Influence of acylation sites of influenza B virus hemagglutinin on fusion pore formation and dilation

Abstract

The cytoplasmic tail (CT) of hemagglutinin (HA) of influenza B virus (BHA) contains at positions 578 and 581 two highly conserved cysteine residues (Cys578 and Cys581) that are modified with palmitic acid (PA) through a thioester linkage. To investigate the role of PA in the fusion activity of BHA, site-specific mutagenesis was performed with influenza B virus B/Kanagawa/73 HA cDNA. All of the HA mutants were expressed on Cos cells by an expression vector. The membrane fusion ability of the HA mutants at a low pH was quantitatively examined with lipid (octadecyl rhodamine B chloride) and aqueous (calcein) dye transfer assays and with the syncytium formation assay. Two deacylation mutants lacking a CT or carrying serine residues substituting for Cys578 and Cys581 promoted full fusion. However, one of the single-acylation-site mutants, C6, in which Cys581 is replaced with serine, promoted hemifusion but not pore formation. In contrast, four other single-acylation-site mutants that have a sole cysteine residue in the CT at position 575, 577, 579, or 581 promoted full fusion. The impaired pore-forming ability of C6 was improved by amino acid substitution between residues 578 and 582 or by deletion of the carboxy-terminal leucine at position 582. Syncytium-forming ability, however, was not adequately restored by these mutations. These facts indicated that the acylation was not significant in membrane fusion by BHA but that pore formation and pore dilation were appreciably affected by the particular amino acid sequence of the CT and the existence of a single acylation site in CT residue 578.

FIG. 1.

Schematic diagram of HA-wt (WT) and HA mutants. The expanded region shows amino acid sequences of the CT. The nomenclature for the mutants was derived by sequentially assigning the numbers 1 to 10 to residues 573 to 582, respectively. Amino acid substitutions or deletions in the HA mutants are summarized. The asterisk indicates the introduction of translational stop codons.

FIG. 2.

Expression and rate of acylation of HA-wt (WT) and HA mutants. Expressed HA mutants and HA-wt were metabolically labeled with [³⁵S]methionine-cysteine (A) and with [³H]PA (B) and treated with TPCK-trypsin to cleave HA0 into HA1 and HA2. The HA proteins were immunoprecipitated and subjected to SDS-PAGE followed by fluorography.

FIG. 3.

Hemifusion and fusion pore formation by HA-wt and HA mutants. After hemadsorption with R18- and calcein-labeled HRBCs, Cos cells expressing HA-wt and HA mutants were exposed to acidic fusion medium (pH 5.0) to induce membrane fusion. wt uncleaved, HA-wt without trypsin treatment. Magnification, ×144.

FIG. 4.

Efficiency of pore formation by HA-wt and HA mutants. The efficiency of pore formation was estimated from the ratio of the number of calcein-transferred cells to the number of R18-transferred cells. The mean and standard deviation determined from three independent experiments are shown.

FIG. 5.

Syncytium formation by HA-wt and HA mutants. Cos cells expressing HA-wt and HA mutants were exposed to acidic fusion medium (pH 5.0) to induce membrane fusion. wt (uncleaved), HA-wt without trypsin treatment. Magnification, ×85.

FIG. 6.

Efficiency of syncytium formation by HA-wt and HA mutants. The efficiency of syncytium formation by wt-HA was estimated from the total number of nuclei in a syncytium as described in Materials and Methods. The efficiency of syncytium formation by HA mutants was calculated relative to that of HA-wt (100%). The mean and standard deviation determined from five independent experiments are shown.