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 Cholesterol is required in the exit pathway of Semliki Forest virus.

1993 ◽  
Vol 123 (1) ◽  
pp. 57-65 ◽  
Author(s):  
M T Marquardt ◽  
T Phalen ◽  
M Kielian
Keyword(s):  
Semliki Forest Virus ◽  
Fusion Reaction ◽  
Virus Production ◽  
Virus Particles ◽  
Target Membrane ◽  
Vesicular Stomatitis ◽  
Lipid Requirement

The enveloped alphavirus Semliki Forest virus (SFV) infects cells via a membrane fusion reaction triggered by low pH. For fusion to occur cholesterol is required in the target membrane, as demonstrated both in in vitro fusion assays and in vivo for virus infection of a host cell. In this paper we examine the role of cholesterol in postfusion events in the SFV life cycle. Cholesterol-depleted insect cells were transfected with SFV RNA or infected at very high multiplicities to circumvent the fusion block caused by the absence of cholesterol. Under these conditions, the viral spike proteins were synthesized and transported to the site of p62 cleavage with normal kinetics. Surprisingly, the subsequent exit of virus particles was dramatically slowed compared to cholesterol-containing cells. The inhibition of virus production could be reversed by the addition of cholesterol to depleted cells. In contrast to results with SFV, no cholesterol requirement for virus exit was observed for the production of either the unrelated vesicular stomatitis virus or a cholesterol-independent SFV fusion mutant. Thus, cholesterol was only critical in the exit pathway of viruses that also require cholesterol for fusion. These results demonstrate a specific and unexpected lipid requirement in virus exit, and suggest that in addition to its role in fusion, cholesterol is involved in the assembly or budding of SFV.

scholarly journals Furin Processing and Proteolytic Activation of Semliki Forest Virus

2003 ◽  
Vol 77 (5) ◽  
pp. 2981-2989 ◽  
Author(s):  
Xinyong Zhang ◽  
Martin Fugère ◽  
Robert Day ◽  
Margaret Kielian
Keyword(s):  
Conformational Changes ◽  
Secretory Pathway ◽  
Semliki Forest Virus ◽  
Fusion Reaction ◽  
Low Ph ◽  
Protein Fusion ◽  
Proteolytic Activation ◽  
Control Virus

ABSTRACT The alphavirus Semliki Forest virus (SFV) infects cells via a low-pH-dependent membrane fusion reaction mediated by the E1 envelope protein. Fusion is regulated by the interaction of E1 with the receptor-binding protein E2. E2 is synthesized as a precursor termed “p62,” which forms a stable heterodimer with E1 and is processed late in the secretory pathway by a cellular furin-like protease. Once processing to E2 occurs, the E1/E2 heterodimer is destabilized so that it is more readily dissociated by exposure to low pH, allowing fusion and infection. We have used FD11 cells, a furin-deficient CHO cell line, to characterize the processing of p62 and its role in the control of virus fusion and infection. p62 was not cleaved in FD11 cells and cleavage was restored in FD11 cell transfectants expressing human furin. Studies of unprocessed virus produced in FD11 cells (wt/p62) demonstrated that the p62 protein was efficiently cleaved by purified furin in vitro, without requiring prior exposure to low pH. wt/p62 virus particles were also processed during their endocytic uptake in furin-containing cells, resulting in more efficient virus infection. wt/p62 virus was compared with mutant L, in which p62 cleavage was blocked by mutation of the furin-recognition motif. wt/p62 and mutant L had similar fusion properties, requiring a much lower pH than control virus to trigger fusion and fusogenic E1 conformational changes. However, the in vivo infectivity of mutant L was more strongly inhibited than that of wt/p62, due to additional effects of the mutation on virus-cell binding.

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 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.

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 Fusion of Semliki forest virus with the plasma membrane can be induced by low pH.

