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A Helenius

Publications and source records attributed to A Helenius.

At least 145 records · Page 8Linked to original sources
PubMed1982

Monensin inhibits Semliki Forest virus penetration into culture cells.

The carboxylic ionophores monensin and nigericin, at concentrations higher than 10 and 6 muM, respectively, prevent the penetration of the Semliki Forest virus (SFV) genome into the cytosol of baby hamster kidney (BHK-21) cells and thereby inhibit viral replication. In the absence of inhibitors, the entry of SFV is known to proceed by adsorptive endocytosis in coated vesicles, followed by acid-triggered membrane fusion in intracellular vacuoles or lysosomes. The results show that binding of the virus to the cell surface, adsorptive endocytosis, and intracellular transport of viruses to the lysosomes are only marginally affected by the ionophores. No direct virucidal effect is observed, nor is the membrane fusion activity of the virus at low pH directly affected. Sequential addition of monensin and ammonium chloride (a non-related lysosomotropic inhibitor of SFV entry) indicates that both inhibitors affect the same step in the entry pathway. On the basis of these data and the known effects of carboxylic ionophores and lysosomotropic weak bases on cellular pH gradients, we conclude that monensin inhibits penetration by increasing the pH in endocytic vacuoles and lysosomes above pH 6, which is the pH threshold for the viral membrane fusion activity.

Animals↗
PubMed1982

Inhibition of Semliki forest virus penetration by lysosomotropic weak bases.

The effect of five lysosomotropic weak bases (chloroquine, amantadine, tributylamine, methylamine and NH4C1) on Semliki Forest virus (SFV) infection has been studied in BHK-21 cells. When present at concentrations equal to or greater than 0.1, 0.5, 2, 15 and 15 mM respectively, the agents inhibited SFV infection by more than 90%. The effect was reversible and involved a process occurring within the first 60 min of virus-cell contact. The agents did not have a direct virucidal effect nor did they affect virus binding to the cells, receptor-mediated endocytosis of prebound virus, intracellular distribution of virus after endocytosis, or the low pH-induced membrane fusion activity of the virus spike glycoproteins. The step blocked by chloroquine and NH4C1 occurred intracellularly and was identified as the release of the virus nucleocapsid into the cytoplasm or the uncoating process. On the basis of these results, our previous studies on SFV entry, and the known effects of lipophilic amines on lysosomes, we conclude that the agents affect entry by a common mechanism: they prevent the transfer of the virus nucleocapsid into the cytoplasm by increasing the lysosomal pH above the critical value needed to trigger a low pH-dependent fusion reaction between the membranes of the lysosome and the virus.

Amantadine↗
PubMed1981

Cell fusion by Semliki Forest, influenza, and vesicular stomatitis viruses.

Representatives of three families of enveloped viruses were shown to fuse tissue culture cells together. These were: Semliki Forest virus (SFV, a togavirus), vesicular stomatitis virus (a rhabdovirus), and two myxoviruses, fowl plaque virus and Japan influenza virus (Japan)/A/305/57). Unlike paramyxoviruses, whose fusion activity is known to occur over a broad pH range, fusion by these viruses was restricted to mildly acidic pH. The pH thresholds for the four viruses were 6.0, 6.1, 5.5, and 5.1, respectively. The precursor form of Japan influenza, which is not infectious and which contains the uncleaved hemagglutinin, had no fusion activity. This result suggested a role for the influenza hemagglutinin in the low-pH-dependent membrane fusion activity. Taken together, our results show that low-pH-induced fusion is a widespread property of enveloped animal viruses and that it may play a role in the infective process. The fusion reactions with all four viruses were fast, efficient, and easy to induce. With UV-inactivated SFV, the fusion was shown to be nonlytic and the polykaryons were viable for at least 12 h. 30 ng of SFV/1 x 10(6) BHK-21 cells were required for 50% fusion, and 250 ng sufficed to fuse the entire culture into a single polykaryon.

Animals↗
PubMed1981

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

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.

Animals↗
PubMed1981

Antibody response to spike protein vaccines prepared from Semliki Forest virus.

Subunit vaccines, containing the spike glycoproteins of Semliki Forest virus (SFV) in three different forms, have been prepared: detergent-solubilized monomers, detergent- and lipid-free octamers, and virosomes in which the spike proteins are reconstituted into phospholipid vesicles. Previous studies have shown that the octamers and the virosomes are very efficient in protecting mice against the encephalitis caused by virulent SFV (Morein, et al., 1978). In this study we have characterized the specific antibody responses in mice vaccinated with the three SFV vaccines and correlated them with the protection against SFV encephalitis. The multimeric forms induced high humoral antibody titres; two doses of only 1 microgram protein gave rise to specific antibody titres of 0.6 mg/ml. The monomeric form was much less immunogenic.

Animals↗
PubMed1981

Asymmetric and symmetric membrane reconstitution by detergent elimination. Studies with Semliki-Forest-virus spike glycoprotein and penicillinase from the membrane of Bacillus licheniformis.

The dissociation and reconstitution of the Semliki Forest virus membrane using the nonionic detergent octyl beta-D-glucoside was studied by sucrose density gradient centrifugation. The dissociation occurred in three stages: lysis at a free equilibrium octyl glucoside concentration of 14--18 mM, solubilization at 18--20 mM, and delipidation of the spike glycoproteins at the critical micellar concentration (22 mM) or higher. After solubilization the spike glycoproteins were present as soluble complexes with sedimentation coefficients of 19 S and 6 S. The 6-S form probably corresponded to a glycoprotein monomer complexed to detergent and the 19-S form consisted of oligomeric detergent-protein complexes. The two forms were in slow equilibrium with each other. When the soluble spike protein complexes and egg lecithin solubilized with octyl glucoside were mixed and the octyl glucoside concentration lowered either by dialysis or by dilution, reconstitution occurred. Three types of products were obtained: vesicles with 30% of the spike protein facing inwards and 70% facing outwards, vesicles with virtually all (95%) of the spike proteins pointing outwards, and small protein-rich soluble aggregates [Helenius et al. (1977) J. Cell Biol. 75, 866]. It was demonstrated that during reconstitution the symmetric vesicles were formed at 19 mM free equilibrium octyl glucoside by the association of the 6-S protein complexes with the phospholipids, and the asymmetric vesicles were formed at 10--16 mM octyl glucoside when the 19-S complexes associated with the lipids. Asymmetric membrane vesicles were also obtained when membrane penicillinase from Bacillus licheniformis was reconstituted with egg lecithin using octyl glucoside. It could be shown that the penicillinase was oligomeric at the octyl glycoside concentration where the reconstitution occurred. The results demonstrate that different mechanisms of reconstitution give rise to the symmetric and the asymmetric vesicles. The critical factor in determining the mechanism is the state of aggregation of the proteins at the octyl glucoside concentration where membranes begin to form from the solubilized lipids.

Bacillus↗
PubMed1980

pH-dependent fusion between the Semliki Forest virus membrane and liposomes.

Semliki Forest virus was mixed with liposomes containing phosphatidylcholine,phosphatidylethanolamine, sphingomyelin, and cholesterol. When the pH of the mixture was dropped to 6 or below, rapid fusion between the membranes of the virus and the liposomes occurred, resulting in the transfer of viral nucleocapsids into the liposomes. Fusion was demonstrated biochemically by trapping RNase or trypsin within the liposomes. Trapped RNase digested the viral RNA into acid-soluble form, providing a simple quantitative assay for fusion. Trapped trypsin digested the viral capsid protein. Fusion was also demonstrated by electron microscopy as the formation of large vesicles containing viral glycoproteins on the surface and nucleocapsids inside. The efficiency of fusion was 91 +/- 6%. In addition to low pH, it required that the viral glycoproteins be intact. In the target liposomes, cholesterol (but none of the individual phospholipids) was essential. Divalent cations were not required. Our previous studies with tissue culture cells indicated that the final step in the penetration of the Semliki Forest virus genome into host cells might involve a fusion event between the membrane of lysosomally trapped viruses and the lysosomal membrane [Helenius, A., Kartenbeck, J., Simons, K. & Fries, E. (1980) J. Cell Biol, 84, 404--420]. The data presented here are fully compatible with this hypothesis.

Capsid↗
PubMed1980

On the entry of Semliki forest virus into BHK-21 cells.

The pathway by which semliki forest virus (SFV), a membrane-containing animal virus, enters BHK-21 cells was studied morphologically and biochemically. After attaching to the cell surface, the majority of viruses was rapidly trapped into coated pits, internalized by endocytosis in coated vesicles, and sequestered into intracellular vacuoles and lysosomes. Direct penetration of viruses through the plasma membrane was never observed. To assess the possible involvement of lysosomes in the release of the genome into the cytoplasm, the effect of five lysosomotropic agents, known to increase the lysosomal pH, was tested. All of these agents inhibited SFV infectivity and one, chloroquine (the agent studied in most detail), inhibited a very early step in the infection but had no effect on binding, endocytosis, or intracellular distribution of SFV. Thus, the inhibitory effect was concluded to be either on penetration of the nucelocapsid into the cytoplasm or on uncoating of the viral RNA. Possible mechanisms for the penetration of the genome into the cytoplasm were studied in vitro, using phospholipids-cholesterol liposomes and isolated SFV. When the pH was 6.0 or lower, efficient fusion of the viral membranes and the liposomal membranes occurred, resulting in the transfer of the nucleocapsid into the liposomes. Infection of cells could also be induced by brief low pH treatment of cells with bound SFV under conditions where the normal infection route was blocked. The results suggest that the penetration of the viral genome into the cytosol takes place intracellularly through fusion between the limiting membrane of intracellular vacuoles and the membrane of viruses contained within them. The low pH required for fusion together with the inhibitory effect of lysosomotropic agents implicate lysosomes, or other intracellular vacuoles with sufficiently low pH, as the main sites of penetration.

Adsorption↗
PubMed1980

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.

Animals↗
PubMed1980

The effects of octylglucoside on the Semliki forest virus membrane. Evidence for a spike-protein--nucleocapsid interaction.

Evidence is presented for a non-covalent interaction between the spike glycoprotein and the nucleocapsid of Semliki Forest virus. When isolated viruses were treated with the non-ionic detergent beta-D-octylglucoside at neutral pH and low ionic strength the lipid bilayer membrane could be removed leaving most of the spike glycoproteins attached to the nucleocapsids. The interaction between capsids and membrane protein was sensitive to elevated pH and ionic strength, but at least partially reversible at neutral pH and low ionic strength. The possible role of this interaction in the viral structure and in the mechanism of budding of the virus from the host cell is discussed.

Animals↗
PubMed1979

Binding of Semliki Forest virus and its spike glycoproteins to cells.

We have studied the binding of the Semliki Forest virus and its isolated spike glycoproteins, in the form of water-soluble octameric complexes, to various cells at 5 degrees C. The number of viruses bound per cell increased strongly with increasing free concentrations of virus up to about 0.2 nM. At higher concentrations smaller increases in binding were observed but saturation was not achieved. The number of viruses bound at a given free concentration was widely different for different cells. For some cells the binding of the virus was maximal at pH 6.8 with little decrease at lower pH, for other cells it was maximal around pH 6.0. The spike protein complexes were used at 100 times higher molar concentrations than the virus. The binding increased strongly with increasing free concentrations up to about 50 nM and saturation was obtained at higher concentrations. Up to 1.3 X 10(6) spike protein complexes could be bound per cell but great variation could be seen between different cell types. For all cells maximal binding was found below pH 6.0. Together with earlier observations, our results suggest that the virus can bind to a cell by two different modes. Around neutral pH the virus binds to specific glycoproteins and at low pH unspecifically to the lipids of the plasma membrane. The possible physiological roles of these two types of binding are discussed.

Animals↗