Role of trans to cis Transition in Viral Fusion Pore Dilation

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. Previous studies of fusion mediated by influenza A hemagglutinin (HA) revealed ∼2-5 nm pores that flickered before dilating to >10 nm. The mechanisms are unknown.Here we studied HA-mediated fusion pore dynamics using a novel single-pore assay, combined with computational simulations accessing extraordinarily long ms-s 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, use of nanodiscs limited the membrane contact areas and maximum pore sizes, better mimicking the initial phases of virus-endosome fusion. With wild type (WT) HA, fusion pores flickered about a mean pore size ∼1 nm. By contrast, fusion pores formed by GPI-anchored HA nucleated at half the WT rate and were significantly larger.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, but contrary to a common view, cis hairpins accumulated on the “viral” membrane, not the pore waist, due to the low mobility HA transmembrane domains. With GPI-HA the anchoring lipids were far more mobile and the trans-cis transition much accelerated. Once most hairpins had converted to cis, because apposing membranes were released the fusion pore dilated significantly.Our results suggest pore dilation requires the trans-cis transition. We hypothesize that this transition is accelerated in GPI-HA by the more mobile lipid anchor, explaining the larger observed pores.