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J Virol. 2017 Aug 9. pii: JVI.00947-17. doi: 10.1128/JVI.00947-17. [Epub ahead of print]

Palmitoylation contributes to membrane curvature in Influenza A virus assembly and hemagglutinin-mediated membrane fusion.

Author information

1
Section on Integrative Biophysics, Eunice Kennedy Shriver National Institute of Child Health and Human Development, NIH, Bethesda, MD chlandap@mail.nih.gov zimmerbj@mail.nih.gov.
2
Section on Integrative Biophysics, Eunice Kennedy Shriver National Institute of Child Health and Human Development, NIH, Bethesda, MD.
3
Unit on Membrane Chemical Physics, Eunice Kennedy Shriver National Institute of Child Health and Human Development, NIH, Bethesda, MD.
4
Rocky Mountain Laboratories, Electron Microscopy Unit, National Institute of Allergy and Infectious Diseases, NIH, Hamilton, MT.

Abstract

The highly conserved cytoplasmic tail of influenza virus glycoprotein hemagglutinin (HA) contains three cysteines, post-translationally 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. De-acylation interrupted HA-M1 interactions since de-acylated mutant HA fail 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, de-acylated HA is correlated with released particles with flatter 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 amongst H2 (A/Japan/305/57), H3 (A/Aichi/2/68), H3 (A/Udorn/72). 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 air-borne pathogen causing seasonal epidemics and occasional pandemics. Hemagglutinin (HA), a glycoprotein abundant on the virion surface is important both in 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 virus-like particle (VLP) assembly. Our data indicates 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.

PMID:
28794042
DOI:
10.1128/JVI.00947-17
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