Rare earth ions block the ion pores generated by the class II fusion proteins of alphaviruses and allow analysis of the biological functions of these pores

An early step during infection by enveloped viruses is the fusion of the envelope with a cellular target membrane. The fusion pore allows the interior structures of the virus to enter the cytoplasm. This reaction is regulated by viral fusion proteins. One group of fusion proteins are the so-called class II proteins, which comprise the homologous fusion proteins of alphaviruses (reviewed by Schlesinger & Schlesinger, 2001), flaviviruses (reviewed by Lindenbach & Rice, 2001) and probably hepatitis C viruses (reviewed by Major et al., 2001). The atomic structures of the E fusion proteins of the flaviviruses Tick-borne encephalitis virus (Rey et al., 1995) and dengue 2 virus (Modis et al., 2003) and of the E1 fusion protein of the alphavirus Semliki Forest virus (SF virus) (Lescar et al., 2001) have been determined.

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.

Cells and viruses.
HEK-293 cells were grown in medium composed of Dulbecco's medium and Ham's F12 medium (1 : 1) with 10 % (v/v) fetal bovine serum. BHK-21 cells were grown in Dulbecco's medium with 5 % (v/v) fetal bovine serum. The alphaviruses SIN virus and SF virus were grown in BHK-21 cells and purified by centrifugation as described previously (Wengler et al., 1999).

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.

Block of ion channels generated during entry of alphaviruses
Ion-permeable pores have been detected in the plasma membrane during entry of alphaviruses, if cells subjected to a patch-clamp configuration were infected at the plasma membrane (Wengler et al., 2003). For the identification of pore-blocking molecules, test substances were added to patch-clamped HEK-293 cells during the current inflow induced by the entry of SF or SIN viruses and the reduction of current was determined. These viruses have a low sequence similarity and it can be assumed that inhibitors that block both pores are generally active against alphavirus pores. In a first series of experiments, the following ions were analysed: La³⁺, Zn²⁺, Mn²⁺, Ni²⁺, Cd²⁺ and Co²⁺. Each of these ions has been described as an ion-channel inhibitor in specific situations (Hille, 2001). It has been shown that Zn²⁺ at a concentration of 2 mM blocks the pores generated by SF virus (Lanzrein et al., 1993; Spyr et al., 1995). Representative results are shown in Fig. 1. The data obtained in such analyses lead to the following conclusions: Mn²⁺, Ni²⁺, Cd²⁺ and Co²⁺ up to concentrations of 1 mM have no discernible inhibitory effect (data not shown). Zn²⁺ at 1 mM leads to about 70 % inhibition of the current (Fig. 1c) and La³⁺ at 100 µM leads to a complete block of current (Fig. 1b). In view of these results, the additional rare earth ions Ce³⁺, Gd³⁺, Tb³⁺, Yb³⁺ and Lu³⁺ were analysed. Each of these ions at 100 µM blocks the current observed during entry of both viruses within 20 s, as shown as an example for Gd³⁺ and SIN virus pores (Fig. 1d). As, in these experiments, the ion flow from the extracellular buffer into the cytoplasm is determined, the ion pores, which are probably already formed in the virus particle at low pH (see later), can be detected only after fusion has transferred these pores together with the viral membrane into the plasma membrane. A block of fusion of the viral membrane into the target membrane therefore would also inhibit the ion current determined in these analyses. The analyses shown in Fig. 1(e and f) address this question. In these analyses, a capillary is positioned close to the patch-clamped cell, which is decorated with virus as described above, and a series of different solutions is allowed to stream continuously around this cell from the capillary. In Fig. 1(e), fusion of virus into the plasma membrane is induced by low-pH solution from the capillary and, in the next step, the cell is superfused by neutral extracellular solution. At neutral pH, fusion of virus particles is stopped. It can be seen that, at neutral pH, spontaneous closure of the existing pores leads to a slow decrease of the membrane current. In Fig. 1(f), the same analysis is made, but 100 µM La³⁺ is present in the neutral extracellular solution. The membrane current is now blocked rapidly within 20 s. This was confirmed by current voltage analyses of membrane current, which revealed the typical currents through opened pores even 3 min after neutral superfusion, whereas these currents were blocked within 15±5 s (SD; n=6) in the presence of 100 µM La³⁺. These data indicate that the ion pores, which close spontaneously at neutral pH within minutes, are blocked rapidly by the La³⁺ ions. A modified version of the experimental approach shown in Fig. 1 allows determination of the concentrationresponse curve for inhibitors. In this assay, the inhibitor is already present in the low-pH buffer used to induce virus entry at the plasma membrane. Original data obtained in the absence of inhibitors and in the presence of 3 µM La³⁺ and of 100 µM La³⁺ are shown in Fig. 2(a). It can be seen that the current during virus entry depends on the concentration of La³⁺ present in the low-pH buffer. The concentrationresponse curves for La³⁺ and Zn²⁺ for SF virus pores are shown in Fig. 2(b).

