Department of Biological Sciences, National University of Singapore, 117543, Singapore.
Department of Biological Sciences, National University of Singapore, 117543, Singapore; Temasek Life Sciences Laboratory, Singapore, 117604, Singapore.
Prog Biophys Mol Biol. 2019 May;143:5-12. doi: 10.1016/j.pbiomolbio.2018.12.006. Epub 2018 Dec 13.
Viruses are metastable macromolecular assemblies that toggle between multiple conformational states through molecular rearrangements that are critical for mediating viral host entry. Viruses respond to different host specific environmental cues to form disassembly intermediates for the eventual release of genomic material required for replication. Although static snapshots of these intermediates have been captured through structural techniques such as X-ray crystallography and cryo-EM, the mechanistic details of these conformational rearrangements underpinning viral metastability have been poorly understood. Amide hydrogen deuterium exchange mass spectrometry (HDXMS) is a powerful tool that measures hydrogen bonding propensities to probe changes in the dynamics of different macromolecular interactions. Chaotropic agents such as urea can be used to disrupt hydrogen bonds between different subunits, thereby ranking regions of the virus that are critical in maintaining viral stability. By controlled urea denaturation with HDXMS, we have identified specific loci in a Turnip Crinkle Virus (TCV) model showing increased deuterium exchange with even minimally disruptive concentrations of urea. These loci represent dynamic disassembly hotspots. These hotspots are predominantly present at the quaternary contacts at the 3-fold and 5-fold axes. This approach can be applied to detect vulnerabilities in virus icosahedral structures to uncover the molecular mechanism of viral disassembly.
病毒是亚稳态的大分子组装体，通过分子重排在多个构象状态之间切换，这对于介导病毒进入宿主至关重要。病毒会对不同的宿主特异性环境线索做出反应，形成用于最终释放复制所需基因组物质的解体中间体。尽管已经通过 X 射线晶体学和冷冻电镜等结构技术捕获了这些中间体的静态快照，但对支持病毒亚稳态的这些构象重排的机制细节了解甚少。酰胺氢氘交换质谱（HDXMS）是一种强大的工具，可测量氢键倾向，以探测不同大分子相互作用动力学的变化。变性剂如尿素可用于破坏不同亚基之间的氢键，从而确定在维持病毒稳定性方面至关重要的病毒区域。通过使用 HDXMS 进行受控尿素变性，我们在芜菁花叶病毒（TCV）模型中鉴定出了特定的位点，这些位点在即使是最小破坏浓度的尿素存在下，氘交换增加。这些位点代表动态解体热点。这些热点主要存在于 3 倍和 5 倍轴的四元接触处。这种方法可用于检测病毒二十面体结构的脆弱性，以揭示病毒解体的分子机制。