1980 ◽  
Vol 87 (1) ◽  
pp. 264-272 ◽  
Author(s):  
J White ◽  
J Kartenbeck ◽  
A Helenius
Keyword(s):  
Plasma Membrane ◽  
Semliki Forest Virus ◽  
Sendai Virus ◽  
Fusion Reaction ◽  
Low Ph ◽  
Medium Ph ◽  
Viral Membrane ◽  
Artificial Nature ◽  
Viral Nucleocapsid ◽  
Infection Pathway

When BHK-21 cells with Semliki Forest virus (SFV) bound at the plasma membrane are briefly treated with low pH medium (pH 5-6), fusion between the viral membrane and the plasma membrane occurs, releasing the viral nucleocapsid into the cytoplasm. The fusion reaction resembles that described previously for Sendai virus but with one fundamental difference; it is strictly dependent on low pH. The fusion reaction is highly efficient. Up to 86% of bound viruses fuse, and 6 X 10(6) virus spike proteins can be inserted into the plasma membrane of each cell. The process is very rapid (full activity is observed after 5 s) and it occurs over a wide temperature range and equally well with all five cell lines tested (BHK-21, HeLa B, HeLa suspension, Raji, and 3T3). Low pH-induced fusion of the virus at the plasma membrane can lead to infection of susceptible cells. The artificial nature of this infection pathway is, however, demonstrated by the facts that infection through the plasma membrane occurs only at subphysiological pH and that it is insensitive to inhibitors of the normal entry route. Nevertheless, these results indicate that low pH membrane fusion introduces the viral genome into the cytoplasm in a form suitable for replication.

scholarly journals Molecular Dissection of the Semliki Forest Virus Homotrimer Reveals Two Functionally Distinct Regions of the Fusion Protein

2002 ◽  
Vol 76 (3) ◽  
pp. 1194-1205 ◽  
Author(s):  
Don L. Gibbons ◽  
Margaret Kielian
Keyword(s):  
Conformational Changes ◽  
Semliki Forest Virus ◽  
Fusion Reaction ◽  
Fusion Peptide ◽  
Structural Basis ◽  
Reducing Conditions ◽  
Target Membrane ◽  
Membrane Bound ◽  
Harsh Conditions

ABSTRACT Semliki Forest virus (SFV) is an enveloped alphavirus that infects cells via a membrane fusion reaction triggered by the acidic pH of endosomes. In response to low pH, the E1 proteins on the virus membrane undergo a series of conformational changes, resulting in the formation of a stable E1 homotrimer. Little is known about the structural basis of either the E1 conformational changes or the resulting homotrimer or about the mechanism of action of the homotrimer in fusion. Here, the E1 homotrimer was formed in vitro from either virus or soluble E1 ectodomain and then probed by various perturbants, proteases, or glycosidase. The preformed homotrimer was extremely stable to moderately harsh conditions and proteases. By contrast, mild reducing conditions selectively disrupted the N-terminal region of trimeric E1, making it accessible to proteolytic cleavage and producing E1 fragments that retained trimer interactions. Trypsin digestion produced a fragment missing a portion of the N terminus just proximal to the putative fusion peptide. Digestion with elastase produced several fragments with cleavage sites between residues 78 and 102, resulting in the loss of the putative fusion peptide and the release of membrane-bound E1 ectodomain as a soluble trimer. Elastase also cleaved the homotrimer within an E1 loop located near the fusion peptide in the native E1 structure. Mass spectrometry was used to map the C termini of several differentially produced and fully functional E1 ectodomains. Together, our data identify two separate regions of the SFV E1 ectodomain, one responsible for target membrane association and one necessary for trimer interactions.

scholarly journals Low Endocytic pH and Capsid Protein Autocleavage Are Critical Components of Flock House Virus Cell Entry

