Fatty Acids on the A/USSR/77 Influenza Virus Hemagglutinin Facilitate the Transition from Hemifusion to Fusion Pore Formation

Abstract

Influenza virus hemagglutinin (HA) has three highly conserved acylation sites close to the carboxyl terminus of the HA2 subunit, one in the transmembrane domain and two in the cytoplasmic domain. Each site is modified by palmitic acid through a thioester linkage to cysteine. To elucidate the biological significance of HA acylation, the acylation sites of HA of influenza virus strain A/USSR/77 (H1N1) were changed by site-directed mutagenesis, and the membrane fusion activity of mutant HAs lacking the acylation site(s) was examined quantitatively using transfer assays of lipid (R18) and aqueous (calcein) dyes. Lipid mixing, so-called hemifusion, activity was not affected by deacylation, whereas transfer of aqueous dye, so-called fusion pore formation, was dramatically restricted. When the fusion reaction was induced by a lower pH than the optimal one, calcein transfer with the mutant HAs was improved, but simultaneously a considerable calcein leakage into the medium was observed. From these results, we conclude that the palmitic acids on the H1 subtype HA facilitate the transition from hemifusion to fusion pore formation.The influenza virus particle attaches to the host cell and is internalized into the cell by receptor-mediated endocytosis. After entry, the virus membrane fuses to the endosomal membrane by acidification of the endosome, resulting in the transfer of the viral ribonucleoprotein into the cytoplasm. The attachment and membrane fusion are mediated by influenza virus hemagglutinin (HA) (11,24). HA is a homotrimer, and each monomer comprises an ectodomain with about 510 amino acid residues, a transmembrane domain with 27 residues, and a cytoplasmic domain with 10 to 11 residues. The HA monomer is synthesized as a single polypeptide chain and cleaved into two subunits, HA1 and HA2, by proteolytic enzymes after virus budding or during intracellular transport.The HA1 and HA2 subunits are functionally specialized. HA1 carries receptor-binding activity, and HA2 mediates membrane fusion (4, 24). The membrane fusion process can be divided into three steps: lipid mixing between the outer lipid monolayers of the viral and endosomal membranes (hemifusion), aqueous pore formation by connecting the two inner lipid monolayers, and pore dilation (8). The ectodomain of HA2 is thought to mediate hemifusion, and the transmembrane and cytoplasmic domains are thought to play an important role in pore formation (10,12,17). Three cysteine residues in the C-terminal region of HA2 are highly conserved between various subtypes of HA. These residues are posttranslationally modified with palmitic acids through thioester bonds, and these palmitoyl chains bind the cytoplasmic domain to the inner surface of the viral membrane (19,20,23,26). The strict conservation of the cysteine residues implies an important role for the acyl chains in the virus life cycle. However, the role of the acyl chains remains unclear.It remains a matter of debate whether the palmitylation of HA2 is involved in membrane fusion. To examine this subject, man...

Sialidase Activity of Influenza A Virus in an Endocytic Pathway Enhances Viral Replication

“…The low-pH condition under endocytosis, which is known as an internalization pathway for influenza A virus, is necessary for the acquisition of the fusogenic capacity of virus HA into the cell membrane (22,24). In the present study, the low-pH stability of influenza virus sialidase gave rise to a distinct difference in virus replication and plaque size on MDCK cells, and we therefore suspected that this property is associated with some intracellular acidic compartments, such as lysosome, endosome, and the trans Golgi network.…”

Acylation-Mediated Membrane Anchoring of Avian Influenza Virus Hemagglutinin Is Essential for Fusion Pore Formation and Virus Infectivity

“…Thus, we reported in one of the early studies on HA acylation that this modification has no effect on fusion activity of subtype H7 (52). However, when it was recognized that H7 HA required coexpression of M2 for structural integrity, it became clear that the fusion activity of this hemagglutinin depended on acylation (9,36), as is also the case with subtypes H1 and H2 (29,40,58). The only exception is H3 HA, where such an effect has not been observed, regardless of whether the hemagglutinin was expressed alone (27,47,50) or as recombinant virus (15,47).…”

“…Restrictions in syncytium formation were also detected with acylation mutants of the fowl plague virus (FPV) HA (subtype H7) expressed in CV1 cells (9). More recently, it was shown in a study employing vector-expressed H1 HA that acylation is required for the formation of aqueous fusion pores (40). However, studies with vector-expressed H3 HA failed to uncover any involvement of acyl chains in the fusion reaction (30,47,50), except for one report on fully deacylated H3 HA, which uncovered a contribution of fatty acids to fusion pore flickering (27).…”

S Acylation of the Hemagglutinin of Influenza Viruses: Mass Spectrometry Reveals Site-Specific Attachment of Stearic Acid to a Transmembrane Cysteine

“…The hydrophobic modification is essential for virus replication, since (depending on the virus strain) virus mutants with more than one acylation site deleted either showed drastically impaired growth or could not be created at all by reverse genetics (1,19,21). S acylation of the HA of influenza A virus (subtypes H1 and H7) and of influenza B virus was shown to be required for membrane fusion, especially for the opening or enlargement of the fusion pore (6,8,12,13,19). In contrast, influenza A mutants containing nonacylated subtype H3 HA show full fusion, but budding of virus particles was reduced (1).…”