Hemagglutinin protein of Peste des Petits Ruminants virus (PPRV) activates the innate immune response via Toll-like receptor 2 signaling.

The toll-like receptor (TLR) family comprises both cell-surface and intracellular receptors that recognize different types of pathogen-associated molecular patterns (PAMPs) leading to the production of pro-inflammatory cytokines and subsequent development of adaptive immunity. TLR2 is a cell-surface receptor initially thought to act as a bacterial sentinel but also shown to recognize a number of viral glycoproteins. In this study, we sought to characterize the role of TLR2 in the activation of the immune response by peste des petits ruminants virus (PPRV), a morbillivirus of the Paramixoviridae family that causes an acute, highly contagious disease in goats and sheep. Using human embryonic kidney (HEK) 293 cells stably expressing human (h)TLR2 but lacking any other TLR, we found that PPRV induces IL-8 production in a dose-dependent manner. That activation is only observed in cells expressing hTLR2 and is greatly reduced when the receptor is blocked by pretreatment with specific antibody. We identified hemagglutinin (H) as the viral protein responsible of TLR2 activation by performing the same assays with purified recombinant mammalian-expressed H protein. Exogenous addition of recombinant H protein to cell culture induces high levels of interleukin (IL)-8 only in TLR2-expressing cells. Moreover, H engagement on TLR2 in the monocytic cell line THP-1 activates extracellular-signal-regulated kinase (ERK) signaling. Stimulation of primary ovine dendritic cells with either inactivated PPRV or purified recombinant H protein results in transcription of pro-inflammatory cytokines and the secretion of the Th1-polarizing cytokine IL-12. The role of these host immune mechanisms in the control of PPR is discussed.

Superior molecularly altered influenza virus hemagglutinin peptide 308-317 inhibits collagen-induced arthritis by inducing CD4+ Treg cell expansion.

OBJECTIVE: To investigate the inhibitory effect and possible mechanism of a novel influenza virus hemagglutinin 308-317 peptide (altered HA308-317 peptide) in collagen-induced arthritis (CIA). METHODS: CIA was induced in DBA/1 mice by immunization with type II collagen (CII). Altered HA308-317 peptide, wild HA308-317 peptide, wild CII263-272 peptide, and irrelevant peptide were administered intranasally beginning at arthritis onset. Clinical and histologic scores were assessed, and cytokine levels were determined in the serum or in supernatants from splenocytes. Characteristics of T cell subsets in response to different peptides were analyzed both in vivo and in vitro. RESULTS: Intranasal administration of wild CII263-272 peptide, wild HA308-317 peptide, or altered HA308-317 peptide could significantly ameliorate CIA, but altered HA308-317 peptide showed greater therapeutic effects than wild CII263-272 peptide and wild HA308-317 peptide. The effect of altered HA308-317 peptide was associated with a substantial decrease in production of interleukin-17 (IL-17) and interferon-γ (IFNγ) and with a marked increase in production of IL-10 and transforming growth factor ß, both in serum and in supernatants from splenocytes treated with altered HA308-317 peptide. Both the number and function of CD4+ Treg cells were significantly up-regulated by altered HA308-317 peptide, with a decreased induction of Th1 cells (CD4+IFNγ+) and Th17 cells (CD4+IL-17+). Adoptive transfer of CD4+CD25+ T cells from altered HA308-317 peptide-treated mice resulted in greater suppressive capacity in ameliorating CIA severity than did adoptive transfer of CD4+CD25+ T cells from wild HA308-317 peptide-treated, wild CII263-272 peptide-treated, or irrelevant peptide-treated mice. CONCLUSION: Intranasal administration of altered HA308-317 peptide potently suppressed the severity of CIA by increasing the number and function of CD4+ Treg cells, suggesting that altered HA308-317 peptide might be a promising candidate for treatment of rheumatoid arthritis.

Heterosubtypic protective immunity against influenza A virus induced by fusion peptide of the hemagglutinin in comparison to ectodomain of M2 protein.

