Solubilization of the semliki forest virus membrane with sodium deoxycholate

The effects of increasing concentrations of sodium deoxycholate on Semliki Forest virus have been studied. Sodium deoxycholate begins to bind to the virus at less than 0.1 mM free equilibrium concentration and causes lysis of the viral membrane at $\text{0.9 ± 0.1}\text{mM}$ free equilibrium concentration when $\text{2.2 ± 0.2 ß 10}{}^3\text{mol}$ of sodium deoxycholate are bound per mol of virus. Liberation of proteins from the membrane begins at $\text{1.5 ± 0.1}\text{mM}$ sodium deoxycholate and the proteins released are virtually free from phospholipid above 2.0 mM sodium deoxycholate. The overall mechanism of sodium deoxycholate solubilization of the viral membrane resembles that of Triton X-100 and sodium dodecyl sulphate except that with sodium deoxycholate the various stages of membrane disruption occur at about 10-fold higher equilibrium free detergent concentrations. At sodium deoxycholate concentrations higher than 2.3 mM the viral spike glycoproteins can be separated by sucrose gradient centrifugation or gel filtration into constituent polypeptides E1, E2 and E3. E1 carries the haemagglutinating activity of the virus.     

Solubilization of the membrane proteins from Semliki Forest virus with Triton X100

Increasing concentrations of Triton X100 have been found to cause stepwise dissociation of the membrane of Semliki Forest virus. The final stage of the breakdown process leads to solubilization of the membrane proteins which can be separated from the membrane lipids and the viral nucleocapsid by density gradient centrifugation in the presence of 0.05% Triton X100. Two different forms of Semliki Forest virus protein have been observed with sedimentation coefficients of approximately 4 S and 23 S. The 4 S aggregate appears to consist of two polypeptide chains complexed with about 75 molecules of Triton X100. The 23 S form is a rosette-like aggregate containing about 16 polypeptide chains and about 260 molecules of Triton X100. Sucrose alters the equilibrium between the 4 S and 23 S forms: removal of sucrose leads to association of the 4 S form to the 23 S form and addition of sucrose to dissociation.A scheme for the dissociation of the Semliki Forest virus membrane is presented which is discussed with reference to other biological membranes. It is suggested that Triton X100 and deoxycholate solubilize amphipathic membrane proteins by binding to the hydrophobic segments of these proteins.     

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.     

Subunit composition of the membrane glycoprotein complex of Semliki Forest virus

The quaternary structure of the membrane glycoproteins E1, E2 and E3 of Semliki Forest virus has been determined in intact virus and in the protein complexes obtained after Triton X100 solubilization. Intact and solubilized virus were treated with a cleavable cross-linking reagent and the covalently cross-linked glycoprotein complexes were isolated and characterized using antibodies specific for the E1 and E2 membrane glycoproteins. The isolation and characterization procedure was done in a low sodium dodecyl sulphate concentration which prevented non-covalent association between glycoprotein species, but did not abolish antigen-antibody binding.The major glycoprotein complex seen after cross-linking of either intact or Triton X100 solubilized virus was an approximately 100,000 molecular weight species composed of E1-E2 heterodimers only. These findings show that E1 and E2 form a complex in the virus and that this complex is retained after solubilization with Triton X100. The smallest membrane glycoprotein E3 was not cross-linked to the other proteins and was therefore lost in the isolation procedure. However, the presence of E3 together with E1 and E2 in complexes obtained after Triton X100 solubilization of intact virus suggests that an E1-E2-E3 trimer is present in the virus. It is likely that this trimer forms the spike-like structures seen on the surface of the virus.We have observed that antibody specific for one component of the virus glycoprotein complex can induce rearrangement of uncross-linked complexes in Triton X100 solubilized form. This fact should be considered when using specific antibody for characterization of protein complexes.     

Fast folding of the two-domain semliki forest virus capsid protein explains co-translational proteolytic activity

The capsid protein of Semliki Forest virus constitutes the N-terminal part of a large viral polyprotein. It consists of an unstructured basic segment (residues 1-118) and a 149 residue serine protease module (SFVP, residues 119-267) comprised of two beta-barrel domains. Previous in vivo and in vitro translation experiments have demonstrated that SFVP folds co-translationally during synthesis of the viral polyprotein and rapidly cleaves itself off the nascent chain. To test whether fast co-translation folding of SFVP is an intrinsic property of the polypeptide chain or whether folding is accelerated by cellular components, we investigated spontaneous folding of recombinant SFVP in vitro. The results show that the majority of unfolded SFVP molecules fold faster than any previously studied two-domain protein (tau=50 ms), and that folding of the N-terminal domain precedes structure formation of the C-terminal domain. This shows that co-translational folding of SFVP does not require additional cellular components and suggests that rapid folding is the result of molecular evolution towards efficient virus biogenesis.     

