scholarly journals Interactions of Semliki Forest virus spike glycoprotein rosettes and vesicles with cultured cells.

1983 ◽  
Vol 96 (2) ◽  
pp. 455-461 ◽  
Author(s):  
M Marsh ◽  
E Bolzau ◽  
J White ◽  
A Helenius
Keyword(s):  
Cell Surface ◽  
Membrane Fusion ◽  
Semliki Forest Virus ◽  
Lipid Vesicles ◽  
Low Ph ◽  
Fusion Activity ◽  
Spike Glycoprotein ◽  
Surface Binding ◽  
Coated Pits ◽  
Intact Virus

Semliki Forest virus (SFV)-derived spike glycoprotein rosettes (soluble octameric complexes), virosomes (lipid vesicles with viral spike glycoproteins), and liposomes (protein-free lipid vesicles) have been used to investigate the interaction of subviral particles with BHK-21 cells. Cell surface binding, internalization, degradation, and low pH-dependent membrane fusion were quantitatively determined. Electron microscopy was used to visualize the interactions. Virosomes and rosettes, but not liposomes, bound to cells. Binding occurred preferentially to microvilli and was inhibited by added SFV; it increased with decreasing pH but was, in all cases, less efficient than intact virus. At 37 degrees C the cell surface-bound rosettes and virosomes were internalized via coated pits and coated vesicles. After a lag period of 45 min the protein components of the internalized ligands were degraded and appeared, as acid-soluble activity, in the medium. The uptake of rosettes and virosomes was found to be similar to the adsorptive endocytosis of SFV except that their average residence times on the cell surface were longer. The rosettes and the liposomes did not show low pH-induced membrane fusion activity. The virosomes, however, irrespective of the lipid compositions used, displayed hemolytic activity at mildly acidic pH and were able to fuse with the plasma membrane of cells with an efficiency of 0.25 that observed with intact viruses. Cell-cell fusion activity was not observed with any of the subviral components. The results indicated that subviral components possess some of the entry properties of the intact virus.

scholarly journals Infectious entry pathway of influenza virus in a canine kidney cell line.

1981 ◽  
Vol 91 (3) ◽  
pp. 601-613 ◽  
Author(s):  
K S Matlin ◽  
H Reggio ◽  
A Helenius ◽  
K Simons
Keyword(s):  
Cell Surface ◽  
Influenza A ◽  
Semliki Forest Virus ◽  
Mdck Cells ◽  
Fusion Reaction ◽  
Low Ph ◽  
Coated Vesicles ◽  
Coated Pits ◽  
Entry Pathway ◽  
Canine Kidney

The entry of fowl plague virus, and avian influenza A virus, into Madin-Darby canine kidney (MDCK) cells was examined both biochemically and morphologically. At low multiplicity and 0 degrees C, viruses bound to the cell surface but were not internalized. Binding was not greatly dependent on the pH of the medium and reached an equilibrium level in 60-90 min. Over 90% of the bound viruses were removed by neuraminidase but not by proteases. When cells with prebound virus were warmed to 37 degrees C, part of the virus became resistant to removal b neuraminidase, with a half-time of 10-15 min. After a brief lag period, degraded viral material was released into the medium. The neuraminidase-resistant virus was capable of infecting the cells and probably did so by an intracellular route, since ammonium chloride, a lysosomotropic agent, blocked both the infection and the degradation of viral protein. When the entry process was observed by electron microscopy, viruses were seen bound primarily to microvilli on the cell surface at 0 degrees C and, after warming at 37 degrees C, were endocytosed in coated pits, coated vesicles, and large smooth-surfaced vacuoles. Viruses were also present in smooth-surfaced invaginations and small smooth-surfaced vesicles at both temperatures. At physiological pH, no fusion of the virus with the plasma membrane was observed. When prebound virus was incubated at a pH of 5.5 or below for 1 min at 37 degrees C, fusion was, however, detected by ferritin immunolabeling. t low multiplicity, 90% of the prebound virus became neuraminidase-resistant and was presumably fused after only 30 s at low pH. These experiments suggest that fowl plague virus enters MDCK cells by endocytosis in coated pits and coated vesicles and is transported to the lysosome where the low pH initiates a fusion reaction ultimately resulting in the transfer of the genome into the cytoplasm. The entry pathway of fowl plague virus thus resembles tht earlier described for Semliki Forest virus.

