Length requirements for membrane fusion of influenza virus hemagglutinin peptide linkers to transmembrane or fusion peptide domains

Abstract

During membrane fusion, the influenza A virus hemagglutinin (HA) adopts an extended helical structure that contains the viral transmembrane and fusion peptide domains at the same end of the molecule. The peptide segments that link the end of this rod-like structure to the membrane-associating domains are approximately 10 amino acids in each case, and their structure at the pH of fusion is currently unknown. Here, we examine mutant HAs and influenza viruses containing such HAs to determine whether these peptide linkers are subject to specific length requirements for the proper folding of native HA and for membrane fusion function. Using pairwise deletions and insertions, we show that the region flanking the fusion peptide appears to be important for the folding of the native HA structure but that mutant proteins with small insertions can be expressed on the cell surface and are functional for membrane fusion. HA mutants with deletions of up to 10 residues and insertions of as many as 12 amino acids were generated for the peptide linker to the viral transmembrane domain, and all folded properly and were expressed on the cell surface. For these mutants, it was possible to designate length restrictions for efficient membrane fusion, as functional activity was observed only for mutants containing linkers with insertions or deletions of eight residues or less. The linker peptide mutants are discussed with respect to requirements for the folding of native HAs and length restrictions for membrane fusion activity.

FIG. 1.

Ribbon diagrams of low-pH HA (top) and neutral-pH HA (bottom). (A) Structure of the rod-shaped low-pH HA trimer, with individual monomers shown in yellow, blue, and green. The viral and host membranes would be located at the top of this structure, as represented. (B) Individual HA2 polypeptide monomer in the same orientation as the green polypeptide from A. Helix 1, helix 2, and the extended chain are labeled. The amino acid sequences of the linker domains are positioned at their respective locations of attachment to the low-pH structure, and the sequence alignments with respect to the fusion peptide and transmembrane domains are shown. The triangles indicate positions at which deletions and insertions were constructed, as detailed at the left (A). (C) Locations of the linker region residues as they reside in the neutral-pH HA. Residues 173, 174, and 175 at the C-terminal end of BHA are labeled, and the locations of residues that become the N-end linker following acidification are shown as red balls within the yellow antiparallel β-sheets. N indicates the location of the fusion peptide. (D) Region of β-structure in larger scale to identify the locations of residues involved in deletion mutants and the locations at which inserted pairs of amino acids were made. (E) Constructs used in this study. At the top is a linear representation of the WT HA2 subunit from the N-terminal fusion peptide (red) through to the C-terminal cytoplasmic tail (CT). The numbers at the top indicate the amino acid positions of HA2. The label N-end indicates the linker between the fusion peptide and helix 1, which is the long central helix that forms the central coiled coil in the low-pH structure. Residues between residues 106 and 112 form a loop in low-pH HA that inverts the polypeptide chain by 180° to locate the fusion peptide and transmembrane domains at the same end of the structure. Helix 2+EC denotes the antiparallel helix and extended chain that trace along the central coiled coil back in the direction of the membrane-associating domains. C-end indicates the linker domain between the trimeric core structure extended-chain region and the transmembrane domain (labeled TM). The nomenclature and sequence details of the mutants analyzed in the N-end and C-end linker regions are represented below the WT representation (these are not to scale).

FIG. 2.

Cell surface expression of HAs as assayed by trypsin cleavage of HA0 into HA1 and HA2. Recombinant vaccinia virus-infected HA-expressing cell monolayers were incubated with or without trypsin, and cell lysates were analyzed by Western blotting following SDS-PAGE under reducing conditions. Lanes labeled VP37 are nonrecombinant vaccinia virus-infected cell controls.

FIG. 3.

Graphs of ELISA data showing the pHs of conformational change for various mutant and WT HAs. Graphs plot the ratios of HC68 to HC3 reactivity as a function of pH. HC68 binds well with neutral-pH HA but poorly to the low-pH structure. HC3 binds equally well with both HA conformations.

FIG. 4.

Polykaryon formation by HA-expressing BHK cells following incubation at pH 5.0.

FIG. 5.

Efficiency of polykaryon formation of WT and mutant HAs. The efficiency of polykaryon formation was estimated by the percentage of nuclei located within syncytia. For WT HA and some of the mutants, this value approached 100%. The means and standard deviations from five independent experiments were determined.

FIG. 6.

Hemifusion and full fusion activities of HA mutants. Human erythrocytes loaded with R18 and calcein were adsorbed to HA-expressing cells and exposed to acidic pH to monitor HA-mediated transfer of R18 (lipid mixing) or the soluble dye calcein (content mixing). WT HA (−), WT HA that has not been treated with trypsin to cleave HA0 into HA1 and HA2.