AbstractHantaviruses 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, the protein-protein interactions leading to the formation of new virions in infected cells are not fully understood yet. In this work, we use quantitative fluorescence microscopy (i.e. Number&Brightness analysis and fluorescence fluctuation spectroscopy) to quantify the interactions that lead to oligomeric spike complex formation in the physiological context of living cells. To this aim, we have analyzed proteins from Puumala and Hantaan orthohantaviruses in several cellular models. For the first time, we quantified the oligomerization state of each protein in relation to its subcellular localization, concentration and the concentration of its interaction partner. Our results indicate that when expressed separately, both glycoproteins can form homo-multimers in a concentration-dependent manner. Fluorescence fluctuation analysis was applied to prove that Gc:Gc contacts observed on virions are also relevant for Gc-Gc interactions in living cells, in the absence of Gn. Furthermore, we proved that the membrane-distal lobes of Gn are not necessary for Gn homo-multimerization. In cells co-expressing both glycoproteins, we observe clear indication of Gn-Gc interactions and the formation of protein complexes with different sizes, while using various labelling schemes to minimize the influence of the fluorescent tags. Our data are compatible with an assembly model according to which hantavirus spikes are formed via the assembly of Gn-Gc hetero-dimers. Furthermore, our results indicate the interconnection of large Gn-Gc hetero-multimers in the Golgi apparatus. Such large glycoprotein multimers may be identified as multiple interacting viral spikes and provide a possible first evidence for the initial assembly steps of the viral envelope, within this organelle, directly in living cells.