Several types of influenza vaccines are available, but due to the highly unpredictable variability of influenza virus surface antigens (hemagglutinin (HA) and neuraminidase) current vaccines are not sufficiently effective against broad spectrum of the influenza viruses. An innovative approach to extend the vaccine efficacy is based on the selection of conserved influenza proteins with a potential to induce inter-subtype protection against the influenza A viruses. A promising new candidate for the preparation of broadly protective vaccine may be a highly conserved N-terminal part of HA2 glycopolypeptide (HA2 gp) called fusion peptide. To study its capacity to induce a protective immune response, we immunized mice with the fusion peptide (aa 1-38 of HA2 gp). The protective ability of fusion peptide was compared with the ectodomain aa 2-23 of M2 protein (eM2) that is antigenically conserved and its immunogenic properties have already been well documented. Corresponding peptides (both derived from A/Mississippi/1/85 (H3N2) virus) were synthesized and conjugated to the keyhole limpet hemocyanin (KLH) and used for the immunization of mice. Both antigens induced a significant level of specific antibodies. Immunized mice were challenged with the lethal dose of homologous (H3N2) or heterologous A/PR/8/34 (H1N1) influenza A viruses. Immunization with the fusion peptide led to the 100% survival of mice infected with 1 LD50 of homologous as well as heterologous virus. Survival rate decreased when infectious dose was raised to 2 LD50. The immunization with eM2 induced effective cross-protection of mice infected even with 3 LD50 of both challenge viruses. The lower, but still effective protection induced by the fusion peptide of HA2 gp suggested that besides ectodomain of M2, fusion peptide could also be considered as a part of cross-protective influenza vaccine. To our knowledge, this is the first report demonstrating that active immunization with the conjugated fusion peptide of HA2 gp provided the effective production of antibodies, what contributed to the cross-protection against influenza infection.

Vaccination with a synthetic peptide from the influenza virus hemagglutinin provides protection against distinct viral subtypes.

Current influenza virus vaccines protect mostly against homologous virus strains; thus, regular immunization with updated vaccine formulations is necessary to guard against the virus' hallmark remodeling of regions that mediate neutralization. Development of a broadly protective influenza vaccine would mark a significant advance in human infectious diseases research. Antibodies with broad neutralizing activity (nAbs) against multiple influenza virus strains or subtypes have been reported to bind the stalk of the viral hemagglutinin, suggesting that a vaccine based on this region could elicit a broadly protective immune response. Here we describe a hemagglutinin subunit 2 protein (HA2)-based synthetic peptide vaccine that provides protection in mice against influenza viruses of the structurally divergent subtypes H3N2, H1N1, and H5N1. The immunogen is based on the binding site of the recently described nAb 12D1, which neutralizes H3 subtype viruses, demonstrates protective activity in vivo, and, in contrast to a majority of described nAbs, appears to bind to residues within a single α-helical portion of the HA2 protein. Our data further demonstrate that the specific design of our immunogen is integral in the induction of broadly active anti-hemagglutinin antibodies. These results provide proof of concept for an HA2-based influenza vaccine that could diminish the threat of pandemic influenza disease and generally reduce the significance of influenza viruses as human pathogens.

Direct ex vivo analyses of HLA-DR1 transgenic mice reveal an exceptionally broad pattern of immunodominance in the primary HLA-DR1-restricted CD4 T-cell response to influenza virus hemagglutinin.

The recent threat of an avian influenza pandemic has generated significant interest in enhancing our understanding of the events that dictate protective immunity to influenza and in generating vaccines that can induce heterosubtypic immunity. Although antigen-specific CD4 T cells are known to play a key role in protective immunity to influenza through the provision of help to B cells and CD8 T cells, little is known about the specificity and diversity of CD4 T cells elicited after infection, particularly those elicited in humans. In this study, we used HLA-DR transgenic mice to directly and comprehensively identify the specificities of hemagglutinin (HA)-specific CD4 T cells restricted to a human class II molecule that were elicited following intranasal infection with a strain of influenza virus that has been endemic in U.S. human populations for the last decade. Our results reveal a surprising degree of diversity among influenza virus-specific CD4 T cells. As many as 30 different peptides, spanning the entire HA protein, were recognized by CD4 T cells, including epitopes genetically conserved among H1, H2, and H5 influenza A viruses. We also compared three widely used major histocompatibility class II algorithms to predict HLA-DR binding peptides and found these as yet inadequate for identifying influenza virus-derived epitopes. The results of these studies offer key insights into the spectrum of peptides recognized by HLA-DR-restricted CD4 T cells that may be the focus of immune responses to infection or to experimental or clinical vaccines in humans.

In vivo biotinylation of the major histocompatibility complex (MHC) class II/peptide complex by coexpression of BirA enzyme for the generation of MHC class II/tetramers.

