Aminopeptidase N purified from gypsy moth brush border membrane vesicles is a specific receptor for Bacillus thuringiensis CryIAc toxin.

We have evaluated the binding of Bacillus thuringiensis Cry toxins to aminopeptidase N (APN) purified from Lymantria dispar (gypsy moth) brush border membrane vesicle (BBMV). CryIAc toxin bound strongly to APN, while either the structurally related CryIAa and CryIAb toxins or CryIC, CryIIA, and CryIIIA toxins showed weak binding to APN. An in vitro competition binding study demonstrated that the binding of CryIAc to L. dispar BBMV was inhibited by APN. Inhibition of short circuit current for CryIAc, measured by voltage clamping of whole L. dispar midgut, was substantially reduced by addition of phosphatidylinositol-specific phospholipase C, which is known to release APN from the midgut membrane. In contrast, addition of phosphatidylinositol-specific phospholipase C had only a marginal effect on the inhibition of short circuit current for CryIAa. These data suggest that APN is the major functional receptor for CryIAc in L. dispar BBMV. A ligand blotting experiment demonstrated that CryIAc recognized a 120-kDa peptide (APN), while CryIAa and CryIAb recognized a 210-kDa molecule in L. dispar BBMV. In contrast, CryIAa and CryIAb bound to both the 120- and 210-kDa molecules in Manduca sexta BBMV, while CryIAc recognized only the 120-kDa peptide. The 120-kDa peptide (APN) in L. dispar BBMV reacted with soybean agglutinin, indicating that N-acetylgalactosamine is a component of this glycoprotein.     

N-Acetylneuraminyllactosylceramide, GM3-NeuAc, a new influenza A virus receptor which mediates the adsorption-fusion process of viral infection. Binding specificity of influenza virus A/Aichi/2/68 (H3N2) to membrane-associated GM3 with different molecular species of sialic acid

Agglutinates of native chicken erythrocytes caused by influenza virus A/Aichi/2/68 (H3N2) at 4 degrees C were potently fused and lysed at low pH (optimum pH 5.3) at 37 degrees C. Exogenous gangliosides GM3 (Sia alpha 2-3Gal beta 1-4Glc beta 1-ceramide) and GM2 (GalNAc beta 1-4(Sia alpha 2-3)-Gal beta 1-4Glc beta 1-ceramide) were integrated into the membranes of chicken asialoerythrocytes within 5-min incubation at 37 degrees C. We found that the incorporation of ganglioside GM3 containing N-acetylneuraminic acid into asialoerythrocytes restored the biological responsiveness to the virus as established by agglutination at 4 degrees C and fusion and hemolysis at 37 degrees C at pH 5.3. Biological responsiveness of GM3-NeuAc-erythrocytes to the virus was considerably higher than that of GM3-NeuGc-erythrocytes under the same experimental conditions. Treatment of the GM3-NeuAc-erythrocytes with neuraminidase again resulted in the complete abolishment of the response to the virus. Erythrocytes containing GM2-NeuAc showed no detectable biological responses toward the virus. The above results indicate that the hemagglutinin of influenza virus A/Aichi/2/68 (H3N2) recognizes the sialyloligosaccharide chain of ganglioside GM3 as its receptor which mediates the adsorption and fusion process on the virus entry into the host cells and has more preferential specificity for binding to N-acetylneuraminic acid-containing GM3 than that to N-glycolyl type in the target cell membranes.     

