TY - JOUR A1 - Petazzi, Roberto Arturo A1 - Koikkarah Aji, Amit A1 - Tischler, Nicole D. A1 - Chiantia, Salvatore T1 - Detection of envelope glycoprotein assembly from old world hantaviruses in the Golgi apparatus of living cells JF - Journal of virology N2 - 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. We used quantitative fluorescence microscopy (i.e., number and 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 coexpressed, we observed in the Golgi apparatus clear indication of GnGc 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 the possible first evidence for the initial assembly steps of the viral envelope within this organelle, and does so directly in living cells.
IMPORTANCE In this work, we investigate protein-protein interactions that drive the assembly of the hantavirus 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 the possible first evidence for the initial assembly steps of the viral envelope, directly in the Golgi apparatus of living cells. KW - fluorescence fluctuation microscopy KW - number and brightness KW - virus KW - assembly KW - fluorescence correlation spectroscopy KW - protein-protein KW - interaction KW - fluorescence microscopy KW - fluorescent image analysis Y1 - 2021 U6 - https://doi.org/10.1128/JVI.01238-20 SN - 1098-5514 VL - 95 IS - 4 PB - American Society for Microbiology CY - Baltimore, Md. ER - TY - JOUR A1 - Dunsing, Valentin A1 - Petrich, Annett A1 - Chiantia, Salvatore T1 - Multicolor fluorescence fluctuation spectroscopy in living cells via spectral detection JF - eLife N2 - Signaling pathways in biological systems rely on specific interactions between multiple biomolecules. Fluorescence fluctuation spectroscopy provides a powerful toolbox to quantify such interactions directly in living cells. Cross-correlation analysis of spectrally separated fluctuations provides information about intermolecular interactions but is usually limited to two fluorophore species. Here, we present scanning fluorescence spectral correlation spectroscopy (SFSCS), a versatile approach that can be implemented on commercial confocal microscopes, allowing the investigation of interactions between multiple protein species at the plasma membrane. We demonstrate that SFSCS enables cross-talk-free cross-correlation, diffusion, and oligomerization analysis of up to four protein species labeled with strongly overlapping fluorophores. As an example, we investigate the interactions of influenza A virus (IAV) matrix protein 2 with two cellular host factors simultaneously. We furthermore apply raster spectral image correlation spectroscopy for the simultaneous analysis of up to four species and determine the stoichiometry of ternary IAV polymerase complexes in the cell nucleus. KW - fluorescence KW - optical microscopy KW - virus assembly KW - protein-protein KW - interactions KW - diffusion KW - Viruses Y1 - 2021 U6 - https://doi.org/10.7554/eLife.69687 SN - 2050-084X VL - 10 PB - eLife Sciences Publications CY - Cambridge ER -