The amphiphilic membrane glycoproteins of Semliki forest virus are attached to the lipid bilayer by their COOH-terminal ends

The membrane location of the Semliki Forest virus glycoproteins E1, E2 and E3 was studied by protease treatment of (1) virus particles and (2) rough micro somes from cells infected with SF virus2. Protease treatment of virus particles removes all but the membrane-associated segments of the glycoproteins. Analyses of protease-treated SF virus membranes in 15% to 22.5% gradient acrylamide gels demonstrate the presence of three distinct peptide species with apparent molecular weights of 9000, 6000 and 5500. The 9000 and the 5500 molecular weight peptides have been aligned to the COOH-terminal end of E2 and the 6000 molecular weight peptide to the COOH-terminal end of El. The mapping of the peptides was done in a “Dintzis”-type of experiment (Dintzis, 1961) where we labelled the proteins of the virus with a gradient of [³⁵S]methionine increasing towards their COOH-terminal end.Protease treatment of microsomes from cells infected with SF virus removes only those parts of the viral glycoproteins that are transversing the lipid bilayer. Analyses of such treated membranes in sodium dodecyl sulphate-containing gels show that a 3000 molecular weight piece is digested from the COOH-terminal end of p62, the cellular precursor of E2 and E3. The COOH-terminus of p62 is shown to be equivalent to that of E2. These results thus demonstrate that the two amphiphilic membrane proteins of SF virus, E1 and E2 (p62) are attached to the lipid bilayer by their COOH-terminal ends. The COOH-terminal end of p62 (E2) spans the microsomal membrane. The third membrane protein, E3, probably does not interact with membrane lipids but is bound to the virus on E1 and (or) E2.     

Studies on the amphipathic nature of the membrane proteins in Semliki Forest virus

The membrane glycoproteins E₁ and E₂ of Semliki Forest virus form spikes protruding from the external surface of the virion. They have been cleaved off by thermolysin or subtilisin leaving peptide segments in the membrane of the spikeless virus particles with a molecular weight of about 5000 enriched in hydrophobic amino acids. These peptides are soluble in chloroform/methanol and are solubilized into mixed micelles with Triton X100, with sodium dodecyl sulphate and with sodium deoxycholate. Peptide mapping studies show that each membrane glycoprotein has its own lipophilic peptide segment which presumably serves to anchor these proteins to the lipid membrane. The hydrophobic segments of the glycoproteins appear to be shielded from proteolysis not only by the lipids in the intact membrane but also by Triton X100 in the detergent-protein complexes obtained when this detergent is used to remove the lipid and solubilize the proteins.     

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.     

Semliki forest virus core protein fragmentation: Its possible role in nucleocapsid disassembly

Semliki Forest virus (SFV) envelope proteins function as proton pores under mildly acidic conditions and translocate protons across the viral membrane [Schlegel, A., Omar, A., Jentsch, P., Morell, A. and Kemp, F. C. (1991) Biosci. Rep. 11, 243–255]. As a consequence, during uptake of SFV by cells via receptor-mediated endocytosis the nucleocapsid is supposed to be exposed to protons. In this paper the effects of mildly acidic pH on SFV nucleocapsids were examined. A partial proteolytic fragmentation of core proteins was observed when nucleocapsids were exposed to mildly acidic pH. A similar proteolytic event was detected when intact SFV virions were exposed to identical conditions. Protease protection assays with exogenous bromelain provided evidence that the capsid protein degradation was due to an endogenous proteolytic activity and not to a proteolytic contamination. Detergent solubilization of virus particles containing degraded nucleocapsids followed by sucrose gradient centrifugation led to a separation of capsid protein fragments and remaining nucleocapsids. These data are discussed in terms of a putative biological significance, namely that the core protein fragmentation may play a role in nucleocapsid disassembly.     

Semliki Forest virus induced cell-cell fusion at neutral extracellular pH

Semliki Forest virus-induced cell-cell fusion from within was considered to exclusively occur at mildly acidic pH (<6.2). Data of this study show that such cell fusion can also be triggered by transient acidification of the cytoplasm of infected cells at an extracellular, neutral pH. Results were obtained by utilizing NH₄Cl pulses combined with covalent modification of cell surface proteins. The observation implies a revision of the current consensus regarding the mechanism of Semliki Forest virus induced cell-cell fusion. We propose a model in which at least two peptide segments of the viral spike protein E₁ may be involved in triggering the fusion event.     

Early onset of virus infection and up-regulation of cytokines in mice treated with cadmium and manganese

A substantial database indicates that a large number of environmental pollutants, chemicals and therapeutic agents to which organisms are exposed cause immunotoxicity. The suppression of immune functions may cause increased susceptibility of the host to a variety of microbial pathogens potentially resulting in a life-threatening state. Evaluation of the immunotoxic potential of chemical xenobiotics is of great concern and, therefore, we have investigated the impact of exposure of inorganic metals, specifically cadmium (Cd) and manganese (Mn) on Encephalomyocarditis virus (EMCV), Semliki Forest virus (SFV), and Venezuelan Equine Encephalitis virus (VEEV) infection. Pretreatment with a single, oral dose of Cd or Mn increased the susceptibility of mice to a sub-lethal infection of these viruses as observed by increased severity of symptoms and mortality compared to untreated controls. An early onset of virus infection was found in brains of Cd and Mn treated animals. Histopathological observations of the brain indicate evidence of inflammation and greater tissue pathology in Cd-or Mn-exposed mice compared to control animals. Meningitis and vascular congestion was seen in virus infected mice in all the metal treated groups, and further, the perivascular inflammation appeared earlier in treated mice compared to control. Encephalitis was maximum in Cd pretreated mice. Widespread environmental contamination of metals and the potential for their exposure and subsequent infection of humans or animals is indicative that further studies of these and all other metals are important to understand the effect of environmental pollution on human health.     

