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

Abstract

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...

Influenza A M2 recruits M1 to the plasma membrane: a fluorescence fluctuation microscopy study

“…Therefore, in order to quantify protein-protein interactions directly at the PM of living cells, we performed fluorescence fluctuation spectroscopy experiments under physiological conditions. Such experimental approaches (i.e., sF(C)CS and (cc)N&B) provide information regarding the oligomeric state, surface concentration, hetero-interactions and dynamics in complex biological systems (51,52,(63)(64)(65)(66).…”

“…It is worth noting that the formation of very large multimers of M1-mEGFP in infected cells might be partially due to i) higher M1 concentrations at the PM or ii) the presence of unlabeled M1 molecules which more efficiently support protein-protein interactions. It was in fact reported that fluorescent viral proteins might not be able to oligomerize on a very large scale (65). Alternatively, other viral proteins or altered lipid metabolism (and, consequently, modification of PM composition) in infected cells might play a role and these possibilities are currently under investigation.…”