Dynamic changes in plasma membrane properties of semliki forest virus infected cells related to cell fusion

The mechanism of the processes leading to membrane fusion is as yet unknown. In this report we demonstrate that changes in membrane potential and potassium fluxes correlate with Semliki Forest virus induced cell-cell fusion at mildly acidic pH. The changes observed occur only at pH's below 6.2 corresponding to values required to trigger the fusion process. A possible role of these alterations of the plasma membrane related to membrane fusion phenomena is discussed.     

Can viral envelope proteins act as or induce proton channels?

The mechanism of the process leading to cell-cell fusion induced by enveloped viruses at a mildly acidic pH is as yet unknown. In this report we demonstrate that the fusion events induced by three viruses of different families, namely Semliki Forest (togavirus), vesicular stomatitis (rhabdovirus) and influenza (orthomyxovirus), share common features. In all three systems a sudden drop of the intracellular pH—below the critical eextracellular pH required to trigger fusion from within (FFWI)—is observed. This influx of protons is specific and not due to a general leakiness of the plasma membrane, and therefore might be caused by the opening of a proton channel.     

Changes in membrane permeability during semliki forest virus induced cell fusion

The infection of Aedes albopictus cells by Semliki Forest virus (SFV) is a non lytic event. Exposure of infected cells to mildly acidic pH (<6.2) leads to syncytium formation. This polykaryon formation is accompanied by an influex of protons into the cells (Kempfet al. Biosci. Rep. 7, 761–769, 1987). We have further investigated this permeability change using various fluorescent or radiolabeled compounds. A significant, pH dependent increase of the membrane permeability to low molecular weight compounds (Mr<1000) was observed when infected cells were exposed to a pH<6.2. The pH dependence of the peremability change was very similar to the pH dependence of cell-cell fusion. The permeability change was sensitive to divalent cations, protons and anionic antiviral drugs such as trypan blue. The nature of this virus induced, pH dependent permeability change is discussed.     

Fusion function of the Semliki Forest virus spike is activated by proteolytic cleavage of the envelope glycoprotein precursor p62.

The precursor protein p62 of the prototype alphavirus Semliki Forest virus (SFV) undergoes during transport to the cell surface a proteolytic cleavage to form the mature envelope glycoprotein E2. To investigate the biological significance of this cleavage event, single amino acid substitutions were introduced at the cleavages site through mutagenesis of cDNA corresponding to the structural region of the SFV genome. The phenotypes of the cleavage site mutants were studied in BHK cells by using recombinant vaccinia virus vectors. Nonconservative substitutions completely abolished p62 cleavage. Uncleaved p62 was transported with normal kinetics to the cell surface, where it became accessible to low concentrations of exogenous trypsin. The proteolytic cleavage of envelope glycoprotein precursors has been shown to activate the membrane fusion potential of viral spikes in several virus families. Here we demonstrate that the fusion function of the SFV spike is activated by the cleavage of p62. Cleavage-deficient p62 expressed at the cell surface did not function in low-pH-triggered (pH 5.5) cell-cell membrane fusion; however, cleavage of the mutated p62 with exogenous trypsin restored the fusion function. We discuss a model for SFV assembly and fusion where p62 cleavage plays a crucial role in the stability of the multimeric association of the viral envelope glycoproteins.     

Semliki Forest virus penetration from endosomes: a morphological study

The low pH dependent membrane fusion reaction responsible for the delivery of the Semliki Forest virus genome into the host cell for replication was visualized by electron microscopy. In order to increase the frequency at which fusion images could be detected a reversible inhibitor, ammonium chloride, was used to synchronize endosomal acidification, and 20 degrees C incubation was employed to concentrate virus particles into the endosomal compartment.     

