Structural Dynamics upon directed Virus Glycan interaction

原生质谱法（MS）在区分不同病毒组装状态和监测病毒稳定性方面非常有效。此外，这种配体结合介导的结构变化可以通过氢/氘交换（HDX） ms来监测。我们假设聚糖作为细胞表面的多价附着因子，可以触发病毒的构象转变。这些变化可以启动细胞摄取，为次级受体打开新的结合位点或改变稳定性，促进细胞内脱膜。最近的研究结果表明，聚糖与诺如病毒的结合是一个复杂的过程，涉及到结合位点的扩大，导致整个衣壳蛋白的进一步结构变化。由于人类诺如病毒已被MS很好地研究过，我们将使用该系统在天然MS中建立结合亲和力和稳定性测定，然后，我们将这些协议应用于小鼠诺如病毒（MNV），我们也可以研究感染性颗粒（Peters和Taube实验室）。我们还将研究多价聚糖模拟物的影响（Hartmann实验室）并参与配体筛选。人类诺如病毒将是建立HDX质谱的第一个目标，原生质谱研究将为HDX质谱分析提供信息，并提供最有希望的聚糖和浓度来探测结构变化。这两种技术将在不同的条件下进行，以比较颗粒的组装状态和基因组结合如何影响结合和结构转变。我们还将把我们的技术池应用于与唾液酸和硫酸肝素结合的默克尔细胞多瘤病毒（MCPyV, Blaum）和16型人乳头瘤病毒（HPV16, Schelhaas）。众所周知，HPV16经历了聚糖诱导的结构转变。聚糖诱导的MCPyV结构变化迄今尚不清楚，但硫酸肝素与HPV16的类似结合似乎是可能的。在所有情况下，我们都可以提供糖聚糖结合时病毒动力学的详细结构信息，这可以为抑制剂和诊断工具的设计提供信息。

Native mass spectrometry (MS) is very powerful in discriminating between different virus assembly states and monitoring virus stability. Additionally, binding affinities of viral assemblies to glycans can be derived by MS. Structural changes mediated by such ligand binding can be monitored by hydrogen/deuterium exchange (HDX) MS. We hypothesize that glycans, being multivalent attachment factors on cell surfaces, can trigger conformational transitions in viruses. These changes can prime for cellular uptake, open up new binding sites for secondary receptors or modify stability, facilitating intracellular uncoating. Recent findings indicate that glycan binding to noroviruses is a complex process involving widening of the binding site leading to further structural changes throughout the capsid protein. Since human noroviruses are well-studied by MS, we will use this system to establish binding affinity and stability assays in native MS. Then, we apply these protocols to murine noroviruses (MNV) where we can also study the infectious particle (Peters and Taube labs). We will also study the impact of multivalent glycan mimetics (Hartmann lab) and participate in ligand screens. Human noroviruses will be the first target to establish HDX MS. Native MS studies will inform HDX MS analysis and provide most promising glycans and concentrations to probe structural changes. Both techniques will be carried out under different conditions to compare how the assembly state and genome incorporation of the particles affect binding and structural transitions. We will also apply our pool of techniques to Merkel Cell Polyomavirus (MCPyV, Blaum) and human papillomavirus type 16 (HPV16, Schelhaas) binding to sialic acids and heparan sulfates. It is known that HPV16 undergoes glycan induced structural transitions. Glycan induced structural changes in MCPyV are so far unknown but appear possible for heparan sulfate binding by analogy with HPV16. In all cases, we can provide detailed structural information on virus dynamics upon glycan binding, which can inform design of inhibitors and diagnostic tools.