2009 ◽  
Vol 83 (17) ◽  
pp. 8628-8637 ◽  
Author(s):  
Amy L. Odegard ◽  
Maggie H. Kwan ◽  
Hanna E. Walukiewicz ◽  
Manidipa Banerjee ◽  
Anette Schneemann ◽  
...  
Keyword(s):  
Host Cell ◽  
Active Regions ◽  
Low Ph ◽  
Endocytic Pathway ◽  
Cell Entry ◽  
Flock House Virus ◽  
Membrane Penetration ◽  
Endosomal Membrane

ABSTRACT The process by which nonenveloped viruses cross cell membranes during host cell entry remains poorly defined; however, common themes are emerging. Here, we use correlated in vivo and in vitro studies to understand the mechanism of Flock House virus (FHV) entry and membrane penetration. We demonstrate that low endocytic pH is required for FHV infection, that exposure to acidic pH promotes FHV-mediated disruption of model membranes (liposomes), and particles exposed to low pH in vitro exhibit increased hydrophobicity. In addition, FHV particles perturbed by heating displayed a marked increase in liposome disruption, indicating that membrane-active regions of the capsid are exposed or released under these conditions. We also provide evidence that autoproteolytic cleavage, to generate the lipophilic γ peptide (4.4 kDa), is required for membrane penetration. Mutant, cleavage-defective particles failed to mediate liposome lysis, regardless of pH or heat treatment, suggesting that these particles are not able to expose or release the requisite membrane-active regions of the capsid, namely, the γ peptides. Based on these results, we propose an updated model for FHV entry in which (i) the virus enters the host cell by endocytosis, (ii) low pH within the endocytic pathway triggers the irreversible exposure or release of γ peptides from the virus particle, and (iii) the exposed/released γ peptides disrupt the endosomal membrane, facilitating translocation of viral RNA into the cytoplasm.

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 Multistep Regulation of Membrane Insertion of the Fusion Peptide of Semliki Forest Virus

2004 ◽  
Vol 78 (7) ◽  
pp. 3312-3318 ◽  
Author(s):  
Don L. Gibbons ◽  
Anna Ahn ◽  
Maofu Liao ◽  
Lena Hammar ◽  
R. Holland Cheng ◽  
...  
Keyword(s):  
Fusion Proteins ◽  
Semliki Forest Virus ◽  
Fusion Reaction ◽  
Fusion Peptide ◽  
Low Ph ◽  
Membrane Insertion ◽  
Neutral Ph ◽  
Influenza Virus Hemagglutinin ◽  
Target Membrane ◽  
Membrane Fusion Proteins

ABSTRACT A prevailing model for virus membrane fusion proteins has been that the hydrophobic fusion peptide is hidden in the prefusion conformation, becomes exposed once the fusion reaction is triggered, and then either inserts into target membranes or is rapidly inactivated. This model is in general agreement with the structure and mechanism of class I fusion proteins, such as the influenza virus hemagglutinin. We here describe studies of the class II fusion protein E1 from the alphavirus Semliki Forest virus (SFV). SFV fusion is triggered by low pH, which releases E1 from its heterodimeric interaction with the E2 protein and induces the formation of a stable E1 homotrimer. The exposure and target membrane interaction of the E1 fusion peptide (residues 83 to 100) were followed using a monoclonal antibody (MAb E1f) mapping to E1 residues 85 to 95. In agreement with the known structure of SFV and other alphaviruses, the fusion peptide was shielded in native SFV particles and exposed when E1-E2 dimer dissociation was triggered by acidic pH. In contrast, the fusion peptide on purified E1 ectodomains (E1*) was fully accessible at neutral pH. Functional assays showed that MAb E1f binding at neutral pH prevented subsequent low-pH-triggered E1* interaction with target membranes and trimerization. E1* was not inactivated by low pH when treated either in the absence of target membranes or in the presence of fusion-inactive cholesterol-deficient liposomes. Thus, the membrane insertion of the E1 fusion peptide is regulated by additional low-pH-dependent steps after exposure, perhaps involving an E1-cholesterol interaction.

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