An antibody that prevents the hemagglutinin low pH fusogenic transition.

We have determined the structure of a complex of influenza hemagglutinin (HA) with an antibody that binds simultaneously to the membrane-distal domains of two HA monomers, effectively cross-linking them. The antibody prevents the low pH structural transition of HA that is required for its membrane fusion activity, providing evidence that a rearrangement of HA membrane-distal domains is an essential component of the transition.

Electron microscopy of the human respiratory syncytial virus fusion protein and complexes that it forms with monoclonal antibodies.

Full-length fusion (F) glycoprotein of human respiratory syncytial virus (HRSV) and a truncated anchorless mutant lacking the C-terminal 50 amino acids were expressed from vaccinia recombinants and purified by immunoaffinity chromatography and sucrose gradient centrifugation. Electron microscopy of full-length F protein in the absence of detergents revealed micelles, (i.e., rosettes) containing two distinct types of protein rods, one cone-shaped and the other lollipop-shaped. Analysis of membrane anchorless F molecules indicated that they were similar to the cone-shaped rods and that rosettes, which they formed on storage, were made up of lollipop-shaped rods. The two forms of F protein may represent different structures that the molecule may adopt before and after activation for its role in membrane fusion. Studies of complexes of these structures with monoclonal antibodies of known specificity provide information on the three-dimensional organization of antigenic sites on the F protein and confirm the oligomeric structure, possibly trimeric, of both full-length F and membrane anchorless F.

Structural basis for membrane fusion by enveloped viruses.

Enveloped viruses such as HIV-1, influenza virus, and Ebola virus express a surface glycoprotein that mediates both cell attachment and fusion of viral and cellular membranes. The membrane fusion process leads to the release of viral proteins and the RNA genome into the host cell, initiating an infectious cycle. This review focuses on the HIV-1 gp41 membrane fusion protein and discusses the structural similarities of viral membrane fusion proteins from diverse families such as Retroviridae (HIV-1), Orthomyxoviridae (influenza virus), and Filoviridae (Ebola virus). Their structural organization suggests that they have all evolved to use a similar strategy to promote fusion of viral and cellular membranes. This observation led to the proposal of a general model for viral membrane fusion, which will be discussed in detail.

Membrane fusion by surrogate receptor-bound influenza haemagglutinin.

In influenza infections, haemagglutinin (HA) mediates the fusion of virus and cellular membranes at endosomal pH, between pH 5 and 6. In vitro, when reconstituted into virosomes, efficient fusion requires target membranes to contain sialic acid receptors or receptor analogues. In the experiments reported, lipid-associated anti-HA monoclonal Fab' fragments were used as surrogate receptors to investigate the fusion capacity of receptor-bound HA compared with unbound HA. The conclusions are drawn, in contrast to those from previous studies, that bound HA can mediate fusion and that fusion mainly involves bound HA when the liposome targets are densely packed with surrogate receptors.

The central structural feature of the membrane fusion protein subunit from the Ebola virus glycoprotein is a long triple-stranded coiled coil.

The ectodomain of the Ebola virus Gp2 glycoprotein was solubilized with a trimeric, isoleucine zipper derived from GCN4 (pIIGCN4) in place of the hydrophobic fusion peptide at the N terminus. This chimeric molecule forms a trimeric, highly alpha-helical, and very thermostable molecule, as determined by chemical crosslinking and circular dichroism. Electron microscopy indicates that Gp2 folds into a rod-like structure like influenza HA2 and HIV-1 gp41, providing further evidence that viral fusion proteins from diverse families such as Orthomyxoviridae (Influenza), Retroviridae (HIV-1), and Filoviridae (Ebola) share common structural features, and suggesting a common membrane fusion mechanism.

Assembly of a rod-shaped chimera of a trimeric GCN4 zipper and the HIV-1 gp41 ectodomain expressed in Escherichia coli.

The HIV-1 envelope subunit gp41 plays a role in viral entry by initiating fusion of the viral and cellular membranes. A chimeric molecule was constructed centered on the ectodomain of gp41 without the fusion peptide, with a trimeric isoleucine zipper derived from GCN4 (pIIGCN4) on the N terminus and part of the trimeric coiled coil of the influenza virus hemagglutinin (HA) HA2 on the C terminus. The chimera pII-41-HA was overexpressed as inclusion bodies in bacteria and refolded to soluble aggregates that became monodisperse after treatment with protease. Either trypsin or proteinase K, used previously to define a protease-resistant core of recombinant gp41 [Lu, M., Blacklow, S. C. & Kim, P. S. (1995) Nat. Struct. Biol. 2, 1075-1082], removed about 20-30 residues from the center of gp41 and all or most of the HA2 segment. Evidence is presented that the resulting soluble chimera, retaining the pIIGCN4 coiled coil at the N terminus, is an oligomeric highly alpha-helical rod about 130 A long that crystallizes. The chimeric molecule is recognized by the Fab fragments of mAbs specific for folded gp41. A similar chimera was assembled from the two halves of the molecule expressed separately in different bacteria and refolded together. Crystals from the smallest chimera diffract x-rays to 2.6-A resolution.

The ectodomain of HIV-1 env subunit gp41 forms a soluble, alpha-helical, rod-like oligomer in the absence of gp120 and the N-terminal fusion peptide.