scholarly journals Regulatory Role of the Morbillivirus Attachment Protein Head-to-Stalk Linker Module in Membrane Fusion Triggering

2018 ◽  
Vol 92 (18) ◽  
Author(s):  
Michael Herren ◽  
Neeta Shrestha ◽  
Marianne Wyss ◽  
Andreas Zurbriggen ◽  
Philippe Plattet
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Cell Surface ◽  
Membrane Fusion ◽  
Measles Virus ◽  
Cell Entry ◽  
Regulatory Role ◽  
Fusion Activity ◽  
Animal Pathogens

ABSTRACTMorbillivirus (e.g., measles virus [MeV] and canine distemper virus [CDV]) host cell entry is coordinated by two interacting envelope glycoproteins, namely, an attachment (H) protein and a fusion (F) protein. The ectodomain of H proteins consists of stalk, connector, and head domains that assemble into functional noncovalent dimer-of-dimers. The role of the C-terminal module of the H-stalk domain (termed linker) and the connector, although putatively able to assume flexible structures and allow receptor-induced structural rearrangements, remains largely unexplored. Here, we carried out a nonconservative mutagenesis scan analysis of the MeV and CDV H-linker/connector domains. Our data demonstrated that replacing isoleucine 146 in H-linker (H-I146) with any charged amino acids prevented virus-mediated membrane fusion activity, despite proper trafficking of the mutants to the cell surface and preserved binding efficiency to the SLAM/CD150 receptor. Nondenaturing electrophoresis revealed that these charged amino acid changes led to the formation of irregular covalent H tetramers rather than functional dimer-of-dimers formed when isoleucine or other hydrophobic amino acids were present at residue position 146. Remarkably, we next demonstrated that covalent H tetramerizationper sewas not the only mechanism preventing F activation. Indeed, the neutral glycine mutant (H-I146G), which exhibited strong covalent tetramerization propensity, maintained limited fusion promotion activity. Conversely, charged H-I146 mutants, which additionally carried alanine substitution of natural cysteines (H-C139A and H-C154A) and thus were unable to form covalently linked tetramers, were fusion activation defective. Our data suggest a dual regulatory role of the hydrophobic residue at position 146 of the morbillivirus head-to-stalk H-linker module: securing the assembly of productive dimer-of-dimers and contributing to receptor-induced F-triggering activity.IMPORTANCEMeV and CDV remain important human and animal pathogens. Development of antivirals may significantly support current global vaccination campaigns. Cell entry is orchestrated by two interacting glycoproteins (H and F). The current hypothesis postulates that tetrameric H ectodomains (composed of stalk, connector, and head domains) undergo receptor-induced rearrangements to productively trigger F; these conformational changes may be regulated by the H-stalk C-terminal module (linker) and the following connector domain. Mutagenesis scan analysis of both microdomains revealed that replacing amino acid 146 in the H-linker region with nonhydrophobic residues produced covalent H tetramers which were compromised in triggering membrane fusion activity. However, these mutant proteins retained their ability to traffic to the cell surface and to bind to the virus receptor. These data suggest that the morbillivirus linker module contributes to the folding of functional pre-F-triggering H tetramers. Furthermore, such structures might be critical to convert receptor engagement into F activation.

Low pH induced membrane fusion of lipid vesicles containing proton-sensitive polymer

Biochemistry ◽  
1987 ◽  
Vol 26 (25) ◽  
pp. 8145-8150 ◽  
Author(s):  
Naoto Oku ◽  
Sayumi Shibamoto ◽  
Fumiaki Ito ◽  
Hisanori Gondo ◽  
Mamoru Nango
Keyword(s):  
Membrane Fusion ◽  
Lipid Vesicles ◽  
Low Ph ◽  
Induced Membrane

scholarly journals Membrane fusion process of Semliki Forest virus. I: Low pH-induced rearrangement in spike protein quaternary structure precedes virus penetration into cells.

