Interaction of influenza virus hemagglutinin with a lipid monolayer. A comparison of the surface activities of intact virions, isolated hemagglutinins, and a synthetic fusion peptide

In the infectious entry pathway of influenza virus, the low pH of the endosomal compartment induces an irreversible conformational change in influenza virus hemagglutinin, leading to fusion of viral and endosomal membranes. In the current report, we characterized the low-pH-induced activation of hemagglutinin of influenza strain X31 by studying its interaction with a lipid monolayer. The surface activities of virions, of isolated hemagglutinins and its proteolytic fragments, and of a synthetic peptide mimicking the amino terminus of subunit 2 of hemagglutinin are compared. The data indicate that the surface activity of both virions and isolated hemagglutinin develop as a result of the low-pH-induced conformational change in hemagglutinin. The surface activity of isolated hemagglutinin is mainly caused by penetration into the lipid monolayer of protein domains other than the amino terminus of subunit 2 of hemagglutinin; domains in subunit 1 may be involved. The surface activity of virions appears to be a secondary effect of the conformational change and is explained by assuming a net transfer of viral lipids to the lipid monolayer.     

Effect of Antibody to Neuraminidase on the Maturation and Hemagglutinating Activity of an Influenza A2 Virus

Antiserum to a recombinant between an A(o) and an A(2) influenza virus had no detectable antibody against an A(2) virus in standard hemagglutination-inhibition tests, and inhibited 95% of viral neuraminidase activity at a 1 to 400 dilution. However, on mixing virus with antiserum, a drop of up to 90% in hemagglutinin titer was observed. The effects of ultrasonication and direct electron microscopic examination indicated that the antiserum caused aggregation of virus particles. When antiserum was added to A(2) virus-infected chick embryo fibroblasts, release of virus appeared markedly inhibited. After ultrasonication to disrupt aggregates, an increase in released hemagglutinin was observed, but the resulting level was considerably lower than that in control cultures containing normal rabbit serum. In thin sections of infected cells, similar numbers of virus profiles were observed in control and antiserum-treated cultures. A marked increase in release of hemagglutinin was noted if receptor-destroying enzyme was added to antiserum-treated cultures. The results indicate that antibody to neuraminidase does not exert a direct effect on viral maturation, but inhibits the detachment of viral progeny from cell surface receptors.     

Role of receptor-binding activity of the viral hemagglutinin molecule in the presentation of influenza virus antigens to helper T cells.

The concentration of antigen required to stimulate influenza virus-specific helper T cells was observed to be dependent upon the antigenic form bearing the relevant determinant: intact, nonreplicative virus was needed only in picomolar amounts, while denatured proteins, protein fragments, or synthetic peptides were required in micromolar concentrations for a threshold level of stimulation. Antigenic efficiency of intact virus was found to result from the attachment of virus to sialic acid residues on the surface of the antigen-presenting cell since spikeless viral particles lacking the hemagglutinin molecule were much less efficient antigens for helper T cells and continuous presence of hemagglutination-inhibiting antihemagglutinin antibodies reduced efficiency of stimulation by intact virus approximately 100-fold for both hemagglutinin and internal virion proteins. Influenza virus associated rapidly with antigen-presenting cells; less than 10 min at 20 degrees C was sufficient to introduce virus for a maximal level of T-cell stimulation. This rapid attachment was blocked by antibodies to the hemagglutinin or by pretreatment of the antigen-presenting cells with neuraminidase to remove the cellular virus receptor. Following viral adsorption by antigen-presenting cells, a lag period of 30 min at 37 degrees C was required for the expression of helper T-cell determinants. One early event identified was the movement of the virus to a neuraminidase-insensitive compartment, which can occur at 10 degrees C, but which was not equivalent to expression of helper T-cell determinants. Preincubation of cells with virus at 10 degrees C for 4 h reduced the lag period of helper T-cell determinant expression to 15 min when these cells were shifted to 37 degrees C, suggesting that transition of the virus to a neuraminidase-resistant state is a required step in presentation of T-cell antigenic determinants.     