Fusion induced by a class II viral fusion protein, semliki forest virus E1, is dependent on the voltage of the target cell

Cells expressing the low pH-triggered class II viral fusion protein E1 of Semliki Forest virus (SFV) were fused to target cells. Fusion was monitored by electrical capacitance and aqueous dye measurements. Electrical voltage-clamp measurements showed that SFV E1-induced cell-cell fusion occurred quickly after acidification for a trans-negative potential across the target membrane (i.e., negative potential inside the target cell) but that a trans-positive potential eliminated all fusion. Use of an ionophore to control potentials for a large population of cells confirmed the dependence of fusion on voltage polarity. In contrast, fusion induced by the class I fusion proteins of human immunodeficiency virus, avian sarcoma leukosis virus, and influenza virus was independent of the voltage polarity across the target cell. Initial pore size and pore growth were also independent of voltage polarity for the class I proteins. An intermediate of SFV E1-induced fusion was created by transient acidification at low temperature. Membranes were hemifused at this intermediate state, and raising the temperature at neutral pH allowed full fusion to occur. Capacitance measurements showed that maintaining a trans-positive potential definitely blocked fusion at steps following the creation of the hemifusion intermediate and may have inhibited fusion at prior steps. It is proposed that the trans-negative voltage across the endosomal membrane facilitates fusion after low-pH-induced conformational changes of SFV E1 have occurred.     

Spike protein oligomerization control of Semliki Forest virus fusion.

We have recently shown, using cleavage-deficient mutants of the p62-E1 membrane protein complex of Semliki Forest virus that p62 cleavage to E2 is necessary for the activation of the fusion function of the complex at pH 5.8 (a pH optimal for virus fusion) (M. Lobigs and H. Garoff, J. Virol. 64:1233-1240, 1990). In this study, we show that the mutant precursor complexes can be induced to activate membrane fusion when treated with more acidic buffers (pH 5.0 and 4.5), which also appear to dissociate most of the p62-E1 complexes and change the conformation of the E1 subunit (the supposed fusion protein of Semliki Forest virus into a form which is resistant to trypsin digestion. These data suggest that p62 cleavage is not essential for membrane fusion per se but that the crucial event activating this process seems to be the apparent dissociation of the heterodimer, which in turn is facilitated by the spike precursor cleavage.     

Mechanisms of mutations inhibiting fusion and infection by Semliki Forest virus

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.     

Mutagenesis of the putative fusion domain of the Semliki Forest virus spike protein.

Semliki Forest virus (SFV), an alphavirus, infects cells via a low pH-triggered membrane fusion reaction that takes place within the cellular endocytic pathway. Fusion is mediated by the heterotrimeric virus spike protein, which undergoes conformational changes upon exposure to low pH. The SFV E1 spike subunit contains a hydrophobic domain of 23 amino acids that is highly conserved among alphaviruses. This region is also homologous to a domain of the rotavirus outer capsid protein VP4. Mutagenesis of an SFV spike protein cDNA was used to evaluate the role of the E1 domain in membrane fusion. Mutant spike proteins were expressed in COS cells and assayed for cell-cell fusion activity. Four mutant phenotypes were identified: (i) substitution of Gln for Lys-79 or Leu for Met-88 had no effect on spike protein fusion activity; (ii) substitution of Ala for Asp-75, Ala for Gly-83, or Ala for Gly-91 shifted the pH threshold of fusion to a more acidic range; (iii) mutation of Pro-86 to Asp, Gly-91 to Pro, or deletion of amino acids 83 to 92 resulted in retention of the E1 subunit within the endoplasmic reticulum; and (iv) substitution of Asp for Gly-91 completely blocked cell-cell fusion activity without affecting spike protein assembly or transport. These results argue that the conserved hydrophobic domain of SFV E1 is closely involved in membrane fusion and suggest that the homologous region in rotavirus VP4 may be involved in the entry pathway of this nonenveloped virus.     

Effects of monovalent cations on Semliki Forest virus entry into BHK-21 cells

Infection of mammalian cells with Semliki Forest virus requires the endocytosis of the virus, its delivery to prelysosomal endosomes, and fusion of the viral envelope with the endosome membrane. Previous studies have indicated that the low endosomal pH triggers a conformational change in the viral spike glycoproteins rendering them fusogenic. In this paper, we demonstrate an additional factor(s) which regulates virus fusion in endosomes. We found that Semliki Forest virus is unable to penetrate or infect baby hamster kidney (BHK-21) cells grown in medium containing reduced Na+ concentrations. Virus endocytosis and degradation are nearly normal, the virus is transported to endosomes where a characteristic low pH-induced loss of trypsin-sensitivity of the E1 spike glycoprotein occurs. Nevertheless, the viral envelope fails to fuse with the endosomal membrane and the viral RNA is not released into the cytosol. As judged by the uptake of the voltage-sensitive probe [3H]triphenylmethyl phosphonium we observed a close correlation between conditions which inhibit virus infection and which cause depolarization of the cells. We propose that in intact cells, the fusion of Semliki Forest virus with the endosome membrane depends not only on acidic endosomal pH, but also on the maintenance of the potential.     

