Membrane Permeability Changes at Early Stages of Influenza Hemagglutinin-Mediated Fusion

Abstract

While biological membrane fusion is classically defined as the leak-free merger of membranes and contents, leakage is a finding in both experimental and theoretical studies. The fusion stages, if any, that allow membrane permeation are uncharted. In this study we monitored membrane ionic permeability at early stages of fusion mediated by the fusogenic protein influenza hemagglutinin (HA). HAb2 cells, expressing HA on their plasma membrane, fused with human red blood cells, cultured liver cells PLC/PRF/5, or planar phospholipid bilayer membranes. With a probability that depended upon the target membrane, an increase of the electrical conductance of the fusing membranes (leakage) by up to several nS was generally detected. This leakage was recorded at the initial stages of fusion, when fusion pores formed. This leakage usually accompanied the "flickering" stage of the early fusion pore development. As the pore widened, the leakage reduced; concomitantly, the lipid exchange between the fusing membranes accelerated. We conclude that during fusion pore formation, HA locally and temporarily increases the permeability of fusing membranes. Subsequent rearrangement in the fusion complex leads to the resealing of the leaky membranes and enlargement of the pore.

Detection of Proton Movement Directly across Viral Membranes To Identify Novel Influenza Virus M2 Inhibitors

“…The M2-VLP assay described here provides a safe, rapid, and robust biochemical format for discovering novel influenza virus inhibitors using cell-free HTS campaigns. Our work also provides, to the best of our knowledge, the first measurements of membrane potential directly across viral membranes and the first evidence that viral membranes are impermeable to ions and so may enable a better understanding of the electrochemical and biophysical properties of viral membranes (19,24,25,39), which have been underexplored areas of virology.…”

“…Taken together, these data demonstrate the functionality of M2 ion channels in VLPs and validate their utility as a noninfectious, cell-free, in vitro biochemical platform capable of assessing M2 ion channel activity. The ability of VLPs to maintain a membrane potential also demonstrates for the first time that lipid viral membranes are structurally intact and impermeable to ions, a matter of previous uncertainty (23), and offers the possibility that membrane potential itself, rather than just pH changes, could play a role in some parts of the virus life cycle (24,25).…”

Cardiomyocyte deletion of mitofusin-1 leads to mitochondrial fragmentation and improves tolerance to ROS-induced mitochondrial dysfunction and cell death

“…Nevertheless, there is experimental evidence to suggest that fusion may involve the formation of holes on membranes (16). For example, it has been observed that during the early steps of hemagglutinin-induced fusion the membranes exhibit a transient increase in permeability that is resolved later as fusion progresses (20). This observation is in agreement with theoretical models that predict the opening of a hole on the area near the hemifusion stalk, (i.e., an intermediate structure that connects the apposed membranes during early fusion; Ref.…”

Shallow Boomerang-shaped Influenza Hemagglutinin G13A Mutant Structure Promotes Leaky Membrane Fusion

“…A shallower angle such as the one observed here for the G13A mutant leads to a weaker membrane interaction and softens the membrane to become permeable to small molecules like carboxyfluorescein. The steep angle by which the wild-type fusion domain inserts allows it to interact with the transmembrane domain of hemagglutinin in the viral membrane and thus fuse the two membranes in a clean non-leaky fashion (28), which cannot happen with the leaky fusion mutant. Therefore, the fusion domain must reach a certain critical angle and depth of membrane insertion to prevent content leakage into the environment during membrane fusion.…”