A conformational change in the hemagglutinin glycoprotein of influenza virus has been observed to occur at pH values corresponding to those optimal for the membrane fusion activity of the virus. CD, electron microscopic, and sedimentation analyses show that, in the pH range 5.2-4.9, bromelain-solubilized hemagglutinin (BHA) aggregates as protein-protein rosettes and acquires the ability to bind both lipid vesicles and nonionic detergent. Trypsin treatment of BHA in the pH 5.0-induced conformation indicates that aggregation is a property of the BHA2 component and that the conformational change also involves BHA1. The implications of these observations for the role of the glycoprotein in membrane fusion are discussed.Viruses such as influenza that contain lipid membranes appear to enter cells during infection by a process involving the fusion of the viral membrane with a cellular membrane. The results of recent investigations have indicated that, for a number of viruses, fusion occurs optimally over narrow ranges of pH, in the case of influenza viruses between pH 5.0 and pH 5.5 (1-4), and it has been proposed that this correlates with the pH at the site of cell entry in intracellular vesicles such as lysosomes (5).Evidence that the hemagglutinin (HA) glycoprotein is involved in influenza virus-mediated fusion includes the observations that post-translational cleavage ofa precursor HA, HAO, to HA1 and HA2 is required for both virus infectivity (6, 7) and in vitro virus-mediated fusion (4,8) and that the hydrophobic amino-terminal sequence of HA2 is analogous to that of the amino terminus ofthe F1 component of Sendai virus fusion glycoprotein (9-11). Furthermore, the findings that the amino-terminal sequence of HA2 consists of 10 uncharged hydrophobic amino acids (9) and is the most highly conserved sequence in the hemagglutinin (12) suggest that the terminal region may be directly involved in the membrane-fusion reaction. Analysis of the three-dimensional structure (12) of bromelain-released HA (BHA), which lacks the carboxyl-terminal hydrophobic region through which the complete HA is associated with the lipid membrane of the virus particle, suggests that a conformational .change may be required before this can occur.In this investigation, a conformational change of BHA from X-31 (H3N2) influenza virus has been observed, which occurs at pH values corresponding to those optimal 'for the in vitro membrane-fusion activity of the virus. CD, sedimentation, electron microscopy, and proteolytic susceptibility studies have been. used to characterize the pH 5.0-induced transition. The experiments lead to the conclusion that, after incubation at low pH, BHA can form hydrophobic associations with other BHA molecules, with lipid vesicles, or with nonionic detergent micelles. These results are discussed with reference to the threedimensional structure of HA and in relation to its possible role in virus-mediated fusion. METHODSVirus and HA Purification. X-31 (H3N2) influenza virus (13) was grown in embryonated hens' e...

Influenza virus haemagglutinin mediates infection of cells by fusion of viral and endosomal membranes, triggered by low pH which induces a conformational change in the protein. We report studies of this change by electron microscopy, neutron scattering, sedimentation and photon correlation on X‐31 (H3N2) haemagglutinin, both intact and bromelain cleaved, in various assemblies. HAs in all preparations showed a thinning at low pH, and a marked elongation which was removed on tryptic digestion, revealing altered features in the remaining stem portion of the molecule. A tentative model of the change is proposed, with reference to the known X‐ray structure at neutral pH, in which major changes occur in the stem tertiary structure, while the top portion is only affected in its quaternary structure.

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SUMMARY High resolution (< 2 nm) electron microscopy of biological specimens requires three exacting conditions to be met simultaneously: (a) fine specimen detail must be protected from destruction by the electron beam (low dose), (b) the electron optics must be adjusted to be capable of imaging that detail interpretably (accurate defocus), and (c) a suitable field of interest must be identified. We describe a method encompassing all three with an 80% success rate using only minor modifications to a transmission electron microscope, and no expensive on‐line computing.