Full-length influenza hemagglutinin HA2 refolds into the trimeric low-pH-induced conformation

Abstract

The influenza virus uses hemagglutinin (HA) to fuse the viral and cellular membranes. As part of an effort to study the membrane-interacting elements of HA, the fusion peptide, and the C-terminal transmembrane anchor, we have expressed in Escherichia coli the full-length HA(2) chain with maltose-binding protein fused at its N-terminus. The chimeric protein can be refolded in vitro in the presence of specific detergents to yield stable, homogeneous trimers, as determined by analytical ultracentrifugation. The trimers have the so-called "low pH" conformation-the rearranged HA(2) conformation obtained when intact HA(1)/HA(2) is induced to refold by exposure to low pH-as detected by electron microscopy and monoclonalantibody reactivity. These results provide further evidence for the notion that the neutral-pH structure of intact HA is metastable and that binding of protons lowers the kinetic barriers that prevent rearrangement to the minimum-free-energy conformation. The refolded chimeric protein described here is a suitable species for undertaking studies of how the fusion peptide inserts into membranes and assessing the nature of possible intermediates in the fusion process.