Real-Hohn Antonio, Groznica Martin, Löffler Nadine, Blaas Dieter, Kowalski Heinrich
Center for Medical Biochemistry, Max Perutz Labs, Vienna Biocenter, Medical University of Vienna, Vienna, Austria.
Front Microbiol. 2020 Jun 26;11:1442. doi: 10.3389/fmicb.2020.01442. eCollection 2020.
Thermal shift assays measure the stability of macromolecules and macromolecular assemblies as a function of temperature. The Particle Stability Thermal Release Assay (PaSTRy) of picornaviruses is based on probes becoming strongly fluorescent upon binding to hydrophobic patches of the protein capsid (e.g., SYPRO Orange) or to the viral RNA genome (e.g., SYTO-82) that become exposed upon heating virus particles. PaSTRy has been exploited for studying the stability of viral mutants, viral uncoating, and the effect of capsid-stabilizing compounds. While the results were usually robust, the thermal shift assay with SYPRO Orange is sensitive to surfactants and EDTA and failed at least to correctly report the effect of excipients on an inactivated poliovirus 3 vaccine. Furthermore, interactions between the probe and capsid-binding antivirals as well as mutual competition for binding sites cannot be excluded. To overcome these caveats, we assessed differential scanning fluorimetry with a nanoDSF device as a label-free alternative. NanoDSF monitors the changes in the intrinsic tryptophan fluorescence (ITF) resulting from alterations of the 3D-structure of proteins as a function of the temperature. Using rhinovirus A2 as a model, we demonstrate that nanoDFS is well suited for recording the temperature-dependence of conformational changes associated with viral uncoating with minute amounts of sample. We compare it with orthogonal methods and correlate the increase in viral RNA exposure with PaSTRy measurements. Importantly, nanoDSF correctly identified the thermal stabilization of RV-A2 by pleconaril, a prototypic pocket-binding antiviral compound. NanoDFS is thus a label-free, high throughput-customizable, attractive alternative for the discovery of capsid-binding compounds impacting on viral stability.
热位移分析可测量大分子和大分子组装体的稳定性随温度的变化。微小核糖核酸病毒的颗粒稳定性热释放分析（PaSTRy）基于这样的原理：探针在与蛋白质衣壳的疏水区域（如SYPRO Orange）或病毒RNA基因组（如SYTO - 82）结合时会发出强烈荧光，而这些区域在加热病毒颗粒时会暴露出来。PaSTRy已被用于研究病毒突变体的稳定性、病毒脱壳以及衣壳稳定化合物的作用。虽然结果通常很可靠，但使用SYPRO Orange的热位移分析对表面活性剂和EDTA敏感，并且至少未能正确报告辅料对灭活脊髓灰质炎病毒3疫苗的影响。此外，不能排除探针与衣壳结合抗病毒药物之间的相互作用以及对结合位点的相互竞争。为了克服这些问题，我们评估了使用纳米差示扫描荧光法（nanoDSF）设备作为无标记替代方法。nanoDSF监测蛋白质三维结构变化导致的内在色氨酸荧光（ITF）随温度的变化。以鼻病毒A2为模型，我们证明nanoDFS非常适合记录与病毒脱壳相关的构象变化的温度依赖性，所需样品量极少。我们将其与正交方法进行比较，并将病毒RNA暴露的增加与PaSTRy测量结果相关联。重要的是，nanoDSF正确地识别了pleconaril（一种典型的口袋结合抗病毒化合物）对RV - A2的热稳定作用。因此，nanoDSF是一种无标记、高通量可定制的、有吸引力的替代方法，可用于发现影响病毒稳定性的衣壳结合化合物。
