Transient Changes of the Conformation of Hemagglutinin of Influenza Virus at Low pH Detected by Time-resolved Circular Dichroism Spectroscopy

Abstract

Membrane fusion of influenza virus is mediated by a conformational change of the viral membrane protein hemagglutinin (HA) triggered by low pH. By near UV CD spectroscopy, which is sensitive to the arrangement and mobility of aromatic amino acids in proteins, we have monitored continuously with a time resolution of 5 s the kinetics of structural alterations of the ectodomain of HA isolated from different influenza virus strains (H1 (A/PR 8/34), H2 (A/Japan), and H3 (X31)). To establish a functional correlation to structural alterations of the HA ectodomain reflected by the CD, we have measured the kinetics of the virus-erythrocyte fusion and of the inactivation of fusion by low pH preincubation of viruses. At acidic pH we found a multiphasic behavior of the CD signal recorded at 283 nm. Upon lowering the pH we detected first an increase of the CD amplitude, which is associated with the formation of a fusion-competent state of HA. The initial increase was followed by a continuous decline of CD amplitude, which can be ascribed to a transformation into a fusion-inactivated conformation that is in its early phase reversible as found for A/Japan. The half-time of the different phases of the CD signal depended on the virus strain, the temperature, and the acidic pH. The results support recent hypotheses that the fusion-competent conformation is an intermediate of the fusion-inactivated structure of HA.The fusion of the viral and the endosomal membranes subsequent to the endocytic uptake of influenza virus is mediated by the integral membrane protein hemagglutinin (HA). 1 HA is a homotrimeric glycoprotein that consists of the two subunits, HA1, bearing the receptor binding site, and HA2, anchored with its C terminus in the viral membrane. At the acidic pH of endosomes, a conformational change of the hemagglutinin ectodomain is triggered (1) that activates its fusion capacity (2, 3). The three-dimensional structure of the ectodomain of HA (X31) at neutral pH is known from x-ray crystallography with a resolution of 3 Å (4). Although the fusion active conformation is still unknown, one of its characteristics is, as has been shown, e.g. by anit-peptide-antibodies (1, 5), the exposure of the highly conserved N terminus of the HA2 subunit, the "fusion sequence," which is originally buried within the stem of the trimer (4). Based on the x-ray crystal structure of a fragment of the HA ectodomain from X31 in its low pH form, Wiley and co-workers (6) suggested that the fusion sequence moves toward the top of the ectodomain by the formation of a long ␣-helix in the HA2 subunit at low pH, thereby extending the trimer stem straight up. Studies with synthetic peptides (7) predicted that this long ␣-helix is formed by a transition of a loop region of HA into an ␣-helix at low pH.However, when incubated at low pH in the absence of target membranes, influenza virus can rapidly lose its fusogenic properties (8 -11). The degree and kinetics of this inactivation is virus strain-specific (9, 12, 13). Clustering of conformationally altered ...

Lysolipids Do Not Inhibit Influenza Virus Fusion by Interaction with Hemagglutinin

“…21). Because the conformational change and exposure of hydrophobic sequences of HA is very rapid, particularly at optimal conditions of pH 5.0 at 37°C (4,39,40), viruses can interact with remaining lysolipid micelles via those hydrophobic sequences before or instead of binding to target membranes. Even if the interaction is reversible, it may be responsible for a conformational transition of HA to a fusion-inactivated state by interaction with lysolipids and/or be due to the delayed contact with the target membrane.…”

Autocatalytic Activation of Influenza Hemagglutinin

“…Amino acid residues 343 to 352, 344 to 353, or 353 to 362 (i.e. fusion peptide residues 1-10, 2-11, or [11][12][13][14][15][16][17][18][19][20] were replaced with the c-myc epitope tag sequence via PCR using a SalI site introduced by a silent mutation just upstream of the fusion peptide region. Wild-type HA and the mutants were cloned into a pIRES (BD Biosciences, Mountain View, CA) based mammalian expression vector containing the CMV promoter after the IRES sequence was removed.…”

“…Acidic pH-activated, refolded HA catalyzes refolding of non-activated HA directly or through alteration of lipid bilayer structure and membrane tension. Steps in the shaded region represent putative intermediates suggested in studies of X-31 HA[12][13][14][15] and include exposure of the fusion peptide and clustering into cooperative units (however, reversible intermediates have not been demonstrated for HA from FPV). Cooperative units might include both "primed" and inactive trimers.…”

“…[7][8][9] The X-ray crystal structures of neutral pH (inactive) and pH-activated HA fragments indicate that the fusion peptide relocates from a site largely buried within the trimer to the membrane-distal end of the structure upon activation. 6,10 Experimental evidence indicates this refolding pathway contains reversible intermediate steps, [10][11][12][13] and cooperative Abbreviations used: HA, hemagglutinin; FPV, fowl plague virus; IRES, internal ribosome entry site; CMV, cytomegalovirus; mAb, monoclonal antibody.…”

Isolation of Influenza Virus A Hemagglutinin C-Terminal Domain by Hemagglutinin Proteolysis in Octylglucoside Micelles

“…The method used was a routine procedure for HA/NA solubilization, and such preparations had been characterized in different ways [11,[17][18][19][20][21]. Transmission electron microscopy showed formation of rosette-like aggregates made of 7 to 9 HA rods in this micelle solution, and these detergent-solubilized spikes virtually associated at the hydrophobic C-terminal anchoring tails.…”