Identification of a Region in the Stalk Domain of the Nipah Virus Receptor Binding Protein That Is Critical for Fusion Activation

Paramyxoviruses, including the emerging lethal human Nipah virus (NiV) and the avian Newcastle disease virus (NDV), enter host cells through fusion of the viral and target cell membranes. For paramyxoviruses, membrane fusion is the result of the concerted action of two viral envelope glycoproteins: a receptor binding protein and a fusion protein (F). The NiV receptor binding protein (G) attaches to ephrin B2 or B3 on host cells, whereas the corresponding hemagglutinin-neuraminidase (HN) attachment protein of NDV interacts with sialic acid moieties on target cells through two regions of its globular domain. Receptor-bound G or HN via its stalk domain triggers F to undergo the conformational changes that render it competent to mediate fusion of the viral and cellular membranes. We show that chimeric proteins containing the NDV HN receptor binding regions and the NiV G stalk domain require a specific sequence at the connection between the head and the stalk to activate NiV F for fusion. Our findings are consistent with a general mechanism of paramyxovirus fusion activation in which the stalk domain of the receptor binding protein is responsible for F activation and a specific connecting region between the receptor binding globular head and the fusion-activating stalk domain is required for transmitting the fusion signal.     

Measles Virus Fusion Machinery Activated by Sialic Acid Binding Globular Domain

Paramyxoviruses, including the human pathogen measles virus (MV) and the avian Newcastle disease virus (NDV), enter host cells through fusion of the viral envelope with the target cell membrane. This fusion is driven by the concerted action of two viral envelope glycoproteins: the receptor binding protein and the fusion protein (F). The MV receptor binding protein (hemagglutinin [H]) attaches to proteinaceous receptors on host cells, while the receptor binding protein of NDV (hemagglutinin-neuraminidase [HN]) interacts with sialic acid-containing receptors. The receptor-bound HN/H triggers F to undergo conformational changes that render it competent to mediate fusion of the viral and cellular membranes. The mechanism of fusion activation has been proposed to be different for sialic acid-binding viruses and proteinaceous receptor-binding viruses. We report that a chimeric protein containing the NDV HN receptor binding region and the MV H stalk domain can activate MV F to fuse, suggesting that the signal to the stalk of a protein-binding receptor binding molecule can be transmitted from a sialic acid binding domain. By engineering the NDV HN globular domain to interact with a proteinaceous receptor, the fusion activation signal was preserved. Our findings are consistent with a unified mechanism of fusion activation, at least for the Paramyxovirinae subfamily, in which the receptor binding domains of the receptor binding proteins are interchangeable and the stalk determines the specificity of F activation.     

Sendai virus membrane fusion: time course and effect of temperature, pH, calcium, and receptor concentration

The conditions that optimize Sendai virus membrane fusion with liposomes have been studied. No fusion occurs in the absence of ganglioside receptors. Maximum fusion occurs when the molar ratio of ganglioside GD1a to phospholipid is 0.02 or greater. The amount of fusion at 37 degrees C increases with time up to at least 6.5 h. The rate of fusion increases from the lowest temperature tested, 10 degrees C, to 40 degrees C. Above 43 degrees C the amount of fusion decreases because of thermal inactivation of the viral proteins. There is a broad pH maximum between pH 7.5 and pH 9.0. At both ends of the pH range the amount of fusion increases and exceeds that found in the physiologic pH range. Neither ethylenediaminetetraacetic acid nor Ca2+ changes the amount of membrane fusion. The optimal conditions for membrane fusion of Sendai virus membranes with liposomes are the same as the optimal conditions for fusion with host cells and with red blood cells. Since the liposomes contain no proteins, the optimal conditions for Sendai virus membrane fusion must be determined by the viral proteins and be mostly independent of the nature or presence of the host proteins.     

pH-independent HIV entry into CD4-positive T cells via virus envelope fusion to the plasma membrane

CD4 functions as the cell-surface receptor for human immunodeficiency virus (HIV); however, the mechanism of virus entry into susceptible cells is unknown. To explore this question we used a human T lymphoblastic cell line (VB) expressing high levels of surface CD4. Neutralization of endosomal compartments (pH greater than 6.4) with lysosomotropic agents did not effectively inhibit HIV nucleocapsid entry into the cytoplasm, and virus treated at low pH (5.5) failed to induce rapid cell-to-cell fusion in uninfected cells. Electron microscopy of VB cells acutely exposed to HIV at neutral pH revealed direct fusion of the virus envelope with the plasma membrane within minutes at 4 degrees C. No endocytosed virions were visualized upon rewarming the HIV-exposed cells to 37 degrees C for as long as 60 min. These results indicate that HIV penetrates CD4-positive T cells via pH-independent membrane fusion.     