Recognition of noninfectious influenza virus by class I-restricted murine cytotoxic T lymphocytes

We have recently shown that murine target cells can be sensitized for lysis by class I-restricted influenza virus-specific cytotoxic T lymphocytes (CTL) using noninfectious influenza virus. Sensitization is dependent on inactivation of viral neuraminidase activity (which can be achieved by heating virus); and requires fusion of viral and cellular membranes. In the present study, we have examined recognition of antigens derived from heat-treated virus by cloned CTL lines induced by immunization with infectious virus. Target cells sensitized with heat-treated virus were recognized by all 11 CTL clones that were specific for internal virion proteins (nucleoprotein and basic polymerase 1), and by one of six clones specific for the major viral glycoprotein (the hemagglutinin). Immunization of mice with heat-treated virus primed their splenocytes for secondary in vitro CTL responses. CTL generated in this manner recognized target cells infected with recombinant vaccinia virus expressing cloned influenza virus gene products. These findings indicate that both integral membrane proteins and internal proteins that comprise virions can be processed by antigen-presenting cells for recognition by class I-restricted CTL. It also appears that not all hemagglutinin determinants recognized on virus-infected cells are presented by cells sensitized with heat-treated virus.     

Expression of defective measles virus genes in brain tissues of patients with subacute sclerosing panencephalitis.

The persistence of measles virus in selected areas of the brains of four patients with subacute sclerosing panencephalitis (SSPE) was characterized by immunohistological and biochemical techniques. The five measles virus structural proteins were never simultaneously detectable in any of the brain sections. Nucleocapsid proteins and phosphoproteins were found in every diseased brain area, whereas hemagglutinin protein was detected in two cases, fusion protein was detected in three cases, and matrix protein was detected in only one case. Also, it could be shown that the amounts of measles virus RNA in the brains differed from patient to patient and in the different regions investigated. In all patients, plus-strand RNAs specific for these five viral genes could be detected. However, the amounts of fusion and hemagglutinin mRNAs were low compared with the amounts in lytically infected cells. The presence of particular measles virus RNAs in SSPE-infected brains did not always correlate with mRNA activity. In in vitro translations, the matrix protein was produced in only one case, and the hemagglutinin protein was produced in none. These results indicate that measles virus persistence in SSPE is correlated with different defects of several genes which probably prevent assembly of viral particles in SSPE-infected brain tissue.     

Effects of low pH on influenza virus. Activation and inactivation of the membrane fusion capacity of the hemagglutinin

The hemagglutinin of influenza virus undergoes a conformational change at low pH, which results in exposure of a hydrophobic segment of the molecule, crucial to expression of viral fusion activity. We have studied the effects of incubation of the virus at low pH either at 37 or 0 degrees C. Treatment of the virus alone at pH 5.0 induces the virus particles to become hydrophobic, as assessed by measuring the binding of zwitterionic liposomes to the virus. At 37 degrees C this hydrophobicity is transient, electron microscopic examination of the virus reveals a highly disorganized spike layer, and fusion activity toward ganglioside-containing zwitterionic liposomes, measured at 37 degrees C with a kinetic fluorescence assay, is irreversibly lost. By contrast, after preincubation of the virus alone at pH 5.0 and 0 degrees C fusion activity remains unaffected. Yet, the preincubation at 0 degrees C does result in exposure of the hydrophobic segment of hemagglutinin, but now hydrophobicity is sustained and viral spike morphology unaltered. Hydrophobicity also remains to a significant extent upon pH neutralization, but fusion activity is negligible under these conditions. It is concluded that for optimal expression of fusion activity the virus must be bound to the target membrane before exposure to low pH. Furthermore, even after exposure of the hydrophobic segment of hemagglutinin, fusion occurs only at low pH. Finally, fusion occurs only at elevated temperature, possibly reflecting the unfolding of hemagglutinin trimers or the cooperative action of several hemagglutinin trimers in the reaction.     

