Transient Excursions to Membrane Core as Determinants of Influenza Virus Fusion Peptide Activity

Abstract

Fusion of viral and host cell membranes is a critical step in the life cycle of enveloped viruses. In the case of influenza virus, it is mediated by subunit 2 of hemagglutinin (HA) glycoprotein whose N-terminal fragments insert into the target membrane and initiate lipid exchange. These isolated fragments, known as fusion peptides (HAfp), already possess own fusogenic activity towards liposomes. Although they have long been studied with the hope to uncover the details of HA-mediated fusion, their actual mechanism of action remains elusive. Here, we use extensive molecular dynamics simulations combined with experimental studies of three HAfp variants to fully characterize their free energy landscape and interaction with lipid bilayer. In addition to customary assumed peptides localization at lipid–water interface, we characterize membrane-spanning configurations, which turn out to be metastable for active HAfps and unstable for the fusion inactive W14A mutant. We show that, while the degree of membrane perturbation by surface peptide configurations is relatively low and does not show any mutation-related differences, the effect of deeply inserted configurations is significant and correlates with insertion depth of the N-terminal amino group which is the highest for the wild type HAfp. Finally, we demonstrate the feasibility of spontaneous peptide transition to intramembrane location and the critical role of strictly conserved tryptofan residue 14 in this process.

Membrane-Bound Configuration and Lipid Perturbing Effects of Hemagglutinin Subunit 2 N-Terminus Investigated by Computer Simulations

“…Nonetheless, as a general scheme, surface-bound HAfps revealed no tendency to associate, and each of them continuously sampled its sector of a circular area within ∼ nm radius from the apex of the coiled-coil stem. An average insertion depth of their C α atoms was the same as observed for monomers in our previous study ( Worch et al, 2021 ), indicating no influence of linkers or trimeric arrangement on membrane penetration ( Figure 5 ). The perturbation of bilayer structure by surface-bound HAfps included modest thinning by up to 0.5 nm and an increase in average APL by around 50%, with both effects confined within the aforementioned circular area ( Figure 4C ).…”

“…Compared with pure POPC membrane, the probability of protrusion per lipid, per unit time within the area extending up to 0.7 nm from protein atoms was enhanced ∼ 10 times by surface-bound and ∼ 200 times by deeply bound trimers ( Figure 7A ). As discussed in our previous work in the context of HAfp monomers ( Worch et al, 2021 ), the difference stems from membrane indentation caused by membrane-spanning configurations and deeper insertion of the N-terminal, charged amino group in their case ( Figure 5 ). The enhancement of lipid tails protrusions observed here for trimers is of the same order as for HAfp monomers, suggesting that the trimerisation does not introduce any new qualitatively different effects in lipid organisation.…”

“…Assuming the customary proposed peptides localisation at the membrane-water interface, we found no particular effects arising due to their tethering to the coiled-coil stem in the trimeric arrangement. Each unit sampled its own region of the membrane surface, with insertion depth and bilayer perturbing capability similar as previously determined for HAfp monomers ( Worch et al, 2021 ). On the contrary, HAfps trimers in the transmembrane configuration were observed to form distinct, symmetric associates with a central hydrated channel.…”

“…It may be possible due to local membrane thinning induced by a favourable interaction of amphiphilic hairpin poles with an aqueous environment on both sides of the lipid bilayer. Such putative membrane-spanning configuration was shown in MD simulations to cause significant membrane perturbation and, thus, to have a much higher fusogenic potential than the surface configuration ( Worch et al, 2017 ; Worch et al, 2021 ). Notably, the deeply inserted state is apparently stabilised by low pH ( Lousa et al, 2020 ), which may provide a plausible explanation for the long-known enhancement of peptides activity by pH drop from neutral to acidic ( Rafalski et al, 1991 ; Epand et al, 1999 ; Han and Tamm, 2000 ; Lau et al, 2004 ; Ge and Freed, 2009 ).…”

“…It is not entirely clear, however, how to reconcile this picture with the evidence of membrane leakage caused by bound, but only fusion-active peptides ( Wharton et al, 1988 ; Haque et al, 2001 ; Lai et al, 2005 ) or the observation of peptide-induced membrane area expansion that also correlates with the level of activity and not membrane binding per se ( Longo et al, 1998 ; Lau et al, 2004 ). Moreover, NMR spectroscopy ( Jia et al, 2015 ; Ghosh and Weliky, 2020 ) and several molecular dynamics (MD) simulation studies ( Victor et al, 2015 ; Worch et al, 2017 ; Worch et al, 2021 ; Lousa et al, 2020 ) suggest that HAfp in helical hairpin conformation can adopt transmembrane orientation. It may be possible due to local membrane thinning induced by a favourable interaction of amphiphilic hairpin poles with an aqueous environment on both sides of the lipid bilayer.…”