Success in generation of major histocompatibility complex (MHC) tetramer relies on application of a key technique, biotinylation of MHC molecule specifically on a single lysine residue using the BirA enzyme. However, in vitro biotinylation of MHC-BSP (BirA enzyme substrate peptide) fusion protein using BirA enzyme is laborious and is prone to losses of target proteins to unacceptable levels. To circumvent this problem, an in vivo biotinylation strategy was developed where the BirA enzyme was coexpressed with target protein, HLA-DR2BSP/MBP, in an insect cell expression system. Bacterial BirA enzyme expressed in Drosophila melanogaster 2 (D. Mel-2) cell lines was biologically functional and was able to biotinylate secretary target protein (on specific lysine residue present on the BSP tag). Biotinylation efficiency was maximized by providing exogenous d-biotin in the culture medium and optimization of the expression vector ratios for cotransfection. By limiting dilution cloning, a clone was identified where the expressed DR2BSP/MBP protein was completely biotinylated. DR2BSP/MBP protein expressed and purified from such a clone was ready to be tetramerized with streptavidin to be used for staining antigen-specific T cells.

MHC class II tetramers containing influenza hemagglutinin and EBV EBNA1 epitopes detect reliably specific CD4(+) T cells in healthy volunteers.

Tracking antigen specific T cells with major histocompatibility complex (MHC) tetramers has provided us with insights into the dynamics of the adaptive immune system and holds great promise to aid in patient management and drug and vaccine development. Progress has been made primarily using MHC class I tetramers to monitor CD8(+) T cells, whereas corresponding efforts to stain CD4(+) T cells with class II tetramers have not been as successful. Two major reasons have been proposed for this lack of progress: (1). The frequency of antigen-specific CD4(+) T cells is lower than the frequency of CD8(+) T cells and (2). some, but not all, antigen- specific CD4(+) T cells can bind tetramer because of low functional avidity. In this study, we asked if CD4(+) T cells specific for common human viruses (e.g., influenza and Epstein-Barr) can be detected in healthy individuals previously exposed to them. We were able to clearly detect specific CD4(+) T cells in all donors after in vitro expansion of peripheral blood mononuclear cells. Furthermore, we observe a clear separation of tetramer negative and tetramer positive CD4(+) T cells in most samples similar to patterns commonly seen with class I tetramers. The data indicate that MHC class II tetramers can be used reliably for the identification of CD4(+) T cells specific for ubiquitous infectious agents in normal donors.

Differential antigen sensitivity and costimulatory requirements in human Th1 and Th2 antigen-specific CD4+ cells with similar TCR avidity.

The differentiation of naive CD4(+) Th cells into Th1 and Th2 phenotypes is influenced by cytokines, concentration of Ag, accessory molecules, and the affinity of the MHC-TCR interaction. To study these factors in human memory T cells, T cell lines with Th1 or Th2 phenotypes specific for the peptide hemagglutinin (HA)(307-319) in the context of DRB1*0401 were established from the peripheral blood of an individual previously vaccinated for influenza virus. Flow cytometric analysis with fluorescent-labeled MHC class II tetramers was used to analyze TCR avidity: the Th2 line bound the HLA-DR*0401-HA(307-319) tetramers with higher mean avidity, although the range of binding avidity largely overlapped with the Th1 line. High-affinity Th1 and Th2 lines were established for further study by FACS sorting. When activated with plate-bound HLA-DR*0401-HA(307-319) monomers, the Th1 line proliferated and produced IFN-gamma without additional costimulation whereas the Th2 line required the addition of soluble anti-CD28 Ab to induce proliferation and IL-5 production, but this requirement could be overcome with high concentrations of plate-bound monomer alone. IL-2 production was dependent on costimulation in both cell lines. These findings demonstrate that upon antigenic rechallenge, Th1 and Th2 cells differ in their response to Ag-specific stimulation. Th2 cells were sensitive to the strength of signal to a greater degree than Th1 cells and required costimulation through CD28 for maximal proliferation. These distinctions between Th1 and Th2 activation are not consistent with a simple avidity model of Ag recognition and indicate both qualitative and quantitative differences in determining cell lineage commitment.

[ Influenza virus proteins modulate hemostasis in vitro and in vivo ]. / Belki virusa grippa moduliruiut gemostaz in vitro i in vivo.