1992 ◽  
Vol 116 (2) ◽  
pp. 339-348 ◽  
Author(s):  
J M Wahlberg ◽  
H Garoff
Keyword(s):  
Membrane Protein ◽  
Membrane Fusion ◽  
Semliki Forest Virus ◽  
Quaternary Structure ◽  
Low Ph ◽  
Host Cells ◽  
Protein Oligomerization ◽  
Protein Subunit ◽  
New Host ◽  
E1 Protein

The Semliki Forest virus (SFV) directs the synthesis of a heterodimeric membrane protein complex which is used for virus membrane assembly during budding at the surface of the infected cell, as well as for low pH-induced membrane fusion in the endosomes when particles enter new host cells. Existing evidence suggests that the E1 protein subunit carries the fusion potential of the heterodimer, whereas the E2 subunit, or its intracellular precursor p62, is required for binding to the nucleocapsid. We show here that during virus uptake into acidic endosomes the original E2E1 heterodimer is destabilized and the E1 proteins form new oligomers, presumably homooligomers, with altered E1 structure. This altered structure of E1 is specifically recognized by a monoclonal antibody which can also inhibit penetration of SFV into host cells as well as SFV-mediated cell-cell fusion, thus suggesting that the altered E1 structure is important for the membrane fusion. These results give further support for a membrane protein oligomerization-mediated control mechanism for the membrane fusion potential in alphaviruses.

scholarly journals Adaptation of Alphaviruses to Heparan Sulfate: Interaction of Sindbis and Semliki Forest Viruses with Liposomes Containing Lipid-Conjugated Heparin

2002 ◽  
Vol 76 (20) ◽  
pp. 10128-10137 ◽  
Author(s):  
Jolanda M. Smit ◽  
Barry-Lee Waarts ◽  
Koji Kimata ◽  
William B. Klimstra ◽  
Robert Bittman ◽  
...  
Keyword(s):  
Cell Surface ◽  
Heparan Sulfate ◽  
Sindbis Virus ◽  
Semliki Forest Virus ◽  
Model System ◽  
Low Ph ◽  
Wild Type ◽  
Enveloped Viruses ◽  
Neutral Ph ◽  
Receptor Interactions

ABSTRACT Passage of Sindbis virus (SIN) in BHK-21 cells has been shown to select for virus mutants with high affinity for the glycosaminoglycan heparan sulfate (HS). Three loci in the viral spike protein E2 (E2:1, E2:70, and E2:114) have been identified that mutate during adaptation and independently confer on the virus the ability to bind to cell surface HS (W. B. Klimstra, K. D. Ryman, and R. E. Johnston, J. Virol. 72:7357-7366, 1998). In this study, we used HS-adapted SIN mutants to evaluate a new model system involving target liposomes containing lipid-conjugated heparin (HepPE) as an HS receptor analog for the virus. HS-adapted SIN, but not nonadapted wild-type SIN TR339, interacted efficiently with HepPE-containing liposomes at neutral pH. Binding was competitively inhibited by soluble heparin. Despite the efficient binding of HS-adapted SIN to HepPE-containing liposomes at neutral pH, there was no fusion under these conditions. Fusion did occur, however, at low pH, consistent with cellular entry of the virus via acidic endosomes. At low pH, wild-type or HS-adapted SIN underwent fusion with liposomes with or without HepPE with similar kinetics, suggesting that interaction with the HS receptor analog at neutral pH has little influence on subsequent fusion of SIN at low pH. Finally, Semliki Forest virus (SFV), passaged frequently on BHK-21 cells, also interacted efficiently with HepPE-containing liposomes, indicating that SFV, like other alphaviruses, readily adapts to cell surface HS. In conclusion, the liposomal model system presented in this paper may serve as a novel tool for the study of receptor interactions and membrane fusion properties of HS-interacting enveloped viruses.

scholarly journals Membrane Fusion Mediated by Baculovirus gp64 Involves Assembly of Stable gp64 Trimers into Multiprotein Aggregates

1998 ◽  
Vol 143 (5) ◽  
pp. 1155-1166 ◽  
Author(s):  
Ingrid Markovic ◽  
Helena Pulyaeva ◽  
Alexander Sokoloff ◽  
Leonid V. Chernomordik
Keyword(s):  
Cell Surface ◽  
Membrane Fusion ◽  
Structural Unit ◽  
Low Ph ◽  
Cross Linking ◽  
Target Cells ◽  
Sf9 Cells ◽  
Density Gradient Ultracentrifugation ◽  
Sds Page ◽  
Experimental Approaches