Premature activation of the paramyxovirus fusion protein before target cell attachment with corruption of the viral fusion machinery

Paramyxoviruses, including the childhood pathogen human parainfluenza virus type 3, enter host cells by fusion of the viral and target cell membranes. This fusion results from the concerted action of its two envelope glycoproteins, the hemagglutinin-neuraminidase (HN) and the fusion protein (F). The receptor-bound HN triggers F to undergo conformational changes that render it competent to mediate fusion of the viral and cellular membranes. We proposed that, if the fusion process could be activated prematurely before the virion reaches the target host cell, infection could be prevented. We identified a small molecule that inhibits paramyxovirus entry into target cells and prevents infection. We show here that this compound works by an interaction with HN that results in F-activation prior to receptor binding. The fusion process is thereby prematurely activated, preventing fusion of the viral membrane with target cells and precluding viral entry. This first evidence that activation of a paramyxovirus F can be specifically induced before the virus contacts its target cell suggests a new strategy with broad implications for the design of antiviral agents.     

冠状病毒S蛋白的结构和功能

冠状病毒S蛋白具有受体结合活性和膜融合活性,在组织嗜性、细胞融合和毒力等方面具有重要作用。本综述了S蛋白的一般结构特征及其与细胞受体和膜融合的关系,并介绍了最近发现的SARS病毒S蛋白与其他冠状病毒的异同。     

The avian retrovirus avian sarcoma/leukosis virus subtype A reaches the lipid mixing stage of fusion at neutral pH

We previously showed that the envelope glycoprotein (EnvA) of avian sarcoma/leukosis virus subtype A (ASLV-A) binds to liposomes at neutral pH following incubation with its receptor, Tva, at >or=22 degrees C. We also provided evidence that ASLV-C fuses with cells at neutral pH. These findings suggested that receptor binding at neutral pH and >or=22 degrees C is sufficient to activate Env for fusion. A recent study suggested that two steps are necessary to activate avian retroviral Envs: receptor binding at neutral pH, followed by exposure to low pH (W. Mothes et al., Cell 103:679-689, 2000). Therefore, we evaluated the requirements for intact ASLV-A particles to bind to target bilayers and fuse with cells. We found that ASLV-A particles bind stably to liposomes in a receptor- and temperature-dependent manner at neutral pH. Using ASLV-A particles biosynthetically labeled with pyrene, we found that ASLV-A mixes its lipid envelope with cells within 5 to 10 min at 37 degrees C. Lipid mixing was neither inhibited nor enhanced by incubation at low pH. Lipid mixing of ASLV-A was inhibited by a peptide designed to prevent six-helix bundle formation in EnvA; the same peptide inhibits virus infection and EnvA-mediated cell-cell fusion (at both neutral and low pHs). Bafilomycin and dominant-negative dynamin inhibited lipid mixing of Sindbis virus (which requires low pH for fusion), but not of ASLV-A, with host cells. Finally, we found that, although EnvA-induced cell-cell fusion is enhanced at low pH, a mutant EnvA that is severely compromised in its ability to support infection still induced massive syncytia at low pH. Our results indicate that receptor binding at neutral pH is sufficient to activate EnvA, such that ASLV-A particles bind hydrophobically to and merge their membranes with target cells. Possible roles for low pH at subsequent stages of viral entry are discussed.     

