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...

Our previous studies have shown that Semliki Forest virus (SFV), a togavirus, binds to cells in the cold and, when the temperature is raised to 37°C, the virus particles are rapidly internalized into endosomes and secondary lysosomes by adsorptive endocytosis (1-3) . Under normal conditions of infection, fusion between the plasma membrane and the virus envelope does not occur (4, 5) . We observed, however, that fusion between SFV and the plasma membrane could be artificially triggered by mildly acidic medium (pH 5 .0-6 .0) (4); as a result, the nucleocapsid entered the cell directly through the plasma membrane, and the spike glycoproteins were left on the cell surface (4) . Fusion with liposomes containing phospholipids and cholesterol as target membranes can also be induced by a brief drop in pH (6) . On the basis of these and other data, we have proposed that, under normal conditions of infection, the membrane fusion activity of SFV is needed for the penetration of the nucleocapsids through the lysosomal membrane, and that the low lysosomal pH is critical in triggering the reaction (references 1, 2, 4, and 6; footnote 1) .During the course of our studies on virus-cell fusion, we observed the formation of large polykaryons when BHK-21 cells were exposed to SFV and low pH . In some cases, these ' Helenius, A ., M . Marsh, and J . White. Manuscript in preparation. 674JUDY WHITE, KARL MATLIN, and ARI HELENIUS European Molecular Biology Laboratory, 6900 Heidelberg, Federal Republic of Germany ABSTRACT Representatives of three families of enveloped viruses were shown to fuse tissue culture cells together. These were : Semliki Forest virus (SFV, a togavirus), vesicular stomatitis virus (a rhabdovirus), and two myxoviruses, fowl plaque virus and Japan influenza virus (Japan/ A/305/57) . Unlike paramyxoviruses, whose fusion activity is known to occur over a broad pH range, fusion by these viruses was restricted to mildly acidic pH . The pH thresholds for the four viruses were 6.0, 6.1, 5.5, and 5 .1, respectively . The precursor form of Japan influenza, which is not infectious and which contains the uncleaved hemagglutinin, had no fusion activity . This result suggested a role for the influenza hemagglutinin in the low-pH-dependent membrane fusion activity . Taken together, our results show that low-pH-induced fusion is a widespread property of enveloped animal viruses and that it may play a role in the infective process.The fusion reactions with all four viruses were fast, efficient, and easy to induce . With UVinactivated SFV, the fusion was shown to be nonlytic and the polykaryons were viable for at least 12 h. 30 ng of SFV/1 x 106 BHK-21 cells were required for 50% fusion, and 250 ng sufficed to fuse the entire culture into a single polykaryon .encompassed the entire culture . In this paper we have characterized this phenomenon and we have demonstrated that, in addition to SFV, representatives of both the myxovirus and rhabdovirus families induced extensive cell fusion at low pH .

Abstract. Oligonucleotide-directed mutagenesis of a cDNA encoding the hemagglutinin of influenza virus has been used to introduce single base changes into the sequence that codes for the conserved apolar "fusion peptide" at the amino-terminus of the HA2 subunit. The mutant sequences replaced the wild-type gene in SV40-HA recombinant virus vectors, and the altered HA proteins were expressed in simian cells. Three mutants have been constructed that introduce single, nonconservative amino acid changes in the fusion peptide, and three fusion phenotypes were observed: substitution of glutamic acid for the glycine residue at the amino-terminus of HA2 abolished all fusion activity; substitution ofglutamic acid for the glycine residue at position 4 in HA2 raised the threshold pH and decreased the efficiency of fusion; and, finally, extension of the hydrophobic stretch by replacement of the glutamic acid at position 11 with glycine yielded a mutant protein that induced fusion of erythrocytes with cells with the same efficiency and pH profile as the wild-type protein. However, the ability of this mutant to induce polykaryon formation was greatly impaired. Nevertheless, all the mutant proteins underwent a pH-dependent conformational change and bound to liposomes. These results are discussed in terms of the mechanism of HA-induced membrane fusion.

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A simple assay is described to monitor fusion between fowl plague virus (FPV, an avian influenza A virus) and liposomes which allows the simultaneous quantitation of both lytic and non‐lytic fusion events. As in fusion between viruses and the plasma membrane and in FPV‐induced cell‐cell fusion, the reaction only occurs at pH 5.5 or below, and it is fast, highly efficient, and essentially non‐lytic when fresh virus and liposomes are used. The fusion occurs over a broad temperature range, and has no requirement for divalent cations. The fusion factor of influenza virus is a hemagglutinin (HA) spike which protrudes from the virus membrane and which is also responsible for virus binding to the host cell. The finding that fusion occurs as efficiently with liposomes containing or lacking virus receptor structures, further emphasizes the remarkable division of labor in the HA molecule: the receptor‐binding sites are located in the globular HA1 domains and the fusion activation peptide is found at the N‐terminal of HA2 in the stem region of the protein. The mechanism of fusion is discussed in terms of the three‐dimensional structure of the HA and the conformational change which the protein undergoes at the fusion pH optimum.