer-capsid proteins into particles. To extend the use of this system for understanding the core and its assembly, we addressed the hypothesis that the hydrophilic amino-terminal region of lambda1, which adopts an extended arm-like conformation around each threefold axis in the reovirus core crystal structure, plays an important role in assembling the core shell. Using a series of lambda1 deletion mutants, we showed that the amino-terminal 230 residues of lambda1, including its zinc finger, are dispensable for CLP assembly. Residues in the 231-to-259 region of lambda1, however, were required. The core crystal structure suggests that residues in the 231-to-259 region are necessary because they affect the interaction of lambda1 with the threefold and/or fivefold copies of sigma2. An effective system for studies of reovirus core structure, assembly, and functions is hereby established.
呼肠孤病毒核心颗粒是一种分子机器，介导病毒正链RNA转录本的合成、加帽和输出。其组装以及结构与功能的关系仍有待深入了解。遵循先前对其他呼肠孤病毒科成员（最显著的是环状病毒和轮状病毒）研究的思路，我们使用重组杆状病毒在昆虫细胞中共表达呼肠孤病毒核心蛋白λ1、λ2和σ2。对产生的类核心颗粒（CLPs）进行了纯化和表征。发现它们在大小、形态和蛋白质组成方面与核心颗粒相似。与核心颗粒一样，它们在体外也可以被两种主要的外衣壳蛋白μ1和σ3包裹，从而产生病毒样颗粒。核心壳蛋白λ1和核心结节蛋白σ2的共表达足以产生能够经受纯化的CLPs，而单独表达λ1则不行，这表明σ2具有先前研究也表明的必需作用。此外，缺乏λ2的CLPs（仅由λ1和σ2形成）不能被μ1和σ3包裹，这表明λ2在这些外衣壳蛋白组装成颗粒的过程中具有必需作用。为了扩展该系统在理解核心颗粒及其组装方面的应用，我们探讨了以下假设：λ1的亲水性氨基末端区域在呼肠孤病毒核心晶体结构中围绕每个三重轴采用延伸的臂状构象，在核心壳的组装中起重要作用。通过一系列λ1缺失突变体，我们表明λ1的氨基末端230个残基（包括其锌指）对于CLP组装是可有可无的。然而，λ1的231至259区域的残基是必需的。核心晶体结构表明，231至259区域的残基是必需的，因为它们影响λ1与σ2的三重和/或五重拷贝的相互作用。由此建立了一个用于研究呼肠孤病毒核心结构、组装和功能的有效系统。