We studied the effect of influenza virus proteins--hemagglutinin, neuraminidase, nucleoprotein, and membrane protein--on hemostasis in vitro and in vivo. The obtained data demonstrated that the envelope proteins hemagglutinin and neuraminidase increased the plasma fibrinolytic and anticoagulant activities and the activity of human tissue plasminogen activator. Among the core proteins of influenza virus, membrane protein proved to have the highest activity; in contrast to hemagglutinin and neuraminidase, it inhibited fibrinolysis, increased the coagulation activity of the plasma, and decreased the activity of human tissue plasminogen activator. Combined action of hemagglutinin and neuraminidase increased the plasma fibrinolytic and anticoagulant activities exceeding their individual effects. Combined action of an envelope protein hemagglutinin and membrane protein also increased the plasma fibrinolytic and anticoagulant activities although to a lesser extent as compared to hemagglutinin alone. The obtained data indicate that the viral proteins are physiologically active and can induce hemostatic changes specific for influenza.

Antigen-specific elimination of T cells induced by oligomerized hemagglutinin (HA) 306-318.

In a previous study we reported that oligomerized T cell epitopes "superactivated" CD4+ T cells. These oligomers, consisting of 12-16 copies of a peptide epitope derived from the hemagglutinin protein of influenza virus (HA306-318), induced a specific T cell response in amounts as little as 5 pg/ml. We now show that the improved antigenicity of these multimerized epitopes can also be utilized to induce "high zone tolerance". Tolerization, similar to activation, occurred at about 3 logs lower concentration of oligomer than of peptide. HA306-318-specific T cell cultures became nonresponsive to stimulation with peptide after incubation with 0.5-5 microg/ml HA306-318 12-mer. The nonresponsiveness was accompanied by a drastic down-regulation of the TCR and by T cell elimination by apoptotic cell death. In contrast, stimulation with peptide even at 50 microg/ml led to temporary induction of anergy. Consequently, induction of tolerance with the oligomer was permanent and no recovery of the cultures was seen in recall experiments 12-14 days after high zone exposure to the 12-mer.

Intranasal immunization against influenza virus using polymeric particles.

The aim of this study was to evaluate the potential of poly(D,L-lactide-co-glycolide) nano-and microspheres, with a mean diameter of 220 nm and 8 microm, respectively, to enhance the nasal and systemic immune responses against influenza virus antigen. High encapsulation levels of antigen were achieved in all cases. Neither the molecular weight nor the antigenicity of the entrapped antigen were affected by the encapsulation procedure. Following nasal immunization, the nasal washes IgA and the serum IgG responses were evaluated. With the soluble antigen, relatively high immune responses were observed. With nanospheres, nasal washes IgA levels were significantly lower (p<0.01) and serum IgG levels were not significantly different (p>0.05) from those obtained with the soluble antigen. With microspheres, both nasal washes IgA and serum IgG levels were significantly lower (p<0.01 and <0.05, respectively) as compared to the levels found for the soluble antigen. In addition, fluorescent microspheres administered intranasally failed to reach the nasal-associated lymphoid tissue (NALT). This lack of particle uptake by NALT and the high immunogenicity of the antigen used in this study, could explain the absence of enhancement of the immune responses by the polymeric particles.

[The effect of influenza virus hemagglutinin on erythrocytes and thrombocytes]. / Vliianie gemaggliutinina virusa grippa na éritrotsity i trombotsity.

We studied the effect of influenza virus hemagglutinin on the volume of erythrocytes and aggregation activity of thrombocytes. Hemagglutinin proved to increase the erythrocyte volume, which ends in their lysis, and to induce disaggregation of the thrombocytes. The effect of hemagglutinin on the erythrocytes and thrombocytes was dose-dependent.

Induction of T cell anergy by low numbers of agonist ligands.

Engagement of TCR by its ligand, the MHC/peptide complex, causes T cell activation. T cells respond positively to stimulation with agonists, and are inhibited by antagonist MHC/peptide ligands. Failure to induce proper conformational changes in the TCR or fast TCR/MHC dissociation are the leading models proposed to explain anergy induction by antagonist ligands. In this study, we demonstrate that presentation of between 1 and 10 complexes of agonist/MHC II by unfixed APC induces T cell anergy that persists up to 7 days and has characteristics similar to anergy induced by antagonist ligand or TCR occupancy without costimulation. Furthermore, anergy-inducing doses of hemagglutinin 306-318 peptide led to the engagement of less than 1000 TCR/CD3 complexes. Thus, engagement of a subthreshold number of TCR by either a low density of agonist/MHC or a 2-3 orders of magnitude higher density of antagonist/MHC causes anergy. Moreover, we show that anergy induced by low agonist concentrations is inhibited in the presence of IL-2 or cyclosporin A, suggesting involvement of the calcineurin signaling pathway.

Fluoride augments the mitogenic and antigenic response of human blood lymphocytes in vitro.