The baculovirus fusogenic activity depends on the low pH conformation of virally-encoded trimeric glycoprotein, gp64. We used two experimental approaches to investigate whether monomers, trimers, and/or higher order oligomers are functionally involved in gp64 fusion machine. First, dithiothreitol (DTT)- based reduction of intersubunit disulfides was found to reversibly inhibit fusion, as assayed by fluorescent probe redistribution between gp64-expressing and target cells (i.e., erythrocytes or Sf9 cells). This inhibition correlates with disappearance of gp64 trimers and appearance of dimers and monomers in SDS-PAGE. Thus, stable (i.e., with intact intersubunit disulfides) gp64 trimers, rather than independent monomers, drive fusion. Second, we established that merger of membranes is preceded by formation of large (greater than 2 MDa), short-lived gp64 complexes. These complexes were stabilized by cell–surface cross-linking and characterized by glycerol density gradient ultracentrifugation. The basic structural unit of the complexes is stable gp64 trimer. Although DTT-destabilized trimers were still capable of assuming the low pH conformation, they failed to form multimeric complexes. The fact that formation of these complexes correlated with fusion in timing, and was dependent on (a) low pH application, (b) stable gp64 trimers, and (c) cell–cell contacts, suggests that such multimeric complexes represent a fusion machine.

scholarly journals Functional Analysis of the Transmembrane (TM) Domain of the Autographa californica Multicapsid Nucleopolyhedrovirus GP64 Protein: Substitution of Heterologous TM Domains

2008 ◽  
Vol 82 (7) ◽  
pp. 3329-3341 ◽  
Author(s):  
Zhaofei Li ◽  
Gary W. Blissard
Keyword(s):  
Membrane Proteins ◽  
Cell Surface ◽  
Membrane Fusion ◽  
Low Ph ◽  
Autographa Californica ◽  
Virus Infectivity ◽  
Type I ◽  
Gpi Anchor ◽  
Tm Domain ◽  
Hiv 1

ABSTRACT GP64, the major envelope glycoprotein of the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) budded virion, is important for host cell receptor binding and mediates low-pH-triggered membrane fusion during entry by endocytosis. In the current study, we examined the functional role of the AcMNPV GP64 transmembrane (TM) domain by replacing the 23-amino-acid GP64 TM domain with corresponding TM domain sequences from a range of viral and cellular type I membrane proteins, including Orgyia pseudotsugata MNPV (OpMNPV) GP64 and F, thogotovirus GP75, Lymantria dispar MNPV (LdMNPV) F, human immunodeficiency virus type 1 (HIV-1) GP41, human CD4 and glycophorin A (GpA), and influenza virus hemagglutinin (HA), and with a glycosylphosphatidylinositol (GPI) anchor addition sequence. In transient expression experiments with Sf9 cells, chimeric GP64 proteins containing either a GPI anchor or TM domains from LdMNPV F or HIV-1 GP41 failed to localize to the cell surface and thus appear to be incompatible with either GP64 structure or cell transport. All of the mutant constructs detected at the cell surface mediated hemifusion (outer leaflet merger) upon low-pH treatment, but only those containing TM domains from CD4, GpA, OpMNPV GP64, and thogotovirus GP75 mediated pore formation and complete membrane fusion activity. This supports a model in which partial fusion (hemifusion) proceeds by a mechanism that is independent of the TM domain and the TM domain participates in the enlargement or expansion of fusion pores after hemifusion. GP64 proteins containing heterologous TM domains mediated virion budding with dramatically differing levels of efficiency. In addition, chimeric GP64 proteins containing TM domains from CD4, GpA, HA, and OpMNPV F were incorporated into budded virions but were unable to rescue the infectivity of a gp64 null virus, whereas those with TM domains from OpMNPV GP64 and thogotovirus GP75 rescued infectivity. These results show that in addition to its basic role in membrane anchoring, the GP64 TM domain is critically important for GP64 trafficking, membrane fusion, virion budding, and virus infectivity. These critical functions were replaced only by TM domains from related viral membrane proteins.