The human immunodeficiency virus-1 (HIV-1) envelope glycoprotein is composed of a soluble glycopolypeptide gp120 and a transmembrane glycopolypeptide gp41. These subunits form non-covalently linked oligomers on the surface of infected cells, virions and cells transfected with the complete env gene. Two length variants of the extracellular domain of gp41 (aa 21-166 and aa 39-166), that both lack the N-terminal fusion peptide and the C-terminal membrane anchor and cytoplasmic domain, have been expressed in insect cells to yield soluble oligomeric gp41 proteins. Oligomerization was confirmed by chemical cross-linking and gel filtration. Electron microscopy and circular dichroism measurements indicate a rod-like molecule with a high alpha-helical content and a high melting temperature (78 degrees C). The binding of monoclonal antibody Fab fragments dramatically increased the solubility of both gp41 constructs. We propose that gp41 folds into its membrane fusion-active conformation, when expressed alone.

A soluble domain of the membrane-anchoring chain of influenza virus hemagglutinin (HA2) folds in Escherichia coli into the low-pH-induced conformation.

The extensive refolding of the membrane-anchoring chain of hemagglutinin (HA) of influenza virus (termed HA2) in cellular endosomes, which initiates viral entry by membrane fusion, suggests that viral HA is meta-stable. HA2 polypeptide residues 38-175 expressed in Escherichia coli are reported here to fold in vivo into a soluble trimer. The structure appears to be the same as the low-pH-induced conformation of viral HA2 by alpha-helical content, thermodynamic stability, protease dissection, electron microscopy, and antibody binding. These results provide evidence that the structure of the low-pH-induced fold of viral HA2 (TBHA2) observed crystallographically is the lowest-energy-state fold of the HA2 polypeptide. They indicate that the HA2 conformation in viral HA before low pH activation of its fusion potential is metastable and suggest that removal of the receptor-binding chain (HA1) is enough to allow HA2 to adopt the stable state. Further, they provide direct evidence that low pH is not required to form the membrane-fusion conformation but acts to make this state kinetically accessible in viral HA.

Electron microscopy of antibody complexes of influenza virus haemagglutinin in the fusion pH conformation.

Activation of the membrane fusion potential of influenza haemagglutinin (HA) at endosomal pH requires changes in its structure. X-ray analysis of TBHA2, a proteolytic fragment of HA in the fusion pH conformation, indicates that at the pH of fusion the 'fusion peptide' is displaced by > 10 nm from its location in the native structure to the tip of an 11 nm triple-stranded coiled coil, and that the formation of this structure involves extensive re-folding or reorganization of HA. Here we examine the structure of TBHA2 with the electron microscope and compare it with the fusion pH structure of HA2 in virosomes, HA2 in aggregates formed at fusion pH by the soluble, bromelain-released ectodomain BHA and HA2 in liposomes with which BHA associates at fusion pH. We have oriented each HA2 preparation for comparison, using site-specific monoclonal antibodies. We conclude that the structural changes in membrane-anchored and soluble HA preparations at the pH of fusion appear to be the same; that in the absence of a target membrane, the 'fusion peptide' of HA in virosomes associates with the virosome membrane so that HA2 is membrane bound at both N- and C-termini, which implies that inversion of the re-folded HA can occur; and that the structural changes observed by X-ray analysis do not result from the proteolytic digestions used in the preparation of TBHA2.

Fusion characteristics of influenza C viruses.

A number of different influenza C virus strains were tested for their fusion properties using a resonance energy assay which allows direct monitoring of fusion between virus membranes and artificial lipid vesicles. The fusion pH of various strains was found to range between 5.6 and 6.1. Haemolytic activity of the different strains with chicken erythrocytes was observed at slightly lower pH values and varied between 5.1 and 5.7. Studies of the kinetics of influenza C virus fusion showed distinct characteristics in fusion activity. A lag before onset of fusion was found with influenza C virus which was not observed for influenza A or B viruses. In addition, studies on the rate of conformational change of the influenza C virus glycoprotein, as determined by morphological changes and endogenous tryptophan fluorescence, suggest that the conformational change is rate-limiting in the fusion process, whereas for influenza A viruses the glycoprotein conformational change is fast and a later step in the fusion process is rate-limiting. Monitoring the conformational change of influenza C virus glycoprotein by the onset of trypsin susceptibility showed, however, that membrane fusion occurred in some cases without onset of trypsin susceptibility, indicating that the trypsin-susceptible conformation is a post-fusogenic conformation.

Electron microscopy of the influenza virus submembranal structure.

Electron micrographs of stain-penetrated influenza virus particles show a variety of internal structures that all consist of units of 4 X 4 nm. Isolated influenza virus M-protein, that is reassociated with lipid, forms very similar structures on liposomes. We therefore conclude that the 4 X 4 nm unit is M-protein. We further argue that the M-protein is not arranged with icosahedral symmetry.

Studies on the structure of the influenza virus haemagglutinin at the pH of membrane fusion.

At the pH required to trigger the membrane fusion activity of the influenza virus haemagglutinin (HA) the soluble ectodomain of the molecule, BHA, which is released from virus by bromelain digestion, aggregates into rosettes. Analyses of soluble proteolytic fragments derived from the rosettes indicated that aggregation is mediated by association of the conserved hydrophobic amino-terminal region of BHA2, the smaller glycopolypeptide component of each BHA subunit. Further analyses of the structure of the soluble fragments and of HA in its low pH conformation by electron microscopy, spectroscopy and in crosslinking experiments showed that, although the membrane distal globular domains lose their trimer structure at the pH of fusion, the central fibrous stem of the molecule remains trimeric and assumes a more stable conformation. The increase in length of BHA2 at low pH observed microscopically appears to result from movement of the amino-terminal region to the membrane proximal end of the molecule and in virus incubated at low pH the amino terminus may insert into the virus membrane. The consequences of these possibilities for the mechanism of membrane fusion are discussed.

Electron microscopy of the low pH structure of influenza virus haemagglutinin.

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.