Studies on Antiviral and Antitumor Antibiotics

Antiviral activity of geodin obtained from a soil fungus was studied employing the Newcastle disease virus—chick embryo fibroblasts culture system. In a plate assay method, the minimum inhibitory concentration was about 9 μg/ml and cytotoxicity was detected at 36 μg/ml. Hemagglutinin synthesis was completely suppressed in a tube assay method when 4 μg/ml of geodin was added after the infection (50 plaque forming units/cell), but at this concentration cytotoxic effect of the antibiotic was observed. At sub-inhibitory concentrations, a dose response was shown in the yield of hemagglutinin and infective virus at 16 hr after the infection, and at this time cytopathic effect was partially or completely arrested depending on the antibiotic concentrations even when complete inhibition of hemagglutinin synthesis was not observed. Geodin did not have any effect on the activity of free virus particles and their adsorption to host cells. The inhibition site of geodin exists somewhere between viral adsorption and viral maturation.     

Studies on antiviral glycosides. II. Chemical modifications of the envelope of Sendai virus

Treatment of Sendai virus with p-azidophenyl-6-chloro-6-deoxy-beta-D-glucopyranoside (APG) caused chemical modification of the viral envelope under UV irradiation, which did not affect the hemagglutinin activity of the virus but inhibited the hemolytic activity. Also, the transfer of phospholipid from the viral envelope to chicken erythrocytes was measured using a spinlabel technique by electron spin resonance (ESR). In this experiment, the phospholipid transfer was depressed by the treatment with APG under the conditions which inhibited the hemolytic activity of the virus. These results suggest that APG bound covalently to lipid may disturb the specific interaction between the protein and the lipid of the viral envelope, resulting in the inhibition of the hemolytic activity. The effects of APG on the hemolysis and phospholipid transfer were compared with the results for the concanavalin A- and amphotericin B-treated viruses.     

Membrane fusion induced by influenza virus hemagglutinin requires protein bound fatty acids

The low pH-dependent fusion of lipid membranes induced by two types of the fatty acylated influenza viral hemagglutinin has been studied by use of an energy transfer assay. When protein bound fatty acids were released from the hemagglutinin by hydroxylamine treatment viral fusion activity was inhibited. The extent of fusion inhibition correlates with the amount of fatty acids cleaved from the hemagglutinin. Virosomes prepared from fowl plague virus containing fatty acid free hemagglutinin showed a much lower fusion activity than control virosomes containing fatty acylated hemagglutinin. The hydroxylamine treatment applied has no detectable effects on the virus other than fatty acid release from its spike glycoproteins. These results support our previous hypothesis that protein bound fatty acids are involved in the induction of membrane fusion by the influenza hemagglutinin.     

Purification of the influenza hemagglutinin glycoprotein and characterization of its carbohydrate components.

Hemagglutinin from influenza A/PR8 virus was purified after treatment of the virus with sodium deoxycholate followed by extraction with tri-n-butyl phosphate. This fully disrupted the virus while preserving hemagglutinating activity. The hemagglutinin was obtained in the form of small aggregates that could be separated from other viral components. Purified hemagglutinin was hydrolyzed to determine carbohydrate composition and digested with Pronase to analyze oligosaccharide structures. Sugars present in the hemagglutinin were galactose, mannose, fucose, and glucosamine in molar rates of about 6:11:2:5, and these comprised 16% of the hemagglutinin glycoprotein. Oligosaccharides obtained from virus included a major component of a molecular weight of 2,800, composed of glucosamine, galactose, mannose, and fucose, and a minor heterogenous component of a molecular weight of 1,500 to 2,000, containing predominantly mannose. The 2,800-molecular-weight oligosaccharide was a constituent of the hemagglutinin, and treatment of this large oligosaccharide with specific exo-glycosidases demonstrated the presence of terminal galactose and fucose and allowed the deduction of a general structure for this component.     

