Petazzi R A, Koikkarah A A, Tischler N D, Chiantia S
University of Potsdam, Institute of Biochemistry and Biology, Potsdam, Germany.
Fundación Ciencia & Vida, Laboratorio de Virología Molecular, Santiago, Chile.
J Virol. 2021 Feb 15;95(4). doi: 10.1128/JVI.01238-20. Epub 2020 Nov 25.
Hantaviruses are emerging pathogens that occasionally cause deadly outbreaks in the human population. While the structure of the viral envelope has been characterized with high precision, protein-protein interactions leading to the formation of new virions in infected cells are not fully understood yet. We use quantitative fluorescence microscopy (i.e., Number&Brightness analysis and fluorescence fluctuation spectroscopy) to monitor the interactions that lead to oligomeric spike complex formation in the physiological context of living cells. To this aim, we quantified protein-protein interactions for the glycoproteins Gn and Gc from Puumala and Hantaan orthohantaviruses in several cellular models. The oligomerization of each protein was analyzed in relation to subcellular localization, concentration, and the concentration of its interaction partner. Our results indicate that when expressed separately, Gn and Gc form respectively homo-tetrameric and homo-dimeric complexes, in a concentration-dependent manner. Site-directed mutations or deletion mutants showed the specificity of their homotypic interactions. When both glycoproteins were co-expressed, we observed in the Golgi apparatus clear indication of Gn-Gc interactions and the formation of Gn-Gc multimeric protein complexes of different sizes, while using various labeling schemes to minimize the influence of the fluorescent tags. Such large glycoprotein multimers may be identified as multiple Gn viral spikes interconnected via Gc-Gc contacts. This observation provides a possible first evidence for the initial assembly steps of the viral envelope, within this organelle, directly in living cells. In this work, we investigate protein-protein interactions that drive the assembly of the hantaviruses envelope. These emerging pathogens have the potential to cause deadly outbreaks in the human population. Therefore, it is important to improve our quantitative understanding of the viral assembly process in infected cells, from a molecular point of view. By applying advanced fluorescence microscopy methods, we monitored the formation of viral spike complexes in different cell types. Our data support a model for hantavirus assembly according to which viral spikes are formed via the clustering of hetero-dimers of the two viral glycoproteins Gn and Gc. Furthermore, the observation of large Gn-Gc hetero-multimers provide a possible first evidence for the initial assembly steps of the viral envelope, directly in the Golgi apparatus of living cells.
汉坦病毒是新兴病原体，偶尔会在人群中引发致命疫情。虽然病毒包膜的结构已得到高精度表征，但导致感染细胞中形成新病毒粒子的蛋白质 - 蛋白质相互作用尚未完全明确。我们使用定量荧光显微镜技术（即数量与亮度分析和荧光涨落光谱法）来监测在活细胞生理环境中导致寡聚刺突复合物形成的相互作用。为此，我们在几种细胞模型中对普马拉病毒和汉坦病毒的糖蛋白Gn和Gc的蛋白质 - 蛋白质相互作用进行了定量分析。分析了每种蛋白质的寡聚化与亚细胞定位、浓度及其相互作用伴侣浓度的关系。我们的结果表明，当单独表达时，Gn和Gc分别以浓度依赖的方式形成同型四聚体和同型二聚体复合物。定点突变或缺失突变体显示了它们同型相互作用的特异性。当两种糖蛋白共表达时，我们在高尔基体中清晰观察到Gn - Gc相互作用以及不同大小的Gn - Gc多聚体蛋白复合物的形成，同时使用了各种标记方案以尽量减少荧光标签的影响。这种大的糖蛋白多聚体可能被识别为通过Gc - Gc接触相互连接的多个Gn病毒刺突。这一观察结果为病毒包膜在该细胞器内直接在活细胞中的初始组装步骤提供了可能的首个证据。在这项工作中，我们研究了驱动汉坦病毒包膜组装的蛋白质 - 蛋白质相互作用。这些新兴病原体有可能在人群中引发致命疫情。因此，从分子角度加深我们对感染细胞中病毒组装过程的定量理解非常重要。通过应用先进的荧光显微镜方法，我们监测了不同细胞类型中病毒刺突复合物的形成。我们的数据支持一种汉坦病毒组装模型，即病毒刺突是通过两种病毒糖蛋白Gn和Gc的异源二聚体聚集形成的。此外，对大的Gn - Gc异源多聚体的观察为病毒包膜在活细胞高尔基体中的初始组装步骤提供了可能的首个证据。
