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. IMPORTANCE 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.

Hantaviruses are enveloped viruses that possess a tri-segmented, negative-sense RNA genome. The viral S-segment encodes the multifunctional nucleocapsid protein (N), which is involved in genome packaging, intracellular protein transport, immunoregulation, and several other crucial processes during hantavirus infection. In this study, we generated fluorescently tagged N protein constructs derived from Puumalavirus (PUUV), the dominant hantavirus species in Central, Northern, and Eastern Europe. We comprehensively characterized this protein in the rodent cell line CHO-K1, monitoring the dynamics of N protein complex formation and investigating co-localization with host proteins as well as the viral glycoproteins Gc and Gn. We observed formation of large, fibrillar PUUV N protein aggregates, rapidly coalescing from early punctate and spike-like assemblies. Moreover, we found significant spatial correlation of N with vimentin, actin, and P-bodies but not with microtubules. N constructs also co-localized with Gn and Gc albeit not as strongly as the glycoproteins associated with each other. Finally, we assessed oligomerization of N constructs, observing efficient and concentration-dependent multimerization, with complexes comprising more than 10 individual proteins.

13Hantaviruses are emerging pathogens that occasionally cause deadly outbreaks in the human 14 population. While the structure of the viral envelope has been characterized with high precision, 15 the protein-protein interactions leading to the formation of new virions in infected cells are not 16 fully understood yet. In this work, we use quantitative fluorescence microscopy (i.e. 17 Number&Brightness analysis and fluorescence fluctuation spectroscopy) to quantify the 18 interactions that lead to oligomeric spike complex formation in the physiological context of 19 living cells. To this aim, we have analyzed proteins from Puumala and Hantaan 20 orthohantaviruses in several cellular models. For the first time, we quantified the 21 oligomerization state of each protein in relation to its subcellular localization, concentration 22 and the concentration of its interaction partner. Our results indicate that when expressed 23 separately, both glycoproteins can form homo-multimers in a concentration-dependent manner. 24Fluorescence fluctuation analysis was applied to prove that Gc:Gc contacts observed on virions 25 are also relevant for Gc-Gc interactions in living cells, in the absence of Gn. Furthermore, we 26 proved that the membrane-distal lobes of Gn are not necessary for Gn homo-multimerization. 27In cells co-expressing both glycoproteins, we observe clear indication of Gn-Gc interactions 28 and the formation of protein complexes with different sizes, while using various labelling 29 schemes to minimize the influence of the fluorescent tags. Our data are compatible with an 30 assembly model according to which hantavirus spikes are formed via the assembly of Gn-Gc 31 hetero-dimers. Furthermore, our results indicate the interconnection of large Gn-Gc hetero-32 multimers in the Golgi apparatus. Such large glycoprotein multimers may be identified as 33 multiple interacting viral spikes and provide a possible first evidence for the initial assembly 34 steps of the viral envelope, within this organelle, directly in living cells. 35 36 Hantaviruses (HV) are single-stranded, negative sense RNA viruses belonging to the 37 Hantaviridae family of the order Bunyavirales. While usually infecting rodents, insectivorous 38 mammals or bats (1), these emerging pathogens can occasionally cause deadly outbreaks in the 39 human population (2). Their segmented genome encodes at least a nucleocapsid protein, a 40 RNA-dependent RNA polymerase and a glycoprotein precursor (GPC) (3). The assembly of 41 HV particles in infected cells is a complex process that relies on the precise spatial and temporal 42 organization of several viral proteins. The specific molecular mechanisms driving protein-43 protein interactions in this context are not fully characterized yet. 44 While negative-stranded RNA viruses typically assemble and bud at the plasma membrane (4, 45 5), Bunyavirales are mostly characterized by intracellular maturation (6). In all HVs, two 46 glycoproteins (GPs) form surface spikes and are derived from the GPC encoded in t...

Hantaviruses are enveloped viruses that possess a tri-segmented, negative-sense RNA genome. The viral S-segment encodes the multifunctional nucleocapsid protein (N), which is involved in genome packaging, intracellular protein transport, immunoregulation and several other crucial processes during hantavirus infection. In this study we have generated fluorescently tagged N protein constructs derived from Puumalavirus, the dominant hantavirus species in Central, Northern and Eastern Europe. We have comprehensively characterized this protein in the rodent cell line CHO-K1, monitoring the dynamics of N protein complex formation and investigating co-localization with host proteins as well as the viral glycoproteins Gc and Gn. We found a significant spatial correlation of N with vimentin, actin and P-bodies, but not with microtubules. N constructs also co-localized with Gn and Gc, albeit not as strong as the glycoproteins associated with each other. Moreover, we as-sessed oligomerization of N constructs, observing efficient and concentration-dependent multi-merization, with complexes comprising more than 10 individual proteins.