Fusion of influenza virions with a planar lipid membrane detected by video fluorescence microscopy

The fusion of individual influenza virions with a planar phospholipid membrane was detected by fluorescence video microscopy. Virion envelopes were loaded with the lipophilic fluorescent marker octadecylrhodamine B (R18) to a density at which the fluorescence of the probe was self-quenched. Labeled virions were ejected toward the planar membrane from a micropipette in a custom-built video fluorescence microscope. Once a virion fused with the planar membrane, the marker was free to diffuse, and its fluorescence became dequenched, producing a flash of light. This flash was detected as a transient spot of light which increased and then diminished in brightness. The diffusion constants calculated from the brightness profiles for the flashes are consistent with fusion of virus to the membrane with consequent free diffusion of probe within the planar membrane. Under conditions known to be fusigenic for influenza virus (low pH and 37 degrees C), flashes appeared at a high rate and the planar membrane quickly became fluorescent. To further establish that these flashes were due to fusion, we showed that red blood cells, which normally do not attach to planar membranes, were able to bind to membranes that had been exposed to virus under fusigenic conditions. The amount of binding correlated with the amount of flashing. This indicates that flashes signaled the reconstitution of the hemagglutinin glycoprotein (HA) of influenza virus, a well-known erythrocyte receptor, into the planar membrane, as would be expected in a fusion process. The flash rate on ganglioside-containing asolectin membranes increased as the pH was lowered. This is also consistent with the known fusion behavior of influenza virus with cell membranes and with phospholipid vesicles. We conclude that the flashes result from the fusion of individual virions to the planar membrane.     

The Sendai virus membrane fusion mechanism studied using image correlation spectroscopy.

The mechanism of Sendai virus membrane fusion to cultured cell membranes was studied. Viral lipids were labeled with the lipophilic dye, 4-(4-(dihexadecylamino)styryl-N-methylquinolinium iodine) (DiQ), and viral proteins were labeled using fluorescein isothiocyanate (FITC). The redistribution of these probes from the virus to cultured cells was followed using the technique of image correlation spectroscopy. This technique assayed the intensity change and the redistribution of these probes as fusion progressed from a more to less aggregated state. The lipid probe DiQ dispersed into the membrane of the target membrane at both 22 and 37 degrees C, while the FITC-labeled proteins dispersed only at 37 degrees C. Simultaneous labeling of virus with both of these probes showed that at 37 degrees C their redistribution proceeded at different rates. These data were consistent with the formation of a hemifusion intermediate during the fusion process.     

Characterization of murine-specific leukemia virus receptor from L cells.

The host cell receptor for Moloney murine leukemia virus was solubilized from murine L-cell membranes and characterized. In initial studies designed to identify a receptor-rich cell line, different mouse cells were screened for binding to Moloney gp70, the viral envelope glycoprotein which determines host cell-binding specificity. gp70 binding to murine L cells was specific and saturable, with an apparent affinity constant (Ka) of 4 X 10(8) M-1, and the number of receptors per cell (6 X 10(5)) was similar to that of other mouse fibroblast cell lines. Characterization of the gp70 receptor with regard to extraction by detergents, protease sensitivity, and heat denaturation suggests that the receptor is an intrinsic membrane protein. Upon extraction of L-cell membranes with 0.2% deoxycholic acid and precipitation with acetone, specific and saturable binding of gp70 could be detected. The solubilized gp70-binding component was eluted upon gel filtration on Sephacryl S-300 into a species with an approximate molecular weight of 110,000.     

囊膜病毒膜融合分子机制研究进展

囊膜病毒通过病毒与宿主细胞膜融合的方式感染宿主,病毒囊膜蛋白介导了膜融合过程。根据这些囊膜蛋白在病毒囊膜表面的排列、蛋白结构及其在融合肽中的位置不同,可将囊膜病毒分为三类,其利用这些囊膜特殊的蛋白分子与受体相互作用完成膜融合。在分子水平上研究这一过程有助于认识病毒侵染的本质和发现关键环节,达到预防与治疗病毒病的目的。     

Lipid-phase structure in epithelial cell membranes: comparison of renal brush border and basolateral membranes