A dual reporter gene based system to quantitate the cell fusion of avian influenza virus H5N1

Membrane fusion is central to the entry of influenza virus into host cells. To quantitatively determine the fusion activity of hemagglutinin (HA) of avian influenza virus H5N1, we established a cell fusion assay based on a dual luciferase reporter gene. The HA fusion activity was assayed by measuring luciferase expression in fused cells, allowing a rapid, sensitive, and quantitative comparison of HA fusion activities at various pHs and in different cells types. The simplicity and the quantitative nature of this novel assay are ideally suited for identifying viral receptors or screening for inhibitors of viral entry in the future.     

Intracellular neutralization of influenza virus by immunoglobulin A anti-hemagglutinin monoclonal antibodies.

Traditionally, immunoglobulin A (IgA) was thought to neutralize virus by forming complexes with viral attachment proteins, blocking attachment of virions to host epithelial cells. Recently we have proposed an intracellular action for dimeric IgA, which is actively transported through epithelial cells by the polymeric immunoglobulin receptor (pIgR), in that it may be able to bind to newly synthesized viral proteins within the cell, preventing viral assembly. To this effect, we have previously demonstrated that IgA monoclonal antibodies against Sendai virus, a parainfluenza virus, colocalize with the viral hemagglutinin-neuraminidase protein within infected epithelial cells and reduce intracellular viral titers. Here we determine whether IgA can interact with influenza virus hemagglutinin (HA) protein within epithelial cells. Polarized monolayers of Madin-Darby canine kidney epithelial cells expressing the pIgR were infected on their apical surfaces with influenza virus A/Puerto Rico/8-Mount Sinai. Polymeric IgA anti-HA, but not IgG anti-HA, delivered to the basolateral surface colocalized with HA protein within the cell by immunofluorescence. Compared with those of controls, viral titers were reduced in the supernatants and cell lysates from monolayers treated with anti-HA IgA but not with anti-HA IgG. Furthermore, the addition of anti-IgA antibodies to supernatants did not interfere with the neutralizing activity of IgA placed in the basal chamber, indicating that IgA was acting within the cell and not in the extracellular medium to interrupt viral replication. Thus, these studies provide additional support for the concept that IgA can inhibit replication of microbial pathogens intracellularly.     

Carbohydrates of Influenza Virus. I. Glycopeptides Derived from Viral Glycoproteins After Labeling with Radioactive Sugars

The carbohydrate moiety of the influenza glycoproteins NA, HA₁, and HA₂ were analyzed by labeling with radioactive sugars. Analysis of glycopeptides obtained after digestion with Pronase indicated that there are at least two different types of carbohydrate side chains. The side chain of type I is composed of glucosamine, mannose, galactose, and fucose. It is found on NA, HA₁, and HA₂. The side chain of type II contains a high amount of mannose and is found only on NA and HA₂. The molecular weights of the corresponding glycopeptides obtained from virus grown in chicken embryo cells are 2,600 for type I and 2,000 for type II. The glycoproteins of virus grown in MDBK cells have a higher molecular weight than those of virus grown in chicken embryo cells, and there is a corresponding difference in the molecular weights of the glycopeptides. Under conditions of partial inhibition of glycosylation, virus particles were isolated that contained hemagglutinin with reduced carbohydrate content. Glycopeptide analysis indicated that this reduction is due to the lack of whole carbohydrate side chains and not to the incorporation of incomplete ones. This observation suggests that glycosylation of the viral glycoproteins involves en bloc transfer of the core sugars to the polypeptide chains.     

Interaction of reovirus with cell surface receptors. II. Generation of suppressor T cells by the hemagglutinin of reovirus type 3

