Department of Microbiology and Plant Pathology, University of California, Riverside, Riverside, California, USA.
Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, California, USA.
J Virol. 2020 Mar 31;94(8). doi: 10.1128/JVI.01794-19.
Viral capsids are dynamic assemblies that undergo controlled conformational transitions to perform various biological functions. The replication-derived four-molecule RNA progeny of (BMV) is packaged by a single capsid protein (CP) into three types of morphologically indistinguishable icosahedral virions with T=3 quasisymmetry. Type 1 (B1) and type 2 (B2) virions package genomic RNA1 and RNA2, respectively, while type 3 (B3+4) virions copackage genomic RNA3 (B3) and its subgenomic RNA4 (sgB4). In this study, the application of a robust -mediated transient expression system allowed us to assemble each virion type separately Experimental approaches analyzing the morphology, size, and electrophoretic mobility failed to distinguish between the virion types. Thermal denaturation analysis and protease-based peptide mass mapping experiments were used to analyze stability and the conformational dynamics of the individual virions, respectively. The crystallographic structure of the BMV capsid shows four trypsin cleavage sites (K, R, K, and K on the CP subunits) exposed on the exterior of the capsid. Irrespective of the digestion time, while retaining their capsid structural integrity, B1 and B2 released a single peptide encompassing amino acids 2 to 8 of the N-proximal arginine-rich RNA binding motif. In contrast, B3+4 capsids were unstable with trypsin, releasing several peptides in addition to the peptides encompassing four predicted sites exposed on the capsid exterior. These results, demonstrating qualitatively different dynamics for the three types of BMV virions, suggest that the different RNA genes they contain may have different translational timing and efficiency and may even impart different structures to their capsids. The majority of viruses contain RNA genomes protected by a shell of capsid proteins. Although crystallographic studies show that viral capsids are static structures, accumulating evidence suggests that, in solution, virions are highly dynamic assemblies. The three genomic RNAs (RNA1, -2, and -3) and a single subgenomic RNA (RNA4) of Brome mosaic virus (BMV), an RNA virus pathogenic to plants, are distributed among three physically homogeneous virions. This study examines the thermal stability by differential scanning fluorimetry (DSF) and capsid dynamics by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analyses following trypsin digestion of the three virions assembled separately using the -mediated transient expression approach. The results provide compelling evidence that virions packaging genomic RNA1 and -2 are distinct from those copackaging RNA3 and -4 in their stability and dynamics, suggesting that RNA-dependent capsid dynamics play an important biological role in the viral life cycle.
病毒衣壳是动态组装体，通过受控构象转变来执行各种生物学功能。（BMV）的复制衍生的四分子 RNA 前体由单个衣壳蛋白 (CP) 包装成三种形态上无法区分的具有 T=3 准对称性的二十面体病毒。类型 1 (B1) 和类型 2 (B2) 病毒分别包装基因组 RNA1 和 RNA2，而类型 3 (B3+4) 病毒共包装基因组 RNA3 (B3) 和其亚基因组 RNA4 (sgB4)。在这项研究中，应用强大的瞬时表达系统允许我们分别组装每种病毒类型。分析形态、大小和电泳迁移率的实验方法未能区分病毒类型。热变性分析和基于蛋白酶的肽质量图谱实验分别用于分析单个病毒的稳定性和构象动力学。BMV 衣壳的晶体结构显示四个胰蛋白酶切割位点（CP 亚基上的 K、R、K 和 K）暴露在衣壳的外部。无论消化时间如何，B1 和 B2 都保持其衣壳结构完整性，释放一个包含 N 近端富含精氨酸的 RNA 结合基序的氨基酸 2 到 8 的单个肽。相比之下，B3+4 衣壳对胰蛋白酶不稳定，除了释放包含衣壳外部四个预测暴露位点的肽之外，还释放了几个肽。这些结果表明，三种类型的 BMV 病毒粒子表现出不同的动力学特性，这表明它们所含的不同 RNA 基因可能具有不同的翻译时间和效率，甚至可能赋予它们不同的结构。大多数病毒含有由衣壳蛋白保护的 RNA 基因组。尽管晶体学研究表明病毒衣壳是静态结构，但越来越多的证据表明，在溶液中，病毒粒子是高度动态的组装体。Brome mosaic virus (BMV) 的三个基因组 RNA（RNA1、-2 和 -3）和一个单亚基因组 RNA（RNA4）是一种致病植物的 RNA 病毒，分布在三种物理上均匀的病毒中。本研究通过胰蛋白酶消化后使用瞬时表达方法分别组装三种病毒粒子，通过差示扫描荧光法（DSF）分析和基质辅助激光解吸电离飞行时间（MALDI-TOF）分析研究热稳定性。结果提供了令人信服的证据，证明包装基因组 RNA1 和 -2 的病毒粒子在稳定性和动力学方面与共包装 RNA3 和 -4 的病毒粒子不同，这表明 RNA 依赖性衣壳动力学在病毒生命周期中发挥着重要的生物学作用。