Membrane and protein interactions of a soluble form of the Semliki Forest virus fusion protein.

Semliki Forest virus is an enveloped alphavirus that infects cells by a membrane fusion reaction triggered by the low pH present in endocytic vacuoles. Fusion is mediated by the E1 spike protein subunit. During fusion, several conformational changes occur in E1 and E2, the two transmembrane subunits of the spike protein. These changes include dissociation of the E1-E2 dimer, alteration of the trypsin sensitivity and monoclonal antibody binding patterns of E1, and formation of a sodium dodecyl sulfate (SDS)-resistant E1 homotrimer. A critical characteristic of Semliki Forest virus fusion is also its dependence on the presence of both cholesterol and sphingomyelin in the target membrane. We have here examined the conformational changes induced by low pH treatment of E1*, the water-soluble, proteolytically truncated ectodomain of the E1 subunit. Following low pH treatment, E1* was shown to bind efficiently to artificial liposomes. Similar to virus fusion, optimal E1*-liposome binding required low pH, cholesterol, and sphingomyelin. The E1 ectodomain, although monomeric in its neutral pH form, assembled into an SDS-resistant oligomer following treatment at low pH. This low pH-induced oligomerization required target membranes containing both cholesterol and sphingomyelin. Our results demonstrate that the E1 ectodomain responds to low pH similarly to the full-length E1 subunit. The ectodomain facilitates the characterization of conformational changes and membrane binding in the absence of virus fusion or other virus components.     

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

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.     

fus-1, a pH Shift Mutant of Semliki Forest Virus, Acts by Altering Spike Subunit Interactions via a Mutation in the E2 Subunit

Semliki Forest virus (SFV), an enveloped alphavirus, is a well-characterized paradigm for viruses that infect cells via endocytic uptake and low-pH-triggered fusion. The SFV spike protein is composed of a dimer of E1 and E2 transmembrane subunits, which dissociate upon exposure to low pH, liberating E2 and the fusogenic E1 subunit to undergo independent conformational changes. SFV fusion and infection are blocked by agents such as ammonium chloride, which act by raising the pH in the endosome and inhibiting the low-pH-induced conformational changes in the SFV spike protein. We have previously isolated an SFV mutant, fus-1, that requires more acidic pH to trigger its fusion activity and is therefore more sensitive to inhibition by ammonium chloride. The acid shift in the fusion activity of fus-1 was here shown to be due to a more acidic pH threshold for the initial dissociation of the fus-1 spike dimer, thereby resulting in a more acidic pH requirement for the subsequent conformational changes in both fus-1 E1 and fus-1 E2. Sequence analysis demonstrated that the fus-1 phenotype was due to a mutation in the E2 spike subunit, threonine 12 to isoleucine. fus-1 revertants that have regained the parental fusion phenotype and ammonium chloride sensitivity were shown to have also regained E2 threonine 12. Our results identify a region of the SFV E2 spike protein subunit that regulates the pH dependence of E1-catalyzed fusion by controlling the dissociation of the E1/E2 dimer.     

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

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.     

pH-induced alterations in the fusogenic spike protein of Semliki Forest virus

The spike glycoproteins of Semliki Forest virus mediate membrane fusion between the viral envelope and cholesterol-containing target membranes under conditions of mildly acidic pH (pH less than 6.2). The fusion reaction is critical for the infectious cycle, catalyzing virus penetration from the acidic endosome compartment. To define the role of the viral spike glycoproteins in the fusion reaction, conformational changes in the spikes at acid pH were studied using protease digestion and binding assays to liposomes and nonionic detergent. A method was also developed to prepare fragments of both transmembrane subunit glycopolypeptides of the spike, E1 and E2, which lacked the hydrophobic anchor peptides. Unlike the intact spikes the fragments were monomeric and therefore useful for obtaining information on conformational changes in individual subunits. The results showed that both E1 and E2 undergo irreversible conformational changes at the pH of fusion, that the conformational change of E1 depends, in addition to acidic pH, on the presence of cholesterol, and that no major changes in the solubility properties of the spikes takes place. On the basis of these findings it was concluded that fusion involves both subunits of the spike and that E1 confers the stereo-specific sterol requirement. The results indicated, moreover, that acid-induced fusion of Semliki Forest virus differs in important respects from that of influenza virus, another well-defined model system for protein-mediated membrane fusion.     