(20K): Fig. 1. Rare earth ions block the current that is generated during entry of alphaviruses at the plasma membrane. HEK-293 cells were subjected to the whole-cell configuration of the patch-clamp technique followed by adsorption of SF or SIN virus particles containing 1 µg E1 protein for 2 min. In all analyses, virus entry was induced by addition of extracellular solution of pH 5·4 (black arrow). At about 30 s after low-pH treatment, a second treatment for resealing of the membrane with buffer containing the ions to be tested for ion pore-blocking activity was made (open arrow). Experimental details are given in Methods. Original membrane-current recordings obtained in analyses with the following virus and resealing conditions are shown: (a) SF virus, pH 5·4 buffer without inhibitor; (b) SF virus, pH 5·4 buffer containing 100 µM La3+; (c) SF virus, pH 5·4 buffer containing 1 mM Zn2+; (d) SIN virus, pH 5·4 buffer containing 100 µM Gd3+; (e) SF virus, pH 7·4 buffer without inhibitor; (f) SF virus, pH 7·4 buffer containing 100 µM La3+. Note the different timescales of the measurements.

(22K): Fig. 2. Determination of the concentrationresponse relationship for inhibition of ion flow at the plasma membrane. HEK-293 cells were subjected to the whole-cell configuration of the patch-clamp technique, followed by adsorption of SF virus containing 1 µg E1 protein for 2 min. Virus entry was induced by addition of extracellular solution of pH 5·4, containing the ion to be tested at a concentration of 1000, 300, 100, 30, 10 or 3 µM. (a) Original membrane-current recordings obtained in the absence of an inhibiting ion and in the presence of 100 or 3 µM La3+. The time of addition of low-pH solution is indicated by a black arrow. (b) The total charge that flows through virus-induced pores into the cell during the first 2 min after addition of low-pH solution was determined by integration of the corresponding membrane currents. The concentrationresponse curves determined from these data for La3+ and Zn2+ and SF virus-derived pores are shown.

Rare earth ions do not interfere with productive infection and represent specific blockers of alphavirus ion pores
In the experiments reported above, HEK-293 cells were used. The same block of ion current in the presence of 100 µM La³⁺ was also observed at the plasma membrane of BHK cells (data not shown). As SF and SIN viruses form plaques on BHK cell monolayers, the BHK system allows determination of whether the flow of ions through the ion-permeable pores during virus entry is necessary for the establishment of a productive infection, as measured by the development of plaques, provided that entry of virus particles occurs at the plasma membrane. Experimental conditions were therefore developed in which it can be assured in each experiment that the majority of plaques, which develop after entry at low pH, are generated from virus particles that enter the cell at the plasma membrane and not at the endosome. To this end, virus adsorption was performed at 0 °C, entry was induced by incubation at various pH values at 37 °C (White et al., 1980) and the endosomal pathway of infection was inhibited by chloroquine (Helenius et al., 1982). Incubation in the presence of chloroquine suppresses the number of plaques formed following virus uptake via the endosomal pathway by at least 80 % (data not shown). In contrast, virus infection at the plasma membrane is not inhibited by chloroquine. In the presence of chloroquine, therefore, low pH-induced entry of a defined number of virus particles at the plasma membrane should generate more plaques than the entry of the same number of particles at neutral pH via the endosome. The increase in the number of plaques at low pH would directly reflect the entry of virus particles at the plasma membrane. The results obtained in a representative experiment are shown in Fig. 3. The entry at the endosome in the presence of chloroquine generates the plaques that can be seen in the Petri dish shown in the upper left of Fig. 3. The results presented in the left column of Fig. 3 show that, after entry at pH 5·8 or 5·4, the number of plaques is greatly increased. Entry of virus particles at the plasma membrane can therefore be detected efficiently in this system. If the presence of rare earth ions during the step of low-pH entry at the plasma membrane blocks either entry or uncoating, the increase of plaques observed at low pH should be blocked by these ions. The right column of Fig. 3 shows the results obtained in the presence of LaCl₃ (0·5 mM) during the entry steps. The results show that LaCl₃ did not inhibit the increase in the formation of plaques at pH 5·8 and 5·4. In this experiment, induction of virus entry was performed by a 1 min incubation at the desired pH, followed by a 1 min incubation at neutral pH. Both solutions contained the inhibitor to be analysed. This procedure was chosen because it has been reported that virus entry may occur shortly after return from acid to neutral pH (Paredes et al., 2004). The same results were obtained if the 1 min incubation at neutral pH was omitted (data not shown). These results indicate that rare earth ions, even at high concentrations, do not interfere with low pH-induced entry of virus particles at the plasma membrane.

