Nanoscale Electrical and Coarse-grained Molecular Dynamics Studies of Influenza Hemagglutinin-mediated Membrane Fusion Pores

Abstract

Fusion of viral and host membranes is a key step during infection by membrane-enclosed viruses. The fusion pore plays a critical role, and must dilate to release the viral genome. Prior studies of fusion mediated by influenza A hemagglutinin (HA) revealed ~2-5 nm pores that flickered before dilating to >10 nm. The mechanisms involved are unknown. Here we studied HA-mediated fusion pore dynamics using a novel single-pore assay (supported by a novel, robust, single-cell optical assay for fusion between HA-expressing cells and nanodiscs), combined with computational simulations accessing extraordinarily long (ms) timescales. We measured pores between HA-expressing fibroblasts and bilayer nanodiscs. From pore currents we infer pore size with millisecond time resolution. Unlike previous in vitro studies, the use of nanodiscs limited the membrane contact areas and maximum pore sizes, better mimicking the initial phases of virus-endosome fusion. In wild-type (WT) HA-mediated fusion pores, pores flickered about a mean pore size ~1.7 nm. In contrast, fusion pores formed by GPI-anchored HA nucleated at less than half the WT rate; results were consistent with earlier findings that showed that while GPI-HA pores stabilize at larger initial conductances than WT, they were not able to enlarge beyond their initial size. We developed radically coarse-grained, explicit lipid molecular dynamics simulations of the fusion pore reconstituted with post-fusion, trans HA hairpins. With WT HA, fusion pores were small, similar to experiment. Over time hairpins gradually converted from trans to cis. With lipid-anchored HA, the trans → cis transition was much accelerated. Once most hairpins had converted to cis, because apposing membranes were released, the fusion pore was able to dilate to sizes close to protein-free. Additionally, in crowded simulations with HA densities approximating those found in HA clusters, we found that HA aggregation, promoted by TMD-TMD interactions, delayed fusion pore dilation by inhibiting the trans → cis transition. Our results suggest that pore dilation requires the trans → cis transition. We hypothesize that this transition is accelerated in GPI-HA by the more mobile lipid anchor, and may explain the larger observed nascent fusion pores