Fusion of influenza virus with the endosomal membrane is inhibited by monoclonal antibodies to defined epitopes on the hemagglutinin (original) (raw)
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The Journal of general virology, 1998
A monoclonal antibody, LMBH6, was derived from mice which had been sequentially immunized with bromelain-cleaved haemagglutinin (BHA) from influenza virus A/Aichi/2/68, A/Victoria/3/75 and A/Philippines/2/82 (all H3N2). LMBH6 recognizes the haemagglutinin (HA) of all H3N2 influenza A strains tested, which were isolated between 1968 and 1989. HA in the low-pH-induced conformation is not recognized, and cleavage of the HA0 precursor to HA1 and HA2 is needed to obtain efficient binding. Compared to other monoclonal antibodies, binding of LMBH6 to virus and to virus-infected cells is weak, while binding to BHA is comparable. Electron microscopy demonstrates binding to the membrane proximal end of the stem structure. The antibody shows no haemagglutination-inhibition activity, but inhibits polykaryon formation and the low-pH-induced conformational change of BHA. However, LMBH6 cannot prevent infection of MDCK cells but slows the growth of virus when included in a plaque assay overlay.
Journal of Virology, 2000
The fusion activity of chimeras of influenza virus hemagglutinin (HA) (from A/fpv/Rostock/34; subtype H7) with the transmembrane domain (TM) and/or cytoplasmic tail (CT) either from the nonviral, nonfusogenic T-cell surface protein CD4 or from the fusogenic Sendai virus F-protein was studied. Wild-type or chimeric HA was expressed in CV-1 cells by the transient T7-RNA-polymerase vaccinia virus expression system. Subsequently, the fusion activity of the expression products was monitored with red blood cells or ghosts as target cells. To assess the different steps of fusion, target cells were labeled with the fluorescent membrane label octadecyl rhodamine B-chloride (R18) (membrane fusion) and with the cytoplasmic fluorophores calcein (molecular weight [MW], 623; formation of small aqueous fusion pore) and tetramethylrhodamine-dextran (MW, 10,000; enlargement of fusion pore). All chimeric HA/F-proteins, as well as the chimera with the TM of CD4 and the CT of HA, were able to mediate t...
Journal of General Virology, 1983
At the pH optimum for membrane fusion the haemagglutinin glycoprotein (HA) of the influenza virus membrane which is implicated in the fusion activity undergoes a conformational change. We have analysed the effects of this change on the antigenicity of the haemagglutinin by reacting the molecule with monoclonal antibodies of defined specificity. The results obtained indicate that specific changes in antigenicity occur in antigenic sites B and D and are interpreted in terms of the three-dimensional structure of the molecule and the effects of low pH incubation on it. Our results also provide evidence for the antigenic significance of amino acid sequence changes in site B of the HAs of natural isolates and allow clear delineation of this site into two regions.
Structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 Å resolution
Nature, 1981
The fusion activity of influenza hemagglutinin (HA) and of HA proteins altered in the amino terminus of HA2 (fusion peptide) by site-directed mutagenesis (Gething, M.-J., Doms, R. W., York, D., and White, J. (1986) J. Cell Biol. 102, 11-23) was analyzed following expression in CV-1 cells using SV40-HA recombinant virus vectors. Fusion was monitored by the redistribution of lipid and cytoplasmic dyes between fluorescently labeled erythrocytes and HA-expressing CV-1 cells using spectrofluorometry and fluorescence microscopy. The kinetics of lipid redistribution after lowering the pH showed the same pattern for wild type HA and nonlethal mutants, although there were shifts in the pH threshold. The time for commitment to the fusogenic state and the temperature dependence of the processes leading to HA-mediated fusion were also the same for wild type and nonlethal mutants. However, striking differences were observed between wild type HA and the nonlethal mutants in their ability to induce pH-dependent redistribution from erythrocytes to HA-expressing cells of large molecular weight (Mr >10,000) fluorescently labeled dextran molecules. The data indicate that the kinetic processes which are measurable in the time range of seconds are insensitive to the structure of the fusion peptide. Surprisingly, however, the fusion peptide plays an important role in later processes related to pore widening which eventually results in delivery of the nucleocapsid into the cell.
Journal of Virology, 2000
Influenza virus matrix protein (M1), a critical protein required for virus assembly and budding, is presumed to interact with viral glycoproteins on the outer side and viral ribonucleoprotein on the inner side. However, because of the inherent membrane-binding ability of M1 protein, it has been difficult to demonstrate the specific interaction of M1 protein with hemagglutinin (HA) or neuraminidase (NA), the influenza virus envelope glycoproteins. Using Triton X-100 (TX-100) detergent treatment of membrane fractions and floatation in sucrose gradients, we observed that the membrane-bound M1 protein expressed alone or coexpressed with heterologous Sendai virus F was totally TX-100 soluble but the membrane-bound M1 protein expressed in the presence of HA and NA was predominantly detergent resistant and floated to the top of the density gradient. Furthermore, both the cytoplasmic tail and the transmembrane domain of HA facilitated binding of M1 to detergent-resistant membranes. Analysis of the membrane association of M1 in the early and late phases of the influenza virus infectious cycle revealed that the interaction of M1 with mature glycoproteins which associated with the detergent-resistant lipid rafts was responsible for the detergent resistance of membrane-bound M1. Immunofluorescence analysis by confocal microscopy also demonstrated that, in influenza virus-infected cells, a fraction of M1 protein colocalized with HA and associated with the HA in transit to the plasma membrane via the exocytic pathway. Similar results for colocalization were obtained when M1 and HA were coexpressed and HA transport was blocked by monensin treatment. These studies indicate that both HA and NA interact with influenza virus M1 and that HA associates with M1 via its cytoplasmic tail and transmembrane domain.
Two Escape Mechanisms of Influenza A Virus to a Broadly Neutralizing Stalk-Binding Antibody
PLoS pathogens, 2016
Broadly neutralizing antibodies targeting the stalk region of influenza A virus (IAV) hemagglutinin (HA) are effective in blocking virus infection both in vitro and in vivo. The highly conserved epitopes recognized by these antibodies are critical for the membrane fusion function of HA and therefore less likely to be permissive for virus mutational escape. Here we report three resistant viruses of the A/Perth/16/2009 strain that were selected in the presence of a broadly neutralizing stalk-binding antibody. The three resistant viruses harbor three different mutations in the HA stalk: (1) Gln387Lys; (2) Asp391Tyr; (3) Asp391Gly. The Gln387Lys mutation completely abolishes binding of the antibody to the HA stalk epitope. The other two mutations, Asp391Tyr and Asp391Gly, do not affect antibody binding at neutral pH and only slightly reduce binding at low pH. Interestingly, they enhance the fusion ability of the HA, representing a novel mechanism that allows productive membrane fusion e...
Proceedings of the National Academy of Sciences, 1982
A conformational change in the hemagglutinin glycoprotein of influenza virus has been observed to occur to 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 conformation change also involves BHA1. The implications of these observations for the role of the glycoprotein in membrane fusion are discussed.