(57K): Fig. 3. Plaque-assay analyses of the influence of ions on the entry of SF virus at the plasma membrane. The experiment comprises the following steps (see Methods for details): pre-incubation of the BHK cell monolayers in the presence of chloroquine, adsorption of about 200 p.f.u. virus at 0 °C, virus entry by incubation at 37 °C at different pH values, post-incubation in the presence of chloroquine, and development of plaques. The variable parameters are present in the virus-entry step, namely the pH value and the absence or presence of rare earth ions. In this experiment, plaques generated by SF virus particles after entry at pH 7·0, 6·2, 5·8 and 5·4 in the absence (control) or presence of 500 µM La3+ are shown as indicated in the figure.

Whilst the data presented in Fig. 3 show that the rare earth ions have no effect on the formation of the fusion pore at the plasma membrane of BHK cells, the data presented in Fig. 1 indicate that the ion flow through this membrane by way of the viral ion pores should be blocked by rare earth ions. This effect can be seen directly in the experiment shown in Fig. 4. The ion-permeable pores generated in the target membrane during virus entry allow the passage of Ca²⁺ ions and lead to a rapid inflow of Ca²⁺ from the extracellular solution into the cytoplasm if virus entry occurs at the plasma membrane. The increase in intracellular Ca²⁺ concentration can be visualized by fluorescence microscopy using the Ca²⁺ fluorochrome Fura-2. In the experiment presented in Fig. 4, BHK cells were loaded with Fura-2, followed by adsorption of virus particles to the plasma membrane at 0 °C and activation of fusion by incubation at pH 5·4 in the absence or presence of La³⁺. It can be seen that a rapid influx of Ca²⁺ into cells decorated with virus particles, but not into undecorated cells, is observed after exposure to low pH in the absence of inhibitor and that this Ca²⁺ inflow is blocked by the presence of 100 µM La³⁺.

(129K): Fig. 4. Inhibition of Ca2+ inflow during entry of SF virus at the plasma membrane. BHK cell monolayers were loaded with Fura-2 for 20 min at 37 °C, followed by adsorption of about 100 p.f.u. SF virus per cell in extracellular solution for 10 min at 0 °C. The cell layers were then mounted into the fluorescence microscope, virus entry was induced immediately by addition of prewarmed (37 °C) low-pH buffer (pH 5·4) and the intracellular Ca2+ concentration was monitored ratiometrically as described in Methods. The pictures are the resulting false-colour representations of the maximal Ca2+ concentration, which were reached about 30 s after addition of low-pH solution. The colour scale is shown at the bottom of the figure. The following data are shown: (a, a') the intracellular Ca2+ concentration of control cells that were not decorated with virus prior to (a) and after (a') low-pH treatment; (b, b') the intracellular Ca2+ concentration of virus-decorated cells prior to (b) and after (b') low-pH treatment; (c, c') the intracellular Ca2+ concentration of virus-decorated cells prior to (c) and after (c') treatment with low-pH buffer containing 100 µM La3+.

The results reported above allow three major conclusions: (i) rare earth ions do not inhibit the formation of the fusion pore by the E1 protein of alphaviruses; (ii) rare earth ions are specific inhibitors of the alphavirus ion pores that are generated in the target membrane during virus entry; (iii) a flow of ions through these pores is not necessary for the establishment of productive infection by alphaviruses.

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.

(117K): Fig. 5. Inhibition of low pH-induced Ca2+ entry into SF virus-infected cells. HEK-293 cell monolayers were infected with SF virus at an m.o.i. of 10. At 3·5 h post-infection, layers were loaded with Fura-2 for 20 min at 37 °C, washed with extracellular solution and subjected to fluorescence microscopy. Formation of ion-permeable pores was induced by low-pH buffer (pH 5·4) and intracellular Ca2+ concentration was monitored ratiometrically as described in Methods. The pictures are the resulting false-colour representations of the maximal Ca2+ concentrations, which were reached about 30 s after addition of low-pH solution. The colour scale is shown at the bottom of the figure. The pictures shown are derived from the following samples: (a) a monolayer of infected cells prior to low-pH treatment and (b) the same monolayer after low-pH treatment. Monolayers of infected cells were treated with low-pH buffer containing (c) 100 µM La3+, (d) 30 µM La3+ and (e) 100 µM Zn2+.

