Abstract
The lipid-enveloped influenza virus enters host cells during infection by binding cell surface receptors and, following receptor-mediated endocytosis, fusing with the membrane of the endosome, delivering the viral genome and transcription machinery into the host cell. These events are mediated by the haemagglutinin (HA) surface glycoprotein. At the low pH of the endosome, an irreversible conformational change in the HA, including the exposure of the hydrophobic fusion peptide, activates membrane fusion. Here we use electron cryomicroscopy and cryotomography to image influenza virus fusion with target membranes at low pH. We visualize structural intermediates of HA and their interactions with membranes during the course of membrane fusion as well as ultra-structural changes in the virus that accompany membrane fusion. Our observations are relevant to a wide range of protein-mediated membrane fusion processes and demonstrate how dynamic membrane events may be studied by cryomicroscopy.
Highlights
The influenza virus envelope contains two spike glycoproteins, the trimeric hemagglutinin (HA) which mediates receptor binding to sialic acid-containing receptors and membrane fusion with host membranes, and the tetrameric receptor-destorying neuraminidase (NA)
After 1min at low pH, over 80% of virus particles interacted with liposomes, either via contact with the viral glycoprotein layer or where liposome and virus membranes became apposed, including through a previously described conical perturbation of liposomes by influenza virus 18
In our identification of the major structural steps in membrane fusion (Fig. 6a) observed here by cryotomography, interaction of the HA extended intermediate with the target liposome is an early event in the progression toward fusion and forms an extended contact zone (Figure 6a(i))
Summary
The influenza virus envelope contains two spike glycoproteins, the trimeric hemagglutinin (HA) which mediates receptor binding to sialic acid-containing receptors and membrane fusion with host membranes, and the tetrameric receptor-destorying neuraminidase (NA). The virus membrane contains a proton channel, M2. HA is synthesized as a precursor, HA0, which is proteolytically cleaved to generate the disulphide-linked HA1 and HA2 subunits, which potentiates the membrane fusion activity[1]. HA2 subunits contain the Cterminal transmembrane anchors (TM) located in the viral membrane, and the N-terminal hydrophobic fusion peptides (FP) (Figure 1a). The X-ray structure of the soluble HA ectodomain at neutral pH (135 Å long) reveals globular head domains containing the receptor binding site on a helical stalk domain formed largely by HA2 with a long central coiled-coil and with the fusion peptides located 35 Å from the viral membrane[2]
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