scholarly journals Specific Single or Double Proline Substitutions in the “Spring-loaded” Coiled-Coil Region of the Influenza Hemagglutinin Impair or Abolish Membrane Fusion Activity

1998 ◽  
Vol 141 (6) ◽  
pp. 1335-1347 ◽  
Author(s):  
Hui Qiao ◽  
Sandra L. Pelletier ◽  
Lucas Hoffman ◽  
Jill Hacker ◽  
R. Todd Armstrong ◽  
...  
Keyword(s):  
Cell Surface ◽  
Red Blood Cells ◽  
Conformational Change ◽  
Blood Cells ◽  
Ph Dependence ◽  
Coiled Coil ◽  
Low Ph ◽  
Cell Surface Expression ◽  
Fusion Activity ◽  
Influenza Hemagglutinin

We tested the role of the “spring-loaded” conformational change in the fusion mechanism of the influenza hemagglutinin (HA) by assessing the effects of 10 point mutants in the region of high coiled-coil propensity, HA2 54–81. The mutants included proline substitutions at HA2 55, 71, and 80, as well as a double proline substitution at residues 55 and 71. Mutants were expressed in COS or 293T cells and assayed for cell surface expression and structural features as well as for their ability to change conformation and induce fusion at low pH. We found the following: Specific mutations affected the precise carbohydrate structure and folding of the HA trimer. All of the mutants, however, formed trimers that could be expressed at the cell surface in a form that could be proteolytically cleaved from the precursor, HA0, to the fusion-permissive form, HA1-S-S-HA2. All mutants reacted with an antibody against the major antigenic site and bound red blood cells. Seven out of ten mutants displayed a wild-type (wt) or moderately elevated pH dependence for the conformational change. V55P displayed a substantial reduction (∼60– 80%) in the initial rate of lipid mixing. The other single mutants displayed efficient fusion with the same pH dependence as wt-HA. The double proline mutant V55P/ S71P displayed no fusion activity despite being well expressed at the cell surface as a proteolytically cleaved trimer that could bind red blood cells and change conformation at low pH. The impairment in fusion for both V55P and V55P/S71P was at the level of outer leaflet lipid mixing. We interpret our results in support of the hypothesis that the spring-loaded conformational change is required for fusion. An alternate model is discussed.

scholarly journals The Fusion Peptide of Semliki Forest Virus Associates with Sterol-Rich Membrane Domains

2002 ◽  
Vol 76 (7) ◽  
pp. 3267-3275 ◽  
Author(s):  
Anna Ahn ◽  
Don L. Gibbons ◽  
Margaret Kielian
Keyword(s):  
Membrane Fusion ◽  
Semliki Forest Virus ◽  
Strong Association ◽  
Fusion Peptide ◽  
Low Ph ◽  
Membrane Domains ◽  
E1 Protein ◽  
Target Membrane ◽  
Virus Mutant ◽  
Detergent Resistant Membrane

ABSTRACT Semliki Forest virus (SFV) is an enveloped alphavirus whose membrane fusion is triggered by low pH and promoted by cholesterol and sphingolipid in the target membrane. Fusion is mediated by E1, a viral membrane protein containing the putative fusion peptide. Virus mutant studies indicate that SFV's cholesterol dependence is controlled by regions of E1 outside of the fusion peptide. Both E1 and E1*, a soluble ectodomain form of E1, interact with membranes in a reaction dependent on low pH, cholesterol, and sphingolipid and form highly stable homotrimers. Here we have used detergent extraction and gradient floatation experiments to demonstrate that E1* associated selectively with detergent-resistant membrane domains (DRMs or rafts). In contrast, reconstituted full-length E1 protein or influenza virus fusion peptide was not associated with DRMs. Methyl β-cyclodextrin quantitatively extracted both cholesterol and E1* from membranes in the absence of detergent, suggesting a strong association of E1* with sterol. Monoclonal antibody studies demonstrated that raft association was mediated by the proposed E1 fusion peptide. Thus, although other regions of E1 are implicated in the control of virus cholesterol dependence, once the SFV fusion peptide inserts in the target membrane it has a high affinity for membrane domains enriched in cholesterol and sphingolipid.

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