It has been shown that fluoride, the agent responsible for reduction of dental caries worldwide and a recognized proliferative agent, is an adjuvant when given intragastrically to rats. Furthermore, plasma fluoride levels increase in humans after various fluoride treatments. The studies presented here show that fluoride also has the ability to affect the cells of the human immune system. This was tested by measuring the effect of sodium fluoride (NaF) on cytokine production by human whole blood cells stimulated in vitro. These studies revealed that NaF augments the human lymphocyte response from human blood to a mitogen (phytohemagglutinin, PHA) or a specific antigen (morbilli antigen from infected cells, MorbAg). The cytokine interferon-gamma (IFN-gamma), released from activated T and/or NK cells, was significantly (p<0.01) increased when whole blood cells were simultaneously incubated with 0.62 mmol/l NaF and PHA compared to PHA alone. This tendency was also true for NaF and MorbAg. The lymphocyte activation marker interleukin-2 receptor (measured in soluble form) increased after simultaneous stimulation of the cells with PHA and 0.62 mmol/l NaF compared to stimulation with PHA only. However, 0.62 mmol/l NaF did not enhance interleukin-6 release, in blood mainly produced by monocytes. The ability to influence the IFN-gamma release during an immune response could be one of the primary means by which the fluoride ion influences the immune system.

Redirecting the complete T cell receptor/CD3 signaling machinery towards native antigen via modified T cell receptor.

We show that a chimeric T cell receptor (TCR) beta chain consisting of a single-chain Fv portion derived from a monoclonal antibody and the full TCR beta chain is able to assemble functionally with endogenous TCR/CD3 components and transfer the antibody specificity as well as the TCR specificity into TCR beta- as well as into TCR beta+ T cells. This allows the incorporation new non-major histocompatibility complex-restricted ligand specificities into the intact TCR/CD3 complex which can exploit the full range of biological activities of the endogenous TCR signaling machinery. This approach can provide wider opportunities to redirect T cells to virus or tumor antigen-bearing cells.

The roles of Fas/APO-1 (CD95) and TNF in antigen-induced programmed cell death in T cell receptor transgenic mice.

The possible involvement of Fas/APO-1 (CD95) and TNF in antigen-specific AICD of thymocytes and mature T cells has been investigated. Antigenic stimulation in vivo of influenza hemagglutinin (HA)-specific TCRtg mice was used to demonstrate that the kinetics of thymocyte and peripheral CD4+ T cell deletion are similar in mice with normal (+/+) or defective Fas (lpr/lpr) background, indicating that a Fas-independent pathway(s) is responsible for the deletion of activated T cells. TCRtg-+/+ or TCRtg-lpr/lpr mice injected with murine TNF-blocking MAb (TN3) showed rapid apoptosis of thymocytes after HA stimulation, indicating that death signaling through Fas and TNF receptors is not essential for HA-induced thymocyte deletion. CDC peripheral T cells in TCRtg-lpr/lpr mice did not undergo apoptosis following injection with HA and TN3, indicating that TNF-mediated apoptosis is involved in the deletion of mature T cells after antigenic stimulation. However, apoptosis still occurred in TCRtg-+/+ mice injected with TN3, indicating that both Fas- and TNF-mediated cell death can contribute to the deletion of activated peripheral T cells.

Induction of anergy in human T helper 0 cells by stimulation with altered T cell antigen receptor ligands.

CD4+ T cells may become profoundly unresponsive to antigenic restimulation following ligation of TCR by immunogenic peptides bound to MHC class II molecules in the absence of costimulation. Furthermore, it has been reported that anergy can be induced as a consequence of engagement of TCR by analogues of antigenic peptides presented by live APCs. In this study, based on resolution of the crystal structure of an influenza virus hemagglutinin (HA) peptide (HA 306-318) bound to HLA-DRB1*0101, we investigated the potential of analogues with amino acid substitutions at those positions predicted to form interactions with TCR to differentially activate and/or anergize HA-specific human Th0 cells restricted by DR1 class II molecules. For some analogues altering the affinity of peptide/TCR interactions revealed a direct positive correlation between antigenicity and their ability to induce anergy. Nevertheless, certain HA peptide analogues functioned as partial agonists, which although they failed to stimulate clonal expansion, were capable of rendering the Th0 cells unresponsive to immunogenic rechallenge. Furthermore, differences were noticed in the characteristics of the anergic phenotype induced by selected analogues. Restimulation with the native peptide of Th0 cells pre-exposed to the HA analogues in the absence of costimulatory signals failed to uncouple IL-4 and IFN-gamma secretion; however, in some instances, dissociation of proliferation from cytokine production was observed. The ability to differentially signal T cells through changing the affinity of peptide/TCR interactions may have implications in the potential use of altered TCR ligands in immunotherapy.

