Detection of Envelope Glycoprotein Assembly from Old-World Hantaviruses in the Golgi Apparatus of Living Cells.

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.