Studies using double mutants of the conformational transitions in influenza hemagglutinin required for its membrane fusion activity

Abstract

Amino acid substitutions widely distributed throughout the influenza hemagglutinin (HA) influence the pH of its membrane fusion activity. We have combined a number of these substitutions in double mutants and determined the effects on the pH of fusion and on the pH at which the refolding of HA required for fusion occurs. By analyzing combinations of mutations in three regions of the metastable neutral-pH HA that are rearranged at fusion pH we obtain evidence for both additive and nonadditive effects and for an apparent order of dominance in the effects of amino acid substitutions in particular regions on the pH of fusion. We conclude that there are at least three components in the structural transition required for membrane fusion activity and consider possible pathways for the transition in relation to the known differences between neutral and fusion pH HA structures.

Figure 1

(Left) Diagram of the structure of a subunit of the neutral-pH HA trimer (2) indicating the amino and carboxyl termini of the HA₁ and HA₂ chains, N₁ and C₁ and N₂ and C₂, respectively; the site of HA₁ exposed to trypsin digestion at fusion pH, HA₁ 27 (27₁); residue 107 of HA₂ (107₂), the first residue in the turn in HA₂ formed at fusion pH; and the three regions of HA in which the amino acid substitutions described here are located; region [1] includes HA₁ residue 17 (17₁) and HA₂ residue 112 (112₂); region [2] includes HA₂ residues 47, 54, and 58; and region [3] includes residues HA₁ 218 and HA₂ 81. (Right) The structure of a subunit of the fusion-pH trimer constructed by adding the HA₁ 28–328 domain structure (4) to the HA₂ 38–175: HA₁ 1–27 thermolytic fragment structure (3). The locations of the mutations following the fusion-pH conformational changes are indicated by the residue numbers as in Left. The discontinuous lines indicate components of the structure that are unknown. The HA₁ chain is more lightly shaded than HA₂.

Figure 2

Immunoblot analysis of reducing 12% polyacrylamide gels of lysates from monolayers of HA-expressing CV 1 cells following incubation with (+) or without (-) trypsin, to show cell surface expression of HA. Lane pairs: A, wild type; B, R54₂E; C, E81₂G; D, H17₁Q,G218₁E; E, H17₁Q,K58₂I; F, R54₂E,K58₂I; G, Q47₂R,K58₂I; H, E81₂G,K58₂I; I, G218₁E,K58₂I; J, H17₁Q,D112₂G; K, wild type; L, D112₂G,K58₂I; M, G218₁E; N, K58₂I; O, D112₂G; P, H17₁Q; and Q, Q47₂R.

Figure 3

Heterokaryon formation by single mutant HA-expressing BHK cells after incubation at the indicated pH. WT, wild type.

Figure 4

Heterokaryon formation by double-mutant HA-expressing BHK cells after incubation at the indicated pH.

Figure 5

Immunoblot analysis of a 12% polyacrylamide gel run under reducing conditions, showing the trypsin susceptibility of HA₁ of wild-type and mutant BHAs as a function of pH. (Left) From top to bottom, wild type, E81₂G, G218₁E, D112₂G, R54₂E, and Q47₂R. (Right) From top to bottom, K58₂I and double mutants E81₂G,K58₂I, G218₁E,K58₂I, R54₂E,K58₂I, and Q47₂R,K58₂I. The pH of incubation for each BHA was (from left to right) 5.9, 5.7, 5.5, 5.3, 5.1, 4.9, 4.7, 4.5, 4.3, and 4.1.