The lipid-phase structures of brush border membrane vesicles (BBMV) and basolateral membrane vesicles (BLMV) isolated from rabbit renal cortex were compared by steady-state and phase-modulation measurements of diphenylhexatriene (DPH) and trans- and cis-parinaric acid (tPnA and cPnA) fluorescence. A temperature-scanning system was used which gave reproducible temperature profiles of steady-state and dynamic fluorescence parameters with a resolution of 0.1 degrees C. Steady-state anisotropy of DPH showed a triphasic dependence on temperature with slope discontinuities at 22 +/- 4 and 47 +/- 3 degrees C (BBMV) and at 23 +/- 2 and 48 +/- 1 degrees C (BLMV). At all temperatures, DPH anisotropy in BBMV was greater than that in BLMV. Ground-state heterogeneity analysis of tPnA and cPnA fluorescence lifetime data demonstrated the presence of long (greater than 12 ns) and short (less than 5 ns) lifetime components, interpreted in terms of solid-phase and fluid-phase lipid domains. The fraction of solid-phase phospholipid decreased from 0.9 to 0.1 for BBMV and from 0.7 to 0.3 in BLMV with increasing temperature (10-50 degrees C). In both membranes, tryptophan-PnA fluorescence energy-transfer measurements showed that membrane proteins were surrounded by a fluidlike phospholipid phase. These results demonstrate the inadequacy of steady-state DPH anisotropy data in defining the structural characteristics of complex biological membranes. Results obtained with the phase-sensitive parinaric acid probes demonstrate major differences in the phase structure of the two opposing cell membranes in both the bulk lipid and the lipid microenvironment around membrane proteins.     

Over-expression of mammalian sialidase NEU3 reduces Newcastle disease virus entry and propagation in COS7 cells

The paramyxovirus Newcastle Disease Virus (NDV) binds to sialic acid-containing glycoconjugates, sialoglycoproteins and sialoglycolipids (gangliosides) of host cell plasma membrane through its hemagglutinin-neuraminidase (sialidase) HN glycoprotein. We hypothesized that the modifications of the cell surface ganglioside pattern determined by over-expression of the mammalian plasma-membrane associated, ganglioside specific, sialidase NEU3 would affect the virus-host cell interactions. Using COS7 cells as a model system, we observed that over-expression of the murine MmNEU3 did not affect NDV binding but caused a marked reduction in NDV infection and virus propagation through cell-cell fusion. Moreover, since GD1a was greatly reduced in COS7 cells following NEU3-over-expression, we added [(3)H]-labelled GD1a to COS7 cells under conditions that block intralysosomal metabolic processing, and we observed a marked increase of GD1a cleavage to GM1 during NDV infection, indicating a direct involvement of the virus sialidase and host cell GD1a in NDV infectivity. Therefore, the decrease of GD1a in COS7 cell membrane upon MmNEU3 over-expression is likely to be instrumental to NDV reduced infection. Evidence was also provided for the preferential association of NDV-HN at 4 degrees C to detergent resistant microdomains (DRMs) of COS7 cells plasma membranes.     

Fusion of Madin-Darby canine kidney cells by HVJ (Sendai virus): absence of direct association of virus particles with the site of membrane fusion

The process of cell fusion of Madin-Darby canine kidney (MDCK) cells by HVJ (Sendai virus) was investigated to determine whether the HVJ particles were directly associated with the site of membrane fusion. Confluent monolayer cultures of MDCK cells are sealed together by tight junctions on the apices of their lateral membranes, so added virus particles can be adsorbed only to the apical surfaces of the cells. After incubation with HVJ at 37 degrees C for 30 min, the cells still appeared mononucleate and unfused by light microscopy, but electron microscopic examination showed that fusion at the lateral membranes had occurred below the tight junctions. Furthermore, when fluorescein isothiocyanate (FITC)-labeled macromolecules, which cannot pass across the gap junctions, were injected into the cells at this stage, labeled macromolecules were found to diffuse into the adjacent cells. These findings strongly suggest that cell fusion was initiated in the lateral membrane, a region distinct from the site of adsorbed HVJ particles. Thus, the virus particles were not directly associated with the fusion site, but induced fusion of the lateral membranes indirectly.     