In studying reovirus interactions with lymphocytes, we have found that reovirus type 3, but not type 1, inhibits the in vitro proliferative response of murine splenic lymphocytes to concanavalin A (Con A). By analyzing recombinant clones containing genes from both reovirus types 1 and 3, we found that the S1 gene, the gene that encodes the viral hemagglutinin, is responsible for the inhibitory effect. In addition we found that type 3, but not type 1, generates suppressor T cells in vitro capable of suppressing Con A proliferation. By analyzing recombinant clones, we also found that the viral hemagglutinin is responsible for the generation of suppressor T cells by reovirus type 3. These effects were observed whether UV-inactivated or live virus was used. Reovirus type 3 inhibition of the proliferative response of murine splenic lymphocytes to Con A was blocked by anti-reovirus type 3 antibody but not by anti-reovirus type 1 antibody. Antiviral antibody had no effect on the ability of reovirus type 3 induced suppressor cells to inhibit Con A proliferation. We have previously demonstrated a receptor on murine lymphocytes for the hemagglutinin of reovirus type 3, and our results suggest that the in vitro suppression of Con A proliferation of murine lymphocytes by reovirus type 3 is secondary to the interaction of the viral hemagglutinin with a receptor on the surface of murine lymphocytes, which results in the generation of functionally active suppressor T cells.     

An O-glycoside of sialic acid derivative that inhibits both hemagglutinin and sialidase activities of influenza viruses

The compound Neu5Ac3alphaF-DSPE (4), in which the C-3 position was modified with an axial fluorine atom, inhibited the catalytic hydrolysis of influenza virus sialidase and the binding activity of hemagglutinin. The inhibitory activities to sialidases were independent of virus isolates examined. With the positive results obtained for inhibition of hemagglutination and hemolysis induced by A/Aichi/2/68 virus, the inhibitory effect of Neu5Ac3alphaF-DSPE (4) against MDCK cells was examined, and it was found that 4 inhibits the viral infection with IC50 value of 5.6 microM based on the cytopathic effects. The experimental results indicate that compound 4 not only inhibits the attachment of virus to the cell surface receptor but also disturbs the release of the progeny viruses from infected cells by inhibiting both hemagglutinin and sialidase of the influenza viruses. The study suggested that the compound is a new class of bifunctional drug candidates for the future chemotherapy of influenza.     

Monoclonal anti-hemagglutinin antibodies detect irreversible antigenic alterations that coincide with the acid activation of influenza virus A/PR/834-mediated hemolysis.

Exposure of influenza virus to an acidic environment, which is known to be required for viral fusion and hemolysis, has recently been shown to induce a conformational change in the hemagglutinin molecule. In the present study, we examined the effects of acid incubation on the antigenicity, biological activity, and morphology of influenza virus A/PR/8/34 (H1N1). Incubation of PR8 virus at pH 5 in the absence of erythrocytes resulted in a rapid and irreversible loss of viral hemolytic activity and infectivity. Apart from a less distinct appearance of the viral surface projections and slight damage to the envelope structure, acid incubation did not result in gross morphological changes in the viral architecture. The acid-induced change could be detected in the form of greatly increased or decreased binding of many monoclonal antibodies directed to each of the four major antigenic regions of the hemagglutinin. Triggering of viral hemolytic activity and antigenic alterations was similarly pH dependent. In addition, the different pH dependencies of egg-grown and trypsin-treated MDCK-grown viruses coincided with an analogous pH dependence of the antigenic alterations that were observed with these viruses. These observations are compatible with the idea that some of the anti-hemagglutinin antibodies detect conformational changes in the hemagglutinin which are required for the initiation of fusion and hemolysis.     

Analysis of <Emphasis Type="Italic">N</Emphasis>-glycans in embryonated chicken egg chorioallantoic and amniotic cells responsible for binding and adaptation of human and avian influenza viruses

The initial step essential in influenza virus infection is specific binding of viral hemagglutinin to host cell-surface glycan receptors. Influenza A virus specificity for the host is mediated by viral envelope hemagglutinin, that binds to receptors containing glycans with terminal sialic acids. Human viruses preferentially bind to α2→6 linked sialic acids on receptors of host cells, whereas avian viruses are specific for the α2→3 linkage on the target cells. Human influenza virus isolates more efficiently infect amniotic membrane (AM) cells than chorioallantoic membrane (CAM) cells. N-glycans were isolated from AM and CAM cells of 10-day-old chicken embryonated eggs and their structures were analyzed by multi-dimensional HPLC mapping and MALDI-TOF-MS techniques. Terminal N-acetylneuraminic acid contents in the two cell types were similar. However, molar percents of α2→3 linkage preferentially bound by avian influenza virus were 27.2 in CAM cells and 15.4 in AM cells, whereas those of α2→6 linkage favored by human influenza virus were 8.3 (CAM) and 14.2 (AM). Molar percents of sulfated glycans, recognized by human influenza virus, in CAM and AM cells were 3.8 and 12.7, respectively. These results have revealed structures and molar percents of N-glycans in CAM and AM cells important in determining human and avian influenza virus infection and viral adaptation.     

