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Distinct functional determinants of influenza hemagglutinin-mediated membrane fusion.

ABSTRACT: Membrane fusion is the critical step for infectious cell penetration by enveloped viruses. We have previously used single-virion measurements of fusion kinetics to study the molecular mechanism of influenza-virus envelope fusion. Published data on fusion inhibition by antibodies to the 'stem' of influenza virus hemagglutinin (HA) now allow us to incorporate into simulations the provision that some HAs are inactive. We find that more than half of the HAs are unproductive even for virions with no bound antibodies, but that the overall mechanism is extremely robust. Determining the fraction of competent HAs allows us to determine their rates of target-membrane engagement. Comparison of simulations with data from H3N2 and H1N1 viruses reveals three independent functional variables of HA-mediated membrane fusion closely linked to neutralization susceptibility. Evidence for compensatory changes in the evolved mechanism sets the stage for studies aiming to define the molecular constraints on HA evolvability.

SUBMITTER: Ivanovic T

PROVIDER: S-EPMC4755761 | biostudies-literature | 2015 Nov

REPOSITORIES: biostudies-literature

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Distinct functional determinants of influenza hemagglutinin-mediated membrane fusion.

Ivanovic Tijana T Harrison Stephen C SC

eLife 20151127

Membrane fusion is the critical step for infectious cell penetration by enveloped viruses. We have previously used single-virion measurements of fusion kinetics to study the molecular mechanism of influenza-virus envelope fusion. Published data on fusion inhibition by antibodies to the 'stem' of influenza virus hemagglutinin (HA) now allow us to incorporate into simulations the provision that some HAs are inactive. We find that more than half of the HAs are unproductive even for virions with no ...[more]

PMID: 26613408

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Project description:The highly conserved cytoplasmic tail of influenza virus glycoprotein hemagglutinin (HA) contains three cysteines, posttranslationally modified by covalently bound fatty acids. While viral HA acylation is crucial in virus replication, its physico-chemical role is unknown. We used virus-like particles (VLP) to study the effect of acylation on morphology, protein incorporation, lipid composition, and membrane fusion. Deacylation interrupted HA-M1 interactions since deacylated mutant HA failed to incorporate an M1 layer within spheroidal VLP, and filamentous particles incorporated increased numbers of neuraminidase (NA). While HA acylation did not influence VLP shape, lipid composition, or HA lateral spacing, acylation significantly affected envelope curvature. Compared to wild-type HA, deacylated HA is correlated with released particles with flat envelope curvature in the absence of the matrix (M1) protein layer. The spontaneous curvature of palmitate was calculated by molecular dynamic simulations and was found to be comparable to the curvature values derived from VLP size distributions. Cell-cell fusion assays show a strain-independent failure of fusion pore enlargement among H2 (A/Japan/305/57), H3 (A/Aichi/2/68), and H3 (A/Udorn/72) viruses. In contradistinction, acylation made no difference in the low-pH-dependent fusion of isolated VLPs to liposomes: fusion pores formed and expanded, as demonstrated by the presence of complete fusion products observed using cryo-electron tomography (cryo-ET). We propose that the primary mechanism of action of acylation is to control membrane curvature and to modify HA's interaction with M1 protein, while the stunting of fusion by deacylated HA acting in isolation may be balanced by other viral proteins which help lower the energetic barrier to pore expansion.IMPORTANCE Influenza A virus is an airborne pathogen causing seasonal epidemics and occasional pandemics. Hemagglutinin (HA), a glycoprotein abundant on the virion surface, is important in both influenza A virus assembly and entry. HA is modified by acylation whose removal abrogates viral replication. Here, we used cryo-electron tomography to obtain three-dimensional images to elucidate a role for HA acylation in VLP assembly. Our data indicate that HA acylation contributes to the capability of HA to bend membranes and to HA's interaction with the M1 scaffold protein during virus assembly. Furthermore, our data on VLP and, by hypothesis, virus suggests that HA acylation, while not critical to fusion pore formation, contributes to pore expansion in a target-dependent fashion.

Dataset Information

Distinct functional determinants of influenza hemagglutinin-mediated membrane fusion.

Publications

Distinct functional determinants of influenza hemagglutinin-mediated membrane fusion.

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