Full-length trimeric influenza virus hemagglutinin II membrane fusion protein and shorter constructs lacking the fusion peptide or transmembrane domain: Hyperthermostability of the full-length protein and the soluble ectodomain and fusion peptide make significant contributions to fusion of membrane vesicles

Abstract

Influenza virus is a Class I enveloped virus which is initially endocytosed into a host respiratory epithelial cell. Subsequent reduction of the pH to the 5–6 range triggers a structural change of the viral hemagglutinin II (HA2) protein, fusion of the viral and endosomal membranes, and release of the viral nucleocapsid into the cytoplasm. HA2 contains fusion peptide (FP), soluble ectodomain (SE), transmembrane (TM), and intraviral domains with respective lengths of ~25, ~160, ~25, and ~10 residues. The present work provides a straightforward protocol for producing and purifying mg quantities of full-length HA2 from expression in bacteria. Biophysical and structural comparisons are made between full-length HA2 and shorter constructs including SHA2 ≡ SE, FHA2 ≡ FP + SE, and SHA2-TM ≡ SE + TM constructs. The constructs are helical in detergent at pH 7.4 and the dominant trimer species. The proteins are highly thermostable in decylmaltoside detergent with Tm > 90 °C for HA2 with stabilization provided by the SE, FP, and TM domains. The proteins are likely in a trimer-of-hairpins structure, the final protein state during fusion. All constructs induce fusion of negatively-charged vesicles at pH 5.0 with much less fusion at pH 7.4. Attractive protein/vesicle electrostatics play a role in fusion, as the proteins are positively-charged at pH 5.0 and negatively-charged at pH 7.4 and the pH-dependence of fusion is reversed for positively-charged vesicles. Comparison of fusion between constructs supports significant contributions to fusion from the SE and the FP with little effect from the TM.

The Stabilities of the Soluble Ectodomain and Fusion Peptide Hairpins of the Influenza Virus Hemagglutinin Subunit II Protein Are Positively Correlated with Membrane Fusion

“…Data for WT proteins are similar to previous results. 26 For all proteins in SRC and HA2 proteins in DM, there is a large SEC peak at ~250 kDa which is assigned to trimer with ~75 kDa protein and ~175 kDa detergent contributions. This SEC assignment is consistent with earlier observations, including a ~1:120 protein:detergent mole ratio for a HA2 fusion protein trimer, and dominant trimer for the HA2 soluble ectodomain in aqueous solution.…”

“…The SE hairpin maintains membrane apposition during the ~1 minute fusion time, and clustering of hairpin trimers and therefore FP domains in the target membrane perturbs this membrane and lowers the activation barrier to achieve hemifusion and pore formation. 11, 26, 47, 52 For this model, the SE hairpin is a fusogenic state rather than a post-fusion state. This model is supported by the WT-HA2-induced vesicle fusion at pH 5.0, which is observed with <100 HA2 trimers/vesicle, less than the ~400 HA trimers/influenza virion.…”

“…Purifications from cells expressing WT-, G1E-, and I173E-FHA2 were based on earlier work, and is denoted protocol A . 26 Buffers were prepared at 4 °C. Wet cells (5 g) were subjected to four rounds of sonication in 40 mL of buffer A (50 mM sodium phosphate at pH 8.0, 0.5% N -lauroylsarcosine (SRC), 300 mM NaCl, and 10 mM imidazole) in an ice bath.…”

“…The eluent was mixed with an equal volume of ice-cold buffer that contained 10 mM Tris at pH 8.0, 0.17% DM, 2 mM EDTA, and 1 M L -arginine, followed by mixing overnight at 4 °C. 26, 30, 31…”

A rapid method for post-antibiotic bacterial susceptibility testing

“…KanR E . coli was obtained from Dr. David P. Weliky[44] and CmpR B . subtilis was obtained from Dr. Lee R. Kroos,[45] both from Michigan State University.…”