A conserved histidine in the ij loop of the Semliki Forest virus E1 protein plays an important role in membrane fusion

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 beta-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.     

Analysis of alphavirus polypeptides by two dimensional polyacrylamide gel electrophoresis

Semliki Forest, Sindbis and Chikungunya viruses were grown and radio-labeled with [³H]-amino acids in Vero cells. Analysis of virus infected cell lysates by two dimensional polyacrylamide gel electrophoresis resulted in detection of polypeptides of molecular, weights corresponding to those of E1, P62, ns60, ns70/72 for Semliki Forest virus, the C, E1, 6K, 14K, PE2, P97, ns60, ns82 for Sindbis virus and E1. P62, P97, ns70/72 for Chikungunya virus. Charge and molecular weight heterogeneity in the precursor polypeptide P62 of Semliki Forest virus was detected. Structural poly-peptides e.g. E1 and E2 of Semliki Forest virus and C, E1, E2 of Sindbis virus and E1 of Chikungunya virus were detected when purified radiolabeled virus preparations were analyzed by two dimensional polyacrylamide gel-electrophoresis. Membrane glycoprotein E1 and E2 of Semliki Forest and E1 of Sindbis and Chikungunya viruses exhibited charge heterogeneity. In contrast to the marked difference in isoelectric points of E1 and E2 of Sindbis virus; E1 and E2 of Semliki Forest virus had almost identical isoelectric points.     

Role of cholesterol in fusion of Semliki Forest virus with membranes.

The low pH-triggered membrane fusion activity of Semliki Forest virus is dependent on the presence of cholesterol in the target membrane. When liposomes containing phospholipids and cholesterol analogs were used, fusion activity was observed with steroids which did not have a planar nucleus or an isooctyl side chain at C-17, but fusion activity was not observed when analogs which lacked the 3 beta-OH group were used. Binding of virus to liposomes at low pH was similarly, but not totally, dependent on the presence of a 3 beta-OH sterol.     

Large scale transient 5-HT3 receptor production with the Semliki Forest Virus Expression System

The expression of recombinant proteins with the Semliki Forest Virus (SFV) system has been scaled up to bioreactor scale. As a model protein for this study the human 5-HT₃ receptor was chosen. The gene for the receptor was subcloned into the SFV expression plasmid pSFV1. Virus production by in vivo packaging and production of the recombinant protein was scaled up, the latter to a reactor volume of 11.5 l. A Vibromix^TM agitation system was chosen to overcome aggregation problems of BHK cells in suspension. In the process, cells were first grown to a density of 10⁶ cells/ml, the medium was then exchanged with fresh medium and the culture was infected with the recombinant virus at an estimated multiplicity of infection of 30. 24 h post infection we measured an expression level of 3 million functional 5-HT₃ receptors per cell. For harvesting, the cells were pelleted by centrifugation. The receptor protein was purified in a single step (Hovius et al., 1998) by exploiting the hexa-His tag at minimal protein loss (51% yield). Experiments to optimise expression resulted in yields up to 8 million receptors per cell, when the pH of a suspension culture was controlled at pH 7.3. Rapid virus generation and protein production, high protein yields as well as successful large scale application have made the SFV expression system attractive to produce large quantities of recombinant protein in a very short time. After optimisation of the expression conditions (in particular by setting the pH at 7.3), yields were increased twofold.     

Production of Semliki Forest Virus from hamster kidney cells in deep culture vessels

The growth of Semliki Forest Virus in stirred culture vessels at volumes of 4 and 301. is described. Virus can be produced on a large scale in deep culture using industrial type vessels. Control of pH within close limits is important for maximum production of infective virus. With the parent, strain of SFV, virus yields were found to be influenced by an interference phenomenon which was apparently not due to interferon. Growth of a cloned strain of SFV obtained by serial selection of large plaques was not affected by this phenomenon. The cloned strain, when inoculated at a cell/virus input ratio of 1:1, gave maximum virus titers of 10¹⁰ p.f.u./ml., indicating an average yield of 10,000 p.f.u/BHK cell.