Orthohantaviruses are tri-segmented negative-sense RNA viruses that can cause severe pathologies in humans. Currently, limited information exists on the molecular interactions driving orthohantavirus assembly in infected cells. Specifically, it is not clear how its glycoproteins (i.e., Gn and Gc) interact with other viral or host molecules. In this study, we use one- and two-color Number and Brightness fluorescence microscopy approaches to quantitatively characterize the interactions between orthohantavirus glycoproteins and the nucleoprotein in transfected cells. Our results indicate that orthohantavirus nucleoprotein homo-interactions are strongly affected by the host environment. Furthermore, we report evidence of Gc–nucleoprotein interactions, based on (i) the high fluorescence cross-correlation between these two proteins and (ii) the increased Gc-Gc interactions observed in the presence of nucleoprotein. Finally, experiments on a Gc deletion mutant suggest that the observed protein–protein interactions are mediated by the cytoplasmic tail of Gc. In conclusion, this study provides new insights into the role of the interactions between orthohantavirus glycoproteins and nucleoprotein in the context of viral assembly.

Hepatitis C virus (HCV) genome codes for various proteins essential to its replication cycle. Among these, the core protein (HCC) forms the capsid via organised multimerization, thereby guiding viral assembly. This process depends on the dynamic localisation of HCC between the endoplasmic reticulum bilayer membrane and the lipid droplet monolayer. While past studies have examined the role of some HCC structural properties and other viral and host proteins in the assembly process, the contribution of lipid molecules was not thoroughly investigated yet. Since specific lipids are upregulated during HCV infection, it is reasonable that these molecules might play an important role for virus fitness and infection progression. In this study, we have addressed this topic and, specifically, how HCC-lipid interactions might impact HCC-HCC interactions. Using in vitro models and quantitative fluorescence microscopy techniques, we investigated HCC binding affinity to lipid membranes, multimerization and lateral diffusivity. Our results reveal the effect of HCC interactions with anionic phospholipids (PLs) on the binding to monolayers and bilayers. Furthermore, our analysis shows that PI(4)P enhances HCC multimerization and, therefore, might drive lateral assembly for capsid formation. These results not only provide insights into the lipid-driven regulation of HCV assembly and identify anionic PLs as potential modulators of the viral replications cycle, but also highlight a previously unrecognised role of PL composition in HCC-membrane interaction. Significance: Hepatitis C virus (HCV) remains a major global health challenge and is associated with substantial mortality. In certain regions, HCV infection reaches epidemic proportions. Despite its prevalence, targeted and cost-effective therapeutic options remain limited, largely due to an incomplete understanding of the viral life cycle. A critical - yet poorly characterized - step in this cycle is the assembly of the HCV core (HCC) protein into the viral capsid, which is essential for replication. Here, we use quantitative fluorescence microscopy to investigate the role of specific lipid species in HCC assembly on model membranes. Our findings show that distinct lipid environments modulate HCC assembly and that HCC, in turn, induces measurable changes in the biophysical properties of the host membrane.

Hepatitis C virus (HCV) genome codes for various proteins essential to its replication cycle. Among these, the core protein (HCC) forms the capsid via organised multimerization, thereby guiding viral assembly. This process depends on the dynamic localisation of HCC between the endoplasmic reticulum bilayer membrane and the lipid droplet monolayer. While past studies have examined the role of some HCC structural properties and other viral and host proteins in the assembly process, the contribution of lipid molecules was not thoroughly investigated yet. Since specific lipids are upregulated during HCV infection, it is reasonable that these molecules might play an important role for virus fitness and infection progression. In this study, we have addressed this topic and, specifically, how HCC-lipid interactions might impact HCC-HCC interactions. Using in vitro models and quantitative fluorescence microscopy techniques, we investigated HCC binding affinity to lipid membranes, multimerization and lateral diffusivity. Our results reveal the effect of HCC interactions with anionic phospholipids (PLs) on the binding to monolayers and bilayers. Furthermore, our analysis shows that PI(4)P enhances HCC multimerization and, therefore, might drive lateral assembly for capsid formation. These results not only provide insights into the lipid-driven regulation of HCV assembly and identify anionic PLs as potential modulators of the viral replications cycle, but also highlight a previously unrecognised role of PL composition in HCC-membrane interaction.

Cellular membranes are composed of lipids typically organized in a double-leaflet structure. Interactions between these two leaflets – often referred to as interleaflet coupling – play a crucial role in various cellular processes. Despite extensive study, the mechanisms governing such interactions remain incompletely understood. Here, we investigate the effects of interleaflet coupling from a specific point of view, i.e. by comparing diffusive dynamics in bilayers and monolayers, focusing on potential lipid-specific interactions between opposing leaflets. Through quantitative fluorescence microscopy techniques, we characterize lipid diffusion and mean molecular area in monolayers and bilayers composed of different lipids. Our results suggest that the observed decrease in bilayer lipid diffusion compared to monolayers depends on lipid identity. Furthermore, our analysis suggests that lipid acyl chain structure and spatial configuration at the bilayer may strongly influence interleaflet interactions and dynamics in bilayers. These findings provide insights into the role of lipid structure in mediating interleaflet coupling and underscore the need for further experimental investigations to elucidate the underlying mechanisms.

Recent advances in hydrodynamic theory have revealed the severe effect of periodic boundary conditions (PBCs) on the diffusive dynamics of lipid membranes in molecular dynamics simulations. Even when accounting for PBC effects, the corrected lipid diffusion coefficients often severely overshoot the experimental estimates. Here, we investigate the underlying reasons for the exaggerated dynamics, and suggest potential ways for improvement. To this end, we examine the diffusion of four lipid types in both bilayers and monolayers using the CHARMM36 force field. We account for PBC effects using the full hydrodynamic treatment: for bilayers we use non-equilibrium simulations to extract the interleaflet friction parameter used in the correction; whereas monolayer hydrodynamics are treated by setting this parameter to zero. Our results suggest that the dynamics of bilayers are too fast, even if interleaflet friction is accounted for. However, the change of the water model to OPC leads to an excellent agreement with experiments. For monolayers, the dynamics with the TIP3P water model agree well with experiments, whereas they are undershot with OPC. As OPC and TIP3P differ in both shear viscosity and surface tension, we develop two new mass-scaled water models to clarify the roles of the thermodynamic and kinetic properties of the water model on lipid dynamics. Our results indicate that both of these quantities play a major role in lipid dynamics. Moreover, it seems that the accurate description of diffusion in both lipid bilayers and monolayers cannot be accounted for by changes in the water model alone, but likely also requires modifications in the lipid model.