scholarly journals Mechanisms of mutations inhibiting fusion and infection by Semliki Forest virus.

1996 ◽  
Vol 134 (4) ◽  
pp. 863-872 ◽  
Author(s):  
M Kielian ◽  
M R Klimjack ◽  
S Ghosh ◽  
W A Duffus
Keyword(s):  
Membrane Fusion ◽  
Cell Fusion ◽  
Semliki Forest Virus ◽  
Fusion Reaction ◽  
Infectious Clone ◽  
Low Ph ◽  
Spike Protein ◽  
Initial Reaction ◽  
Target Membrane ◽  
Cell Cell

Semliki Forest virus (SFV) infects cells by an acid-dependent membrane fusion reaction catalyzed by the virus spike protein, a complex containing E1 and E2 transmembrane subunits. E1 carries the putative virus fusion peptide, and mutations in this domain of the spike protein were previously shown to shift the pH threshold of cell-cell fusion (G91A), or block cell-cell fusion (G91D). We have used an SFV infectious clone to characterize virus particles containing these mutations. In keeping with the previous spike protein results, G91A virus showed limited secondary infection and an acid-shifted fusion threshold, while G91D virus was noninfectious and inactive in both cell-cell and virus-liposome fusion assays. During the low pH- induced SFV fusion reaction, the E1 subunit exposes new epitopes for monoclonal antibody (mAb) binding and forms an SDS-resistant homotrimer, the virus associates hydrophobically with the target membrane, and fusion of the virus and target membranes occurs. After low pH treatment, G91A spike proteins were shown to bind conformation-specific mAbs, associate with target liposome membranes, and form the E1 homotrimer. However, both G91A membrane association and homotrimer formation had an acid-shifted pH threshold and reduced efficiency compared to wt virus. In contrast, studies of the fusion-defective G91D mutant showed that the virus efficiently reacted with low pH as assayed by mAb binding and liposome association, but was essentially inactive in homotrimer formation. These results suggest that the G91D mutant is noninfectious due to a block in a late step in membrane fusion, separate from the initial reaction to low pH and interaction with the target membrane, and involving the lack of efficient formation of the E1 homotrimer.

scholarly journals E1 Mutants Identify a Critical Region in the Trimer Interface of the Semliki Forest Virus Fusion Protein

2009 ◽  
Vol 83 (21) ◽  
pp. 11298-11306 ◽  
Author(s):  
Catherine Y. Liu ◽  
Margaret Kielian
Keyword(s):  
Membrane Fusion ◽  
Protein Interactions ◽  
Semliki Forest Virus ◽  
Hot Spot ◽  
Fusion Reaction ◽  
Ph Dependence ◽  
Infectious Clone ◽  
Low Ph ◽  
Host Cells ◽  
Target Membrane

ABSTRACT The alphavirus Semliki Forest virus (SFV) uses a membrane fusion reaction to infect host cells. Fusion of the virus and cell membranes is triggered by low pH in the endosome and is mediated by the viral membrane protein E1. During fusion, E1 inserts into the target membrane, trimerizes, and refolds into a hairpin conformation. Formation of the E1 homotrimer is critical to membrane fusion, but the mechanism of trimerization is not understood. The crystal structure of the postfusion E1 trimer shows that an aspartate residue, D188, is positioned in the central core trimer interface. D188 is conserved in all reported alphavirus E1 sequences. We tested the contribution of this amino acid to trimerization and fusion by replacing D188 with alanine (D188A) or lysine (D188K) in an SFV infectious clone. These mutations were predicted to disrupt specific interactions at this position and/or change their pH dependence. Our results indicated that the D188K mutation blocked SFV fusion and infection. At low pH, D188K E1 inserted into target membranes but was trapped as a target membrane-inserted monomer that did not efficiently form the stable core trimer. In contrast, the D188A mutant was infectious, although trimerization and fusion required a lower pH. While there are extensive contacts between E1 subunits in the homotrimer, the D188K mutant identifies an important “hot spot” for protein-protein interactions within the core trimer.

scholarly journals A Conserved Histidine in the ij Loop of the Semliki Forest Virus E1 Protein Plays an Important Role in Membrane Fusion

