Membrane Remodeling in Viral Infection and Viral Assembly

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

Investigators

Linked publications & trials

Abstract

The first project in this report focuses on our studies of the isolated FP of influenza virus both computationally and experimentally. Our recent discovery of the aggregation of FP was investigated through molecular dynamics simulations in four different membrane compositions to examine lipid-mediated clustering and poration free energy. The formation of highly stable antiparallel FP dimers was confirmed and found to be primarily stabilized by peptide-peptide interactions, and was largely insensitive to membrane composition. However, lipid sorting under the FP dimer was strongly dependent on the lipid spontaneous curvature, with a positive spontaneous curvature-generating lipid depleted, and negative spontaneous curvature-generating lipids enriched under the dimer. In simulations of ten FP dimers, the cholesterol-containing membrane promoted higher order clustering, seen as formation of tightly bound tetramers and linear arrangements of dimers that were not observed in the other membrane compositions. These tightly bound tetramers were associated with reduced poration efficiency compared to more loosely associated configurations, consistent with earlier experimental work showing that cholesterol partially inhibits poration by FP. These findings demonstrate how lipids with different curvature-generating propensities modulate peptide aggregation and pore formation, and may have broader implications for other enveloped viruses, such as SARS-CoV-2, where similar fusion peptide clustering has been observed. Complementing these computational studies, experiments involving labeled FP interacting with a membrane model system (supported lipid bilayers) indicate lipid compositional differences in the surface distribution of HA in time. Cholesterol:POPC membranes display a heterogeneous distribution of FP that differs from that observed in pure POPC membranes. These differences recapitulate our previously published observations indicating differences between FP interacting with cholesterol:POPC and POPC vesicles (Rice et al. 2022). In the second project of this report, molecular dynamic simulations (MDS) and single-molecule localization microscopy (SMLM) were used to test the electrostatic hypothesis on the relationship between HA and PIP2 We began by mutating the CTD of HA for charge only (HARE, HARREQ), palmitoylation sites only (HAMAY), or a combination thereof (HAREMAY). In all cases, HA clustering density at the PM was significantly reduced with the largest reduction occurring in double mutated CTDs (HAREMAY). These double mutant HA (HAREMAY) clusters were also found to have larger circularities and perimeters, implying a structural change to the cluster properties. PIP2 co-clustering was also affected, revealing more available free PIP2 at the PM in the double mutant case (HAREMAY). To confirm these observations were directly related to the CTD, we measured no changes to the cluster properties from HA with mutations to the transmembrane domain. By mutating the CTD of both charge and palmitoylation sites, we show a decrease in the co-clustering properties of HA and PIP2. We define this mechanism of interaction as having two parts, (1) electrostatic and (2) hydrophobic.