Low-pH treatment of alphavirus particles leads to formation of ion pores in the viral membrane, which can be blocked by rare earth ions
In the experiments reported above, alphavirus ion pores, which are formed in cellular membranes, have been analysed. Evidence has been presented that ion-permeable pores are also formed in the viral membrane if alphavirus particles are exposed to low pH (Spyr et al., 1995; Käsermann & Kempf, 1996). These pores were detected by measuring the flow of propidium iodide into SF virus particles at low pH, which allows the binding of the membrane-impermeant propidium iodide to the viral genome and leads to an increase in the fluorescence of propidium iodide. If specific pores are generated in these experiments, it might be expected that they can be blocked by rare earth ions. We therefore analysed the influence of rare earth ions on these pores in SF and SIN virus particles. A representative experiment is presented in Fig. 6. It can be seen that adjustment of SF virus to low pH leads to inflow of propidium iodide, which is blocked in the presence of 100 µM La³⁺. The total amount of genome RNA present in the reaction was about 5 µg and the data show that the observed increase in fluorescence corresponds to the reaction of about 1·5 µg RNA with propidium iodide. Similar results are obtained with SIN virus. For this virus, the salt concentration in the reaction had to be increased to 2 M to obtain an efficient suspension to individual particles. The final signal generated by propidium iodide is about half that generated by SF virus and is also blocked by 100 µM La³⁺ (data not shown). These results support the conclusion that the changes of permeability observed in the membrane of isolated alphavirus particles at low pH represent the formation of ion-permeable pores and are not caused by non-specific lesions.

(19K): Fig. 6. Analysis of formation of ion-permeable pores in isolated alphavirus particles at low pH. The formation of ion-permeable pores and a possible inhibition by ions were determined by measurement of the increase in fluorescence that results from the flow of propidium iodide into virus particles. The data obtained in an analysis of pore formation using SF virus particles containing about 20 µg E1 protein in the absence or presence of 100 µM La3+ are shown. As described in detail in Methods, this assay consists of four steps, which are shown as follows: the fluorescence of the two identical 180 µl starting samples containing virus in suspension buffer, pH 8·1, is shown in the left columns. The two samples are identified by the light- and dark-shaded bars. To one of the samples (light-shaded bar), the following three solutions were added consecutively: suspension buffer containing La3+, pH 5·0 buffer, suspension buffer. To the second sample (dark-shaded bar), the same solutions were added in the following sequence: suspension buffer, pH 5·0 buffer, suspension buffer containing La3+. The stable fluorescence that developed after each of these three additions was recorded and is shown in columns t1, t2 and t3, respectively. The amount of RNA that corresponds to the fluorescence intensity shown on the scale on the left is given on the scale on the right.

The class II fusion protein of alphaviruses forms ion-permeable pores at low pH, which can be detected in three different situations: in the target membrane during virus entry, in the plasma membrane of infected cells during viral multiplication and in isolated virus particles. The results reported above show that rare earth ions are specific inhibitors of these ion pores in all three situations. Rare earth ions completely block the ion permeability of the pores at 0·1 mM, but even at 0·5 mM, they have no effect on virus entry at the plasma membrane and therefore on the formation of the viral fusion pore. The transition-element ions Mn²⁺, Co²⁺, Ni²⁺ and Cd²⁺ did not block the alphavirus ion pores, whereas Zn²⁺ at 2 mM blocked the pores, a finding reported previously by Lanzrein et al. (1993). However, an effect of Zn²⁺ on the fusion of SF virus into liposomes has been reported (Corver et al., 1997). Recently, a specific binding of the holmium rare earth ion to a crystallized trimeric form of the SF virus E1 protein has been described (Gibbons et al., 2004). The data presented above indicate that rare earth ions are effective and specific inhibitors of the alphavirus ion pores.

Entry of alphaviruses leads to a co-entry of the 16 kDa protein toxin α-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.

This study was supported by grant RE 1046/1-2 to Holger Repp and by grant WE 518/3-3 to Gerd Wengler from the Deutsche Forschungsgemeinschaft.

Footnotes

^,†, Gerd Wengler² †These authors