Semliki Forest virus envelope proteins function as proton channels

It has been shown that isolated nucleocapsids of Semliki Forest virus (SFV) contract upon low pH exposure (Soederlundet al., 1972). This contraction of the nucleocapsids has been used as an indicator to demonstrate that the spike proteins of SFV can translocate protons into the interior of the virus particle upon low pH (5.8) exposure. Spikeless virus particles obtained after bromelain digestion, which were used as a control, did not translocate protons. This implies that the ectodomain of the spike plays a crucial role for the proton translocation.     

A model of the binding, entry, uncoating, and RNA synthesis of Semliki Forest virus in baby hamster kidney (BHK-21) cells

A quantitative understanding of viral trafficking would be useful in treating viral-mediated diseases, designing protocols for viral gene therapy, and optimizing heterologous protein production. In this article, a model for the trafficking of Semliki Forest virus and its RNA synthesis in baby hamster kidney (BHK-21) cells is presented. This model includes the various steps leading to infection such as attachment, endocytosis, and viral fusion in the endosome. The model estimates a mean fusion time of 4 to 6 min for the wild-type virus, and 38 min for Fus-1, an SFV mutant which requires a lower pH for fusion. These mean fusion times are consistent with the time-scale of endosomal acidification, suggesting viruses fuse almost instantaneously with the endosomal membrane as soon as the pH of the endosome drops below the pH threshold of the virus. Infection is most likely controlled at the level of viral uncoating, as shown by the close agreement between the efficiency of uncoating and the experimentally determined fraction of viruses that is infectious. The viral RNA synthesized per cell is best described by assuming that it depends on the number of uncoated viruses prior to the onset of replication according to a saturation-type expression. A Poisson distribution is used to determine the distribution of uncoated viruses among the cells. Because attachment is the rate-limiting step in the uncoating of the virus, increasing the attachment rate can lead to enhanced RNA synthesis and, hence, new virion production. Such an increase in the attachment rate may be obtained by lowering the medium pH or the addition of a polycation. (c) 1995 John Wiley & Sons, Inc.     

Semliki Forest virus vectors for rapid and high-level expression of integral membrane proteins

Semliki Forest virus (SFV) vectors have been applied for the expression of recombinant integral membrane proteins in a wide range of mammalian host cells. More than 50 G protein-coupled receptors (GPCRs), several ion channels and other types of transmembrane or membrane-associated proteins have been expressed at high levels. The establishment of large-scale SFV technology has facilitated the production of large quantities of recombinant receptors, which have then been subjected to drug screening programs and structure-function studies on purified receptors. The recent Membrane Protein Network (MePNet) structural genomics initiative, where 100 GPCRs are overexpressed from SFV vectors, will further provide new methods and technologies for expression, solubilization, purification and crystallization of GPCRs.     

Fusion of Semliki Forest virus with cholesterol-containing liposomes at low pH: a specific requirement for sphingolipids

Semliki Forest virus (SFV) utilizes a membrane fusion strategy to introduce its genome into the host cell. After binding to cell-surface receptors, virus particles are internalized through receptor-mediated endocytosis and directed to the endosomal cell compartment. Subsequently, triggered by the acid pH in the lumen of the endosomes, the viral envelope fuses with the endosomal membrane. As a result of this fusion reaction the viral RNA gains access to the cell cytosol. Low-pH-induced fusion of SFV, in model systems as well as in cells, has been demonstrated previously to be strictly dependent on the presence of cholesterol in the target membrane. In this paper, we show that fusion of SFV with cholesterol-containing liposomes depends on sphingomyelin (SM) or other sphingolipids in the target membrane, ceramide representing the sphingolipid minimally required for mediating the process. The action of the sphingolipid is confined to the actual fusion event, cholesterol being necessary and sufficient tor low-pH-dependent binding of the virus to target membranes. The 3-hydroxyl group on the sphingosine backbone plays a key role in the SFV fusion reaction, since 3-deoxy-sphingomyelin does not support the process. This, and the remarkably low levels of sphingolipid required for half-maximal fusion (1–2 mol%), suggest that the sphingolipid does not play a structural role in SFV fusion, but rather acts as a co-factor, possibly through activation of the viral fusion protein. Domain formation between cholesterol and sphingolipid, although it may facilitate SFV fusion, is unlikely to play a crucial role in the process.