2004 ◽  
Vol 78 (24) ◽  
pp. 13543-13552 ◽  
Author(s):  
Chantal Chanel-Vos ◽  
Margaret Kielian
Keyword(s):  
Membrane Fusion ◽  
Conformational Changes ◽  
Semliki Forest Virus ◽  
Fusion Reaction ◽  
Infectious Clone ◽  
Critical Role ◽  
Low Ph ◽  
Sequence Comparisons ◽  
E1 Protein ◽  
Fusion Loop

ABSTRACT The enveloped alphavirus Semliki Forest virus (SFV) infects cells via a low pH-triggered membrane fusion reaction mediated by the E1 protein. E1 is a class II fusion protein that contains the hydrophobic fusion peptide loop and converts to a stable homotrimer during the fusion reaction. Intriguingly, the fusion loop is closely associated with a loop connecting the i and j β-strands. This ij loop plays a role in the cholesterol dependence of membrane fusion and is specifically susceptible to proteolysis in the protease-resistant E1 homotrimer. The SFV ij loop contains a histidine residue at position 230. Sequence comparisons revealed that an analogous histidine is completely conserved in all alphavirus and flavivirus fusion proteins. An E1 H230A mutant was constructed using the SFV infectious clone. Although cells infected with H230A RNA produced virus particles, these virions were completely noninfectious and were blocked in both cell-cell fusion and lipid mixing assays. The H230A virions efficiently bound to cell surface receptors and responded to low pH by undergoing acid-dependent conformational changes including dissociation of the E1/E2 dimer, exposure of the fusion loop, association with target liposomes, exposure of acid-conformation-specific epitopes, and formation of the stable E1 homotrimer. Studies with a soluble fragment of E1 showed that the mutant protein was defective in lipid-dependent conformational changes. Our results indicate that the E1 ij loop and the conserved H230 residue play a critical role in alphavirus-membrane fusion and suggest the presence of a previously undescribed late intermediate in the fusion reaction.

scholarly journals Novel Mutations That Control the Sphingolipid and Cholesterol Dependence of the Semliki Forest Virus Fusion Protein

2002 ◽  
Vol 76 (24) ◽  
pp. 12712-12722 ◽  
Author(s):  
Prodyot K. Chatterjee ◽  
Christina H. Eng ◽  
Margaret Kielian
Keyword(s):  
Membrane Fusion ◽  
Semliki Forest Virus ◽  
Fusion Reaction ◽  
Fusion Peptide ◽  
Single Amino Acid ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Novel Mutations ◽  
E1 Protein ◽  
Target Membrane

ABSTRACT The enveloped alphavirus Semliki Forest virus (SFV) infects cells via a membrane fusion reaction mediated by the E1 membrane protein. Efficient SFV-membrane fusion requires the presence of cholesterol and sphingolipid in the target membrane. Here we report on two mutants, srf-4 and srf-5, selected for growth in cholesterol-depleted cells. Like the previously isolated srf-3 mutant (E1 proline 226 to serine), the phenotypes of the srf-4 and srf-5 mutants were conferred by single-amino-acid changes in the E1 protein: leucine 44 to phenylalanine and valine 178 to alanine, respectively. Like srf-3, srf-4 and srf-5 show striking increases in the cholesterol independence of growth, infection, membrane fusion, and exit. Unexpectedly, and unlike srf-3, srf-4 and srf-5 showed highly efficient fusion with sphingolipid-free membranes in both lipid- and content-mixing assays. Both srf-4 and srf-5 formed E1 homotrimers of decreased stability compared to the homotrimers of the wild type and the srf-3 mutant. All three srf mutations lie in the same domain of E1, but the srf-4 and srf-5 mutations are spatially separated from srf-3. When expressed together, the three mutations could interact to produce increased sterol independence and to cause temperature-sensitive E1 transport. Thus, the srf-4 and srf-5 mutations identify novel regions of E1 that are distinct from the fusion peptide and srf-3 region and modulate the requirements for both sphingolipid and cholesterol in virus-membrane fusion.

