Hemolytic activity of influenza virus hemagglutinin glycoproteins activated in mildly acidic environments.

Abstract

Hemagglutinin (HA) glycoproteins isolated from influenza virus caused hemolysis and liposome lysis at pH < 6.0. The pH dependence was similar to that of the parent virus. Hemagglutination and hemolysis titers of HA were comparable with those of virus. The time course of hemolysis by HA was somewhat different from that by virus. HA did not cause fusion of erythrocytes in acidic media, in contrast to virus. Both HA and virus, previously incubated at pH < 6.0, lost their low-pH-induced hemolytic activity. Isolated HA formed rosette-like structures at neutral pH, and these aggregated in acidic media. Virus also aggregated in acidic media and its envelope became leaky to negative stain. HA previously incubated at pH < 6.0 became susceptible to trypsin digestion. Both reversible and irreversible structural changes of HA were observed by fluorescence spectroscopy; a reversible change at a pH between neutral and 6.4 and an irreversible one at pH < 6.0. Bromelain-released HA did not cause hemolysis and liposome lysis in acidic media. The precursor form of HA did not have hemolytic activity in acidic media. The similarity in pH dependence indicates that the structural change in HA induced at pH < 6.0 is the cause of activation and inactivation of hemolysis, HA and virus aggregation, and trypsin susceptibility. We propose that the hydrophobic NH2-terminal segment of HA2 is exposed during the structural change and interacts with the target membranes, causing a permeability increase and leading to hemolysis and lysis. The virus-induced hemolysis can be ascribed for the most part to envelope fusion activated in acidic media.Low pH-induced hemolytic activity of influenza virus has recently been observed (1-3). Based on this finding, we proposed an infectious cell entry mechanism of the virus (4), which is essentially the same as that proposed and developed for Semliki Forest virus (5)-i.e., uptake by endocytosis and fusion of endocytosed vesicles with lysosomes, followed by fusion of the virus envelope with the secondary lysosomal membrane because of the low pH. Recently, studies supporting this entry mechanism for influenza virus have been reported (6,7). Hemagglutinin (HA) glycoproteins on the virus envelope were suggested as the molecules responsible for the low pH-induced activity (1, 4). In the present study, we focused on the function of HA isolated from virus in comparison with that of the parent virus. We also studied functions of bromelain-released HA (BHA), which lacks the COOH-terminal hydrophobic anchoring peptide (8), and of the precursor form of HA, HAo. MATERIALS AND METHODSInfluenza virus AOPR8 was grown in embryonated chicken eggs and purified as described (9). Virus was also grown in a monolayer culture of chicken embryo fibroblasts to obtain HAo (4). The virus was suspended by Pipes/saline (135 mM NaCI/10 mM Pipes, pH 7.4) and stored at -80°C.HA was isolated by solubilization with detergent and centrifugation in sucrose density gradients. Virus was treated with Triton X-100 (4 mg/mg of viral protein...

Haemagglutinin of Influenza A Virus Is a Target for the Antiviral Effect of NorakinR

“…4, trypsinization of haemagglutinin-containing autoliposomes (as in the experiments of Skehel et al, 1982) at pH 7.0 caused no change in HA1 or HA2, but at acid pH the HA1 subunit was markedly affected. As was also found by Skehel et al (1982) and Sato et al (1983), this protein disintegrated to give a product with a mol. wt.…”

“…The data in the literature suggest that, to induce fusion (red blood cell lysis) the influenza virus haemagglutinin must undergo certain conformational changes which take place only at acid pH (Skehel et al, 1982;Sato et al, 1983;Daniels et al, 1983). The experiments with the haemagglutinin of influenza virus performed by Skehel et al (1982) and Sato et al (1983) demonstrated that these changes result in haemagglutinin aggregation which can be revealed by centrifugation in sucrose gradients and by electron microscopy.…”

“…The experiments with the haemagglutinin of influenza virus performed by Skehel et al (1982) and Sato et al (1983) demonstrated that these changes result in haemagglutinin aggregation which can be revealed by centrifugation in sucrose gradients and by electron microscopy. Sato et al (1983) showed that intact virions of influenza virus also formed aggregates at acid pH.…”

“…The experiments with the haemagglutinin of influenza virus performed by Skehel et al (1982) and Sato et al (1983) demonstrated that these changes result in haemagglutinin aggregation which can be revealed by centrifugation in sucrose gradients and by electron microscopy. Sato et al (1983) showed that intact virions of influenza virus also formed aggregates at acid pH. In the experiments of Kawasaki et al (1983) and Hosaka et al (1983), red blood cell lysis could be induced by liposomes containing the influenza virus haemagglutinin; therefore, we studied the influence of Norakin on FPV haemagglutinin aggregation at acid pH using preparations of autoliposomes containing the FPV haemagglutinin whose characteristics are given above.…”

“…The experiments of Skehel et al (1982) and Sato et al (1983) showed that the HA1 subunit of the haemagglutinin of influenza virus becomes sensitive to trypsin as a result of certain conformational changes occurring at acid pH. We studied the effect of Norakin on this process.…”