Spring-loaded model revisited: paramyxovirus fusion requires engagement of a receptor binding protein beyond initial triggering of the fusion protein

During paramyxovirus entry into a host cell, receptor engagement by a specialized binding protein triggers conformational changes in the adjacent fusion protein (F), leading to fusion between the viral and cell membranes. According to the existing paradigm of paramyxovirus membrane fusion, the initial activation of F by the receptor binding protein sets off a spring-loaded mechanism whereby the F protein progresses independently through the subsequent steps in the fusion process, ending in membrane merger. For human parainfluenza virus type 3 (HPIV3), the receptor binding protein (hemagglutinin-neuraminidase [HN]) has three functions: receptor binding, receptor cleaving, and activating F. We report that continuous receptor engagement by HN activates F to advance through the series of structural rearrangements required for fusion. In contrast to the prevailing model, the role of HN-receptor engagement in the fusion process is required beyond an initiating step, i.e., it is still required even after the insertion of the fusion peptide into the target cell membrane, enabling F to mediate membrane merger. We also report that for Nipah virus, whose receptor binding protein has no receptor-cleaving activity, the continuous stimulation of the F protein by a receptor-engaged binding protein is key for fusion. We suggest a general model for paramyxovirus fusion activation in which receptor engagement plays an active role in F activation, and the continued engagement of the receptor binding protein is essential to F protein function until the onset of membrane merger. This model has broad implications for the mechanism of paramyxovirus fusion and for strategies to prevent viral entry.     

Sendai virus-erythrocyte membrane interaction: quantitative and kinetic analysis of viral binding, dissociation, and fusion.

A kinetic and quantitative analysis of the binding and fusion of Sendai virus with erythrocyte membranes was performed by using a membrane fusion assay based on the relief of fluorescence self-quenching. At 37 degrees C, the process of virus association displayed a half time of 2.5 min; at 4 degrees C, the half time was 3.0 min. The fraction of the viral dose which became cell associated was independent of the incubation temperature and increased with increasing target membrane concentration. On the average, one erythrocyte ghost can accommodate ca. 1,200 Sendai virus particles. The stability of viral attachment was sensitive to a shift in temperature: a fraction of the virions (ca. 30%), attached at 4 degrees C, rapidly (half time, ca. 2.5 min) eluted from the cell surface at 37 degrees C, irrespective of the presence of free virus in the medium. The elution can be attributed to a spontaneous, temperature-induced release, rather than to viral neuraminidase activity. Competition experiments with nonlabeled virus revealed that viruses destined to fuse do not exchange with free particles in the medium but rather bind in a rapid and irreversible manner. The fusion rate of Sendai virus was affected by the density of the virus particles on the cell surface and became restrained when more than 170 virus particles were attached per ghost. In principle, all virus particles added displayed fusion activity. However, at high virus-to-ghost ratios, only a fraction actually fused, indicating that a limited number of fusion sites exist on the erythrocyte membrane. We estimate that ca. 180 virus particles maximally can fuse with one erythrocyte ghost.     

Relative affinity of the human parainfluenza virus type 3 hemagglutinin-neuraminidase for sialic acid correlates with virus-induced fusion activity.

The ability of enveloped viruses to cause disease depends on their ability to enter the host cell via membrane fusion events. An understanding of these early events in infection, crucial for the design of methods of blocking infection, is needed for viruses that mediate membrane fusion at neutral pH, such as paramyxoviruses and human immunodeficiency virus. Sialic acid is the receptor for the human parainfluenza virus type 3 (HPF3) hemagglutinin-neuraminidase (HN) glycoprotein, the molecule responsible for binding of the virus to cell surfaces. In order for the fusion protein (F) of HPF3 to promote membrane fusion, the HN must interact with its receptor. In the present report, two variants of HPF3 with increased fusion-promoting phenotypes were selected and used to study the function of the HN glycoprotein in membrane fusion. Increased fusogenicity correlated with single amino acid changes in the HN protein that resulted in increased binding of the variant viruses to the sialic acid receptor. These results suggest that the avidity of binding of the HN protein to its receptor regulates the level of F protein-mediated fusion and begin to define one role of the receptor-binding protein of a paramyxovirus in the membrane fusion process.