Intravenous injection of soluble antigen induces thymic and peripheral T-cells apoptosis.

The mechanism by which tolerance is induced via systemic administration of high doses of aqueous antigen has been analyzed by using mice transgenic for a T-cell receptor specific for the influenza virus hemagglutinin (HA) peptide comprising amino acids 126-138. After intravenous injection of 750 (but not 75) micrograms of HA peptide, a state of hyporesponsiveness was rapidly induced. In the thymus, in situ apoptosis in the cortex and at the corticomedullary junction was responsible for a synchronous and massive deletion of CD4+ CD8+ thymocytes. In secondary lymphoid organs, HA-reactive T cells were initially activated but were hyporesponsive at the single cell level. After 3 days, however, those cells were rapidly deleted, at least partially, through an apoptotic process. Therefore, both thymic and peripheral apoptosis, in addition to T-cell receptor desensitization, contribute to high-dose tolerance.

Oligonucleotide targeting to alveolar macrophages by mannose receptor-mediated endocytosis.

Antisense oligonucleotides (ONs) have proven useful for selective inhibition of gene expression. However, their effective use is limited by inefficient cellular uptake and lack of cellular targeting. In this paper, we report a drug targeting system which utilizes mannose receptor-mediated endocytosis to enhance cellular uptake of ONs in alveolar macrophages (AMs). The system employs a molecular complex consisting of partially substituted mannosylated poly(L-lysine) (MPL), electrostatically linked to a 5' fluorescently labeled ON. Upon recognition by the macrophage mannose receptors, the MPL was internalized by the receptor-mediated pathway, co-transporting the ON. Our results indicate that the AMs treated with the MPL:ON complex exhibited a significant increase in ON uptake (up to 17-fold) over free ON-treated controls. Effective ON uptake was shown to require the recognition of the mannose moiety since unmodified polylysine was much less effective in promoting ON uptake. Specific internalization of the ON:MPL complex by the mannose receptor pathway was verified by competitive inhibition using mannosylated albumin. Under this condition, the ON complex uptake was inhibited. The requirement of mannose receptors for complex uptake was further demonstrated using a macrophage cell line, J774.1, which expresses a low level of mannose receptors. When treated with the complex, these cells showed no susceptibility to ON uptake, thus suggesting the targeting ability of the carrier system to the AMs. Following cellular internalization, the ON complex appeared largely accumulated in endocytic vesicles. Enhanced endosomal exit of the ON was achieved using a fusogenic peptide derived from the amino terminal sequence of influenza virus hemagglutinin HA2. Cytotoxicity studies showed that at the concentrations effectively enhancing ON uptake, both MPL and the fusogenic peptide caused no toxic effects to the cells, thereby suggesting their potential safety and utilization in vivo.

Inhibition of influenza-induced membrane fusion by lysophosphatidylcholine.

Lysolipids have been reported to inhibit various membrane fusion events, and it was suggested that inhibition was due to their "inverted cone" shape, which hinders the formation of intermediate lipid structures required for fusion (Chernomordik, L. V., Vogel, S. S., Sokoloff, A., Onaran, H. O., Leikina, E. A., and Zimmerberg, J. (1993) FEBS Lett. 318, 71-76). Here, the effect of lysophosphatidylcholine (LPC) on fusion mediated by the hemagglutinin (HA) of influenza virus was investigated. Virus-liposome fusion was inhibited by LPC if the lysolipid was added to the membranes from an aqueous stock solution but not if LPC was symmetrically distributed over both leaflets of the liposomal bilayer. These findings would be consistent with an effect of LPC on lipid intermediate formation, but inhibition increased with increasing acyl chain length and thus a less pronounced inverted cone shape of the lysolipids suggesting that the mechanism of inhibition might be different. At low pH, due to the exposure of the fusion peptide of HA, followed by its insertion into the liposomal membrane, virus acquires the ability to bind to zwitterionic liposomes lacking receptors for HA. This type of binding was inhibited by LPC. Moreover, leakage of calcein from receptor-containing liposomes, induced by purified HA at low pH, was inhibited by LPC. Therefore, the inhibition of influenza-induced fusion by LPC was caused by the binding of LPC to fusion peptides, thereby preventing their interaction with the target membrane rather than an effect on intermediate lipid structures.