Labeling of influenza viruses with synthetic fluorescent and biotin-labeled lipids

Direct labeling of virus particles is a powerful tool for the visualization of virus–cell interaction events. However, this technique involves the chemical modification of viral proteins that affects viral biological properties. Here we describe an alternative approach of influenza virus labeling that utilizes Function-Spacer-Lipid(FSL) constructs that can be gently inserted into the virus membrane. We assessed whether labeling with fluorescent(fluo-Ad-DOPE) or biotin-labeled(biot-CMG2-DOPE) probes has any deleterious effect on influenza virus hemagglutinin(HA) receptor specificity, neuraminidase(NA) activity, or replicative ability in vitro. Our data clearly show that neither construct significantly affected influenza virus infectivity or viral affinity to sialyl receptors. Neither construct influenced the NA activities of the influenza viruses tested, except the A/Puerto Rico/8/34(H1N1) strain. Our data indicate that lipid labeling provides a powerful tool to analyze influenza virus infection in vitro.     

Persistent infection of cells in culture by measles virus. II. Effect of measles antibody on persistently infected HeLa sublines and recovery of a HeLa clonal line persistently infected with incomplete virus

Rustigian, Robert (Tufts University School of Medicine, Boston, Mass.). Persistent infection of cells in culture by measles virus. II. Effect of measles antibody on persistently infected HeLa clonal line persistently infected with incomplete virus. J. Bacteriol. 92:1805-1811. 1966.-The effect of viral antibody on persistent infection of HeLa cells by the Edmonston strain of measles virus was investigated by culturing cells from three persistently infected clones in medium supplemented with human immune globulin. The three infected HeLa clones were isolated from a persistently infected parent line. Two sublines which were grown in the presence of measles antibody developed a nonyielder state, wherein there is no detectable virus infectious for normal HeLa cultures. There is, however, continued synthesis of intracellular viral antigen and formation of viral intracytoplasmic inclusion bodies. The development of a nonyielder state was associated with a marked decrease in the degree of hemadsorption in cultures of both sublines. Further studies of the viral properties of non-yielder HeLa cell populations were made with a clone obtained from one of these sublines by plating under antibody. Persistent infection in this line was characterized by synthesis of incomplete virus even when the cells were cultured thereafter in anti-body-free medium. This was evidenced by (i) failure to recover infectious virus from the clonal population despite continued formation of intracellular viral antigen and viral intracytoplasmic inclusion bodies in a majority of the cells, (ii) the presence of only a few cells with surface viral antigen(s) including hemagglutinin, and (iii) the relatively weak antibody response to viral envelope antigen(s) after injection of cells into guinea pigs.     

Antigenic modulation induced by monoclonal antibodies: antibodies to measles virus hemagglutinin alters expression of other viral polypeptides in infected cells

Polyclonal antibody to measles virus can have profound effects on external (outer plasma membrane) as well as internal (cytoplasmic) viral polypeptides expressed in infected cells. The process, termed "antibody-induced antigenic modulation," was further investigated by using monoclonal antibody to several viral polypeptides. Four monoclonal antibodies against the viral hemagglutinin had the ability to decrease the expression of the phosphoprotein, fusion, and membrane protein. A monoclonal antibody to the nucleocapsid protein did not cause these changes. The observed decreases were not due to preferential degradation of viral polypeptides as determined by pulse-chase experiments. Our results indicate that a specific signal to an epitope on the plasma membrane (monoclonal antibody measles virus hemagglutinin) can alter the expression of measles virus phosphoprotein and membrane protein, both polypeptides present in the cytoplasm of infected cells.