scholarly journals An Epitope of the Semliki Forest Virus Fusion Protein Exposed during Virus-Membrane Fusion

1999 ◽  
Vol 73 (12) ◽  
pp. 10029-10039 ◽  
Author(s):  
Anna Ahn ◽  
Matthew R. Klimjack ◽  
Prodyot K. Chatterjee ◽  
Margaret Kielian
Keyword(s):  
Membrane Fusion ◽  
Binding Site ◽  
Semliki Forest Virus ◽  
Fusion Reaction ◽  
Ph Dependence ◽  
Fusion Peptide ◽  
Endocytic Pathway ◽  
Spike Protein ◽  
Viral Fusion ◽  
Wide Range

ABSTRACT Semliki Forest virus (SFV) is an enveloped alphavirus that infects cells via a membrane fusion reaction triggered by acidic pH in the endocytic pathway. Fusion is mediated by the spike protein E1 subunit, an integral membrane protein that contains the viral fusion peptide and forms a stable homotrimer during fusion. We have characterized four monoclonal antibodies (MAbs) specific for the acid conformation of E1. These MAbs did not inhibit fusion, suggesting that they bind to an E1 region different from the fusion peptide. Competition analyses demonstrated that all four MAbs bound to spatially related sites on acid-treated virions or isolated spike proteins. To map the binding site, we selected for virus mutants resistant to one of the MAbs, E1a-1. One virus isolate, SFV 4-2, showed reduced binding of three acid-specific MAbs including E1a-1, while its binding of one acid-specific MAb as well as non-acid-specific MAbs to E1 and E2 was unchanged. The SFV 4-2 mutant was fully infectious, formed the E1 homotrimer, and had the wild-type pH dependence of infection. Sequence analysis demonstrated that the relevant mutation in SFV 4-2 was a change of E1 glycine 157 to arginine (G157R). Decreased binding of MAb E1a-1 was observed under a wide range of assay conditions, strongly suggesting that the E1 G157R mutation directly affects the MAb binding site. These data thus localize an E1 region that is normally hidden in the neutral pH structure and becomes exposed as part of the reorganization of the spike protein to its fusion-active conformation.

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.

scholarly journals Important Role for the Transmembrane Domain of Severe Acute Respiratory Syndrome Coronavirus Spike Protein during Entry

2006 ◽  
Vol 80 (3) ◽  
pp. 1302-1310 ◽  
Author(s):  
Rene Broer ◽  
Bertrand Boson ◽  
Willy Spaan ◽  
François-Loïc Cosset ◽  
Jeroen Corver
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Membrane Fusion ◽  
Cell Fusion ◽  
Transmembrane Domain ◽  
Cytoplasmic Tail ◽  
Transmembrane Domains ◽  
Spike Protein ◽  
Fusion Activity ◽  
Wild Type ◽  
Cell Cell

ABSTRACT The spike protein (S) of severe acute respiratory syndrome coronavirus (SARS-CoV) is responsible for receptor binding and membrane fusion. It contains a highly conserved transmembrane domain that consists of three parts: an N-terminal tryptophan-rich domain, a central domain, and a cysteine-rich C-terminal domain. The cytoplasmic tail of S has previously been shown to be required for assembly. Here, the roles of the transmembrane and cytoplasmic domains of S in the infectivity and membrane fusion activity of SARS-CoV have been studied. SARS-CoV S-pseudotyped retrovirus (SARSpp) was used to measure S-mediated infectivity. In addition, the cell-cell fusion activity of S was monitored by a Renilla luciferase-based cell-cell fusion assay. Svsv-cyt, an S chimera with a cytoplasmic tail derived from vesicular stomatitis virus G protein (VSV-G), and Smhv-tmdcyt, an S chimera with the cytoplasmic and transmembrane domains of mouse hepatitis virus, displayed wild-type-like activity in both assays. Svsv-tmdcyt, a chimera with the cytoplasmic and transmembrane domains of VSV-G, was impaired in the SARSpp and cell-cell fusion assays, showing 3 to 25% activity compared to the wild type, depending on the assay and the cells used. Examination of the oligomeric state of the chimeric S proteins in SARSpp revealed that Svsv-tmdcyt trimers were less stable than wild-type S trimers, possibly explaining the lowered fusogenicity and infectivity.

