1 Rudolf-Buchheim-Institut für Pharmakologie, Justus-Liebig-Universität, D-35392 Giessen, Germany
2 Institut für Virologie, Fachbereich Veterinärmedizin, Justus-Liebig-Universität, D-35392 Giessen, Germany
Correspondence
Gerd Wengler
gerd.wengler{at}gmx.de
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ABSTRACT |
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These authors contributed equally to this work.
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INTRODUCTION |
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In this manuscript, the function of the E1 fusion protein of alphaviruses (reviewed by Garoff et al., 1994; Kielian, 1995
) is analysed. The fusion protein of alphaviruses has two additional functions besides its fusion activity: it generates an icosahedral lattice on the viral surface (Lescar et al., 2001
; Pletnev et al., 2001
) and it forms ion-permeable pores in the target membrane during virus entry (Carrasco, 1995
; Nyfeler et al., 2001
and references cited therein; Wengler et al., 2003
). In vivo, alphaviruses use the endosomal membrane as target membrane, but in vitro, virus can be adsorbed to the plasma membrane and entry at this membrane is induced by low pH (White et al., 1980
). Under these conditions, the ion-permeable pores allow a flow of ions from the extracellular medium into the cytoplasm. This current can be analysed by using an individual cell in the whole-cell patch-clamp configuration as target cell for virus entry (Wengler et al., 2003
).
Recently, we proposed a model that explains whether class II fusion-protein molecules form a fusion pore or ion-permeable pores: for steric reasons, only a fraction of the activated molecules can interact with the endosomal membrane. These molecules form the fusion pore. The rest of the molecules react with the viral membrane, in which they are anchored and form ion-permeable pores (Wengler et al., 2004). As the viral membrane is transferred into the target membrane after fusion, these ion-permeable pores can be detected in the plasma membrane if virus entry is targeted there. If this model is correct, it should be possible to block the ion-permeable pores without interference with the formation of the fusion pore. We have used the pores generated by the alphaviruses SF virus and Sindbis virus (SIN virus) to address this question. Transition-metal ions are inhibitors of ion channels (Aidley & Stanfield, 1996
; Hille, 2001
) and Zn2+ ions at a concentration of 2 mM block the ion pores generated by the SF virus (Lanzrein et al., 1993
; Spyr et al., 1995
). We have analysed a number of further ions as possible inhibitors and have found that rare earth ions specifically block the ion pores of alphaviruses at a concentration of 100 µM. The results of these studies are presented in this report.
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METHODS |
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Patch-clamp analyses of closure of membrane pores.
Patch-clamp measurements were performed as described previously (Wengler et al., 2003). In short, in the standard procedure, virus particles were suspended in extracellular solution [140 mM NaCl, 3 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 10 mM glucose, 10 mM HEPES (pH 7·35), adjusted with NaOH] containing 2 % Ficoll 400 (Sigma) to a concentration of 50 µg fusion protein ml1. A 20 µl aliquot of this suspension was transferred to the surroundings of the patch-clamped cell. After 2 min incubation at 20 °C for virus adsorption, 50 µl low-pH solution consisting of extracellular solution with 3 % Ficoll 400, buffered with 10 mM MES at pH 5·4, was applied to the surroundings of the patch-clamped cell to induce virus entry. In order to assay possible inhibitors, buffer containing the inhibitor was added to the surrounding of the patch-clamped cell during the virus-induced current inflow, as indicated in the description of the experiments. The following salts were analysed as inhibitors: ZnCl2, MnCl2, CoCl2, NiCl2, CdCl2 and the rare earth salts CeCl3, LaCl3, TbCl3, GdCl3, LuCl3 and Yb(CF3SO3)3 (ytterbium trifluoromethanesulfonate). All rare earth ion salts were obtained from Fluka. Stock solutions of 100 mM of the salts were stored at 20 °C.
Determination of intracellular Ca2+ concentration by fluorescence microscopy.
