nanoDSF:In vitroLabel-Free Method to Monitor Picornavirus Uncoating and Test Compounds Affecting Particle Stability
nanoDSF:In vitroLabel-Free Method to Monitor Picornavirus Uncoating and Test Compounds Affecting Particle Stability
复制标题
DOI：
10.3389/fmicb.2020.01442
复制
发表时间：
2020-06-26
影响因子：
5.2
通讯作者：
Kowalski, Heinrich
中科院分区：
文献类型：
--
作者：
Real-Hohn, Antonio;Groznica, Martin;Kowalski, Heinrich
热位移测定测量作为温度的函数的大分子和大分子组装体的稳定性。小核糖核酸病毒的颗粒稳定性热释放测定（PaSTRy）是基于探针在与蛋白质衣壳的疏水补丁（例如，SYPRO橙子）或病毒RNA基因组（例如，SYTO-82），在加热病毒颗粒时暴露。PaSTRy已被用于研究病毒突变体的稳定性、病毒脱壳和稳定caplets的化合物的作用。虽然结果通常具有耐用性，但SYPRO橙子热位移试验对表面活性剂和EDTA敏感，至少未能正确报告辅料对灭活脊髓灰质炎病毒3型疫苗的影响。此外，不能排除探针和captain结合抗病毒剂之间的相互作用以及对结合位点的相互竞争。为了克服这些注意事项，我们评估了差示扫描荧光法与nanoDSF设备作为一个无标记的替代品。NanoDSF监测由蛋白质的3D结构改变引起的内在色氨酸荧光（ITF）的变化作为温度的函数。使用鼻病毒A2作为模型，我们证明了nanoDFS非常适合于记录与微量样品的病毒脱壳相关的构象变化的温度依赖性。我们将其与正交方法进行比较，并将病毒RNA暴露量的增加与PaSTRy测量结果相关联。重要的是，nanoDSF正确地鉴定了通过普来那利（一种原型口袋结合抗病毒化合物）对RV-A2的热稳定作用。因此，NanoDFS是用于发现影响病毒稳定性的captain结合化合物的无标记、高通量定制、有吸引力的替代方案。
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.