scholarly journals Biochemical Consequences of a Mutation That Controls the Cholesterol Dependence of Semliki Forest Virus Fusion

2000 ◽  
Vol 74 (4) ◽  
pp. 1623-1631 ◽  
Author(s):  
Prodyot K. Chatterjee ◽  
Malini Vashishtha ◽  
Margaret Kielian
Keyword(s):  
Lipid Bilayers ◽  
Conformational Changes ◽  
Semliki Forest Virus ◽  
Fusion Reaction ◽  
Low Ph ◽  
Single Amino Acid ◽  
Single Amino Acid Change ◽  
Target Membrane ◽  
Alphavirus Infection

ABSTRACT The enveloped alphavirus Semliki Forest virus (SFV) infects cells via a low-pH-triggered membrane fusion reaction that requires cholesterol and sphingolipid in the target membrane. Cholesterol-depleted insect cells are highly resistant to alphavirus infection and were used to select srf-3, an SFV mutant that is ∼100-fold less cholesterol dependent for infection due to a single amino acid change in the E1 spike subunit, proline 226 to serine. Sensitive lipid-mixing assays here demonstrated that the in vitro fusion of srf-3 and wild-type (wt) virus with cholesterol-containing liposomes had comparable kinetics, activation energies, and sphingolipid dependence. In contrast, srf-3fusion with sterol-free liposomes was significantly more efficient than that of wt virus. Thus, the srf-3 mutation does not affect its general fusion properties with purified lipid bilayers but causes a marked and specific reduction in cholesterol dependence. Upon exposure to low pH, the E1 spike subunit undergoes distinct conformational changes, resulting in the exposure of an acid conformation-specific epitope and formation of an E1 homotrimer. These conformational changes were strongly cholesterol and sphingolipid dependent for wt SFV and strikingly less cholesterol dependent for srf-3. Our results thus demonstrate the functional importance of fusogenic E1 conformational changes in the control of SFV cholesterol dependence.

scholarly journals N- (4-Hydroxyphenyl) retinamide suppresses SARS-CoV-2 spike protein-mediated cell-cell fusion by a dihydroceramide Δ4-desaturase 1-independent mechanism

2021 ◽  
Author(s):  
Yasuhiro Hayashi ◽  
Kiyoto Tsuchiya ◽  
Mizuki Yamamoto ◽  
Yoko Nemoto-Sasaki ◽  
Kazunari Tanigawa ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Viral Infection ◽  
Membrane Fusion ◽  
Membrane Fluidity ◽  
Cell Fusion ◽  
Host Cells ◽  
Spike Protein ◽  
Sphingolipid Metabolism ◽  
Independent Mechanism ◽  
Cell Cell

The membrane fusion between the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and host cells is essential for the initial step of infection; therefore, the host cell membrane components, including sphingolipids, influence the viral infection. We assessed several inhibitors of the enzymes pertaining to sphingolipid metabolism, against SARS-CoV-2 spike protein (S)-mediated cell-cell fusion and viral infection. N -(4-hydroxyphenyl) retinamide (4-HPR), an inhibitor of dihydroceramide Δ4-desaturase 1 (DES1), suppressed cell-cell fusion, and viral infection. The analysis of sphingolipid levels revealed that the inhibition efficiencies of cell-cell fusion and viral infection in 4-HPR-treated cells were consistent with an increased ratio of saturated sphinganine-based lipids to total sphingolipids. We investigated the relationship of DES1 with the inhibition efficiencies of cell-cell fusion. The changes in the sphingolipid profile induced by 4-HPR were mitigated by the supplementation with exogenous cell-permeable ceramide; however, the reduced cell-cell fusion could not be reversed. The efficiency of cell-cell fusion in DES1 knockout (KO) cells was at a level comparable to that in wild-type (WT) cells; however, the ratio of saturated sphinganine-based lipids to the total sphingolipids was higher in DES1 KO cells, compared to that in WT cells. 4-HPR reduced cell membrane fluidity without any significant effects on the expression or localization of angiotensin-converting enzyme 2, the SARS-CoV-2 receptor. Therefore, 4-HPR suppresses SARS-CoV-2 S-mediated membrane fusion through a DES1-independent mechanism, and this decrease in membrane fluidity induced by 4-HPR could be the major cause for the inhibition of SARS-CoV-2 infection. Importance Sphingolipids could play an important role in SARS-CoV-2 S-meditated membrane fusion with host cells. We studied the cell-cell fusion using SARS-CoV-2 S expressing cells and sphingolipid-manipulated target cells, with an inhibitor of the sphingolipid metabolism. 4-HPR (also known as fenretinide) is an inhibitor of DES1 and it exhibits antitumor activity and suppresses cell-cell fusion and viral infection. 4-HPR suppresses membrane fusion through a decrease in membrane fluidity, which could possibly be the cause for the inhibition of SARS-CoV-2 infection. There is accumulating clinical data on the safety of 4-HPR. Therefore, it could be a potential candidate drug against COVID-19.

scholarly journals Cholesterol is required for infection by Semliki Forest virus.

1991 ◽  
Vol 112 (4) ◽  
pp. 615-623 ◽  
Author(s):  
T Phalen ◽  
M Kielian
Keyword(s):  
Semliki Forest Virus ◽  
Fusion Reaction ◽  
Low Ph ◽  
Endocytic Pathway ◽  
Hydroxyl Group ◽  
Target Membrane ◽  
Vesicular Stomatitis ◽  
Infection Pathway

Semliki Forest virus (SFV) and many other enveloped animal viruses enter cells by a membrane fusion reaction triggered by the low pH within the endocytic pathway. In vitro, SFV fusion requires cholesterol in the target membrane, but the role of cholesterol in vivo is unknown. In this paper, the infection pathway of SFV was studied in mammalian and inset cells substantially depleted of sterol. Cholesterol-depleted cells were unaltered in their ability to bind, internalize, and acidify virus, but were blocked in SFV fusion and subsequent virus replication. Depleted cells could be infected by the cholesterol-independent vesicular stomatitis virus, which also enters cells via endocytosis and low pH-mediated fusion. The block in SFV infection was specifically reversed by cholesterol but not by cholestenone, which lacks the critical 3 beta-hydroxyl group. Cholesterol thus is central in the infection pathway of SFV, and may act in vivo to modulate infection by SFV and other pathogens.

scholarly journals Membrane fusion mutants of Semliki Forest virus.

1984 ◽  
Vol 98 (1) ◽  
pp. 139-145 ◽  
Author(s):  
M C Kielian ◽  
S Keränen ◽  
L Kääriäinen ◽  
A Helenius
Keyword(s):  
Membrane Fusion ◽  
Semliki Forest Virus ◽  
Fusion Reaction ◽  
Low Ph ◽  
Endocytic Pathway ◽  
Wild Type ◽  
New Class ◽  
Weak Bases ◽  
Ph Probes

Previous reports have indicated that the entry of Semliki Forest virus (SFV) into cells depends on a membrane fusion reaction catalyzed by the viral spike glycoproteins and triggered by the low pH prevailing in the endosomal compartment. In this study the in vitro pH-dependent fusion of SFV with nuclease-filled liposomes has been used to select for a new class of virus mutants that have a pH-conditional defect. The mutants obtained had a threshold for fusion of pH 5.5 as compared with the wild-type threshold of 6.2, when assayed by polykaryon formation, fusion with liposomes, or fusion at the plasma membrane. They were fully capable of infecting cells under standard infection conditions but were more sensitive to lysosomotropic agents that increase the pH in acidic vacuoles of the endocytic pathway. The mutants were, moreover, able to penetrate and infect baby hamster kidney-21 cells at 20 degrees C, indicating that the endosomes have a pH below 5.5. The results confirm the involvement of pH-triggered fusion in SFV entry, emphasize the central role played by acidic endosomal vacuoles in this reaction, shed further light on the mechanism of SFV inhibition by lysosomotropic weak bases, and demonstrate the usefulness of mutant viruses as biological pH probes of the endocytic pathway.

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