The concentration of Ca2+ was determined ratiometrically by using the Ca2+ dye Fura-2 (Silver, 1998) as described previously (Koschinski et al., 2003
). The intracellular Ca2+ concentration was analysed by using the Metafluor imaging software (Molecular Devices Corporation). Low-pH treatment (pH 5·4) was performed by addition of low-pH extracellular solution, as described above for the patch-clamp analyses.
Analyses of the influence of ions on virus entry at the plasma membrane by plaque assay.
Virus entry at the plasma membrane was analysed by an experiment consisting of five steps. (i) BHK cells grown in 9 cm diameter Petri dishes to about 50 % confluence were pre-incubated in growth medium containing 200 µM chloroquine (Sigma) for 15 min at 37 °C. (ii) The medium was exchanged against 200 µl extracellular solution, pH 7·4, containing about 200 p.f.u. virus at 0 °C. Virus adsorption was performed by 15 min incubation at 0 °C. (iii) Virus entry was induced by addition of 10 ml prewarmed (37 °C) extracellular solution containing 10 mM MES instead of HEPES at the pH indicated in the description of the experiments (pH values between 7·0 and 5·0 were analysed). After incubation for 1 min at 37 °C, this solution was discarded and a further incubation in the presence of extracellular solution of pH 7·4, containing 200 µM chloroquine, was performed for 1 min at 37 °C. In the analyses of the influence of ions on virus entry, both solutions contained the ion at 500 µM. (iv) Cells were incubated in growth medium containing 200 µM chloroquine for 1 h at 37 °C. (v) Cells were washed with growth medium and incubated with growth medium containing 5 % methylcellulose (Methocel MC, medium viscosity; Fluka) for 2 days for development of plaques. Plaques were visualized by fixation of the cell layer with formaldehyde, followed by staining with crystal violet. Pre-incubation, virus adsorption and post-incubation were performed identically in all analyses. Only the conditions of entry differed, as indicated above.
Analysis of propidium iodide entry into virus particles.
The basic technique described by Spyr et al. (1995) was used. A buffer that allows efficient suspension of virus particles was used as starting buffer. In the case of SF and SIN viruses, suspension buffer consisted of 0·5 M NaCl, 2 mM Tris/HCl (pH 8·1) and 2 M NaCl, 2 mM Tris/HCl (pH 8·1), respectively. In the standard experiment, two wells (A and B) were each supplied with 160 µl suspension buffer containing 25 µM propidium iodide and 20 µl virus containing about 20 µg fusion protein. The corresponding fluorescence was taken as starting fluorescence. After each of the following four additions, the resulting stable fluorescence was recorded: (i) 20 µl suspension buffer or suspension buffer containing the inhibitor was added to wells A and B, respectively; (ii) 20 µl 300 mM MES, pH 5·0, was added to both wells; (iii) 20 µl suspension buffer containing the inhibitor or suspension buffer was added to wells A and B, respectively. This is the reverse of the additions made under step (i). At this stage, both wells had the same composition. (iv) rRNA was added in aliquots containing 2·5 µg RNA as an internal control for the availability of free propidium iodide and for an estimation of the fluorescence increase generated by the corresponding amount of RNA. The excitation wavelength was 530 nm and emitted light >612 nm in wavelength was recorded.
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RESULTS |
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Inhibition of ion channels formed by E1 protein accumulating during alphavirus multiplication
E1 protein accumulates during multiplication of alphaviruses in the plasma membrane and forms ion-permeable pores if the cells are exposed to low pH (Dick et al., 1996; Nyfeler et al., 2001
). These channels allow an inflow of Ca2+ into the cells, which have a rather low intracellular Ca2+ concentration. By using the Ca2+-imaging technique, we have shown that, at 3 h post-infection, SF virus-infected HEK-293 cells form ion-permeable pores within seconds following exposure to low pH (Wengler et al., 2004
). The inclusion of inhibitors in the low-pH buffer solution in this experimental setting allows the identification of ions that block these pores (Fig. 5
). It can be seen that a rapid Ca2+ inflow into infected cells occurs at low pH in the absence of inhibitor (Fig. 5a, b
), that this inflow is blocked by the presence of 100 µM La3+ (Fig. 5c
) and that 30 µM La3+ (Fig. 5d
) or 100 µM Zn2+ (Fig. 5e
) does not block Ca2+ entry. The data obtained in these experiments show that a concentration of 100 µM is necessary and sufficient for the rare earth ions to block the pores observed in SF and SIN virus-infected cells and that the other ions mentioned in the first paragraph do not block these pores at a concentration of 100 µM (data not shown). The data indicate that the pores induced by low pH from the virus-specific proteins that accumulate in the plasma membrane during virus multiplication are structurally similar or identical to the pores that are generated during virus entry.
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DISCUSSION |
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Entry of alphaviruses leads to a co-entry of the 16 kDa protein toxin -sarcin (Madan et al., 2005
). As the individual ion-permeable pore has a conductance of 100125 pS (Wengler et al., 2003
), it can be estimated that it will have a diameter of <1 nm and will exclude the passage of molecules with molecular mass greater than about 800 Da (Benz et al., 1989
; Panchal et al., 2002
). However, the conversion of the viral surface proteins into ion-permeable pores is a complex process and it is not unlikely that a failure to perform this process correctly might generate lesions that allow the passage of large molecules. In an analysis of the permeability changes that occur during fusion of alphaviruses with liposomes, Smit et al. (2002)
have concluded that this fusion is a non-leaky process. This conclusion has been drawn from the findings that [3H]inulin (5200 Da) was retained in the liposomes and that about 9 % of the [3H]sucrose (344 Da) was released during fusion. The formation of ion-permeable pores during virus fusion is not inconsistent with these observations.
The ability to block the permeability of the ion pores allows analysis of the biological role of these pores. The results reported above show that, during entry of alphaviruses at the plasma membrane, a flow of ions through the ion-permeable pores generated by the E1 protein in the target membrane after fusion is not necessary for productive infection. This finding was unexpected, as we had suggested a role of these pores in core disassembly. Cores of alphaviruses accumulate in the cytoplasm during virus multiplication. The cores isolated from infected cells late in infection and by detergent treatment from virus particles are stable structures and the genome RNA present in these cores is not accessible for translation in vitro (Koschinski et al., 2003). We have proposed that a flow of protons from the endosome into the cytoplasm through the virus-generated pores might be necessary for the disassembly of cores of alphaviruses by ribosomes (Wengler & Wengler, 2002
). In view of the data reported above, this model is no longer tenable. An alternative role of the viral ion pore in core disassembly has been discussed by Lanzrein et al. (1993)
: the formation of ion-permeable pores in the envelope of alphaviruses in the endosome prior to fusion would allow the flow of protons into the virus and might prepare the viral core for disassembly. The analyses reported above support the finding that ion pores are generated in the envelope of alphavirus particles at low pH (Schlegel et al., 1991
; Spyr et al., 1995
) and indicate that these pores are blocked by rare earth ions at a concentration of 0·1 mM. The finding that alphavirus infection at the plasma membrane was not inhibited in the presence of 0·5 mM rare earth ions indicates that an ion flow through these pores into the virus particle is not necessary for virus entry and core disassembly. Taken together, the data indicate that, in the endosome, low pH activates the fusion activity of the viral surface and the formation of ion-permeable pores in the virus particle, that ions flow through these pores from the endosome into the virus particle and from the endosome into the cytoplasm after viral fusion, but that these ion flows are not necessary for the establishment of productive infection.
Why does the E1 fusion protein of alphaviruses form ion-permeable pores that are not necessary for virus infection? A possible answer is the interpretation that the E1 protein is derived from a family of proteins that have the ability to insert into lipid bilayers at low pH and form ion-permeable pores under these conditions. Furthermore, the lipid specificity of the fusion protein of alphaviruses indicates that proteins that interact specifically with cholesterol (White & Helenius, 1980; Kielian & Helenius, 1984
) and sphingolipids (Nieva et al., 1994
) would be of special interest. The protein could have been modified into the viral fusion protein, retaining a residual pore-forming activity. This idea is illustrated by the following example. The entry of diphtheria toxin has been studied intensively (reviewed by Collier, 2001
). Diphtheria toxin binds to a receptor at the plasma membrane and is taken up by endocytosis. The low pH in the endosome induces a conformational change in the toxin that leads to insertion into the endosomal membrane, followed by transfer of the A fragment of the toxin into the cytoplasm. The same entry can also be induced by low pH at the plasma membrane. Of special interest is the fact that the B fragment of the toxin forms cation-selective channels in the target membrane at low pH. In this example, the assumption is that alphaviruses have used modified diphtheria toxin as fusion protein. If assembled in multiple copies on the surface of a virus particle, the original ability of diphtheria toxin to insert into the target membrane at low pH in the endosome would allow for the transfer of the viral core into the cytoplasm. A large number of protein toxins that enter the cell and act on cytosolic targets or of membrane-damaging protein toxins have been identified (reviewed by Alouf & Freer, 1999
; Sandvig & van Deurs, 2002
; Burns et al., 2003
) and can be used alternatively in this approach. Currently, no sequence nor structural similarity has been identified between the E1 fusion protein and any protein toxin.
The proposal that the class II fusion proteins might be derived from pore-forming proteins is attractive, as it helps to explain not only the low-pH entry, but also several experimental results that currently are not readily understood. Fusion of the viral membrane into the target membrane by class II fusion proteins has a number of unique properties. Under the best-characterized conditions, fusion is activated in the endosome by low pH (reviewed by Kielian, 1995; Heinz & Allison, 2003
). However, conditions have been described under which virus entry can occur at neutral pH, either by chemical reduction of critical disulfides (Abell & Brown, 1993
) or under physiological conditions in insect cells (Hernandez et al., 2001
). Furthermore, experiments have been reported that indicate that alphaviruses deprived of a lipid membrane retain a residual infectivity (Omar & Koblet, 1988
). This finding is further supported by recent experiments that indicate that entry of the SIN virus at the plasma membrane may occur in the absence of membrane fusion and does not require disassembly of the viral protein shell (Paredes et al., 2004
). The assumption that class II fusion proteins are derived from protein toxins may allow the explanation of all of these results. A number of protein toxins exist that are activated by interaction with an appropriate lipid membrane at neutral pH. The finding that the E1 fusion protein of alphaviruses interacts specifically with cholesterol and sphingolipids (reviewed by Kielian et al., 2000
) indicates that it might be derived from a pore-forming protein toxin that is activated by interaction with these lipids at neutral pH. The class II fusion proteins are associated with a second protein in the viral surface (reviewed by Strauss & Strauss, 2001
; Heinz & Allison, 2003
). It is possible that a disruption of this interaction can activate class II fusion proteins at neutral pH. Under many physiological conditions, low pH triggers the disruption, but under special circumstances, disruption might be induced at neutral pH, e.g. by reducing conditions, as described above, or by interaction with specific lipids in the target membrane (Koschinski et al., 2003
). The origin of class II fusion proteins from pore-forming proteins might also explain why the fusion of the viral membrane with the target membrane might not be absolutely necessary for infection: if a lipid membrane is present in the virus, it will be fused into the target membrane, but the assembly of the viral surface proteins might be able to transfer the viral core into the target cytoplasm in the absence of a viral lipid membrane. The entry of viruses containing class II fusion proteins in this case may be basically similar to the entry of non-enveloped viruses.
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ACKNOWLEDGEMENTS |
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Received 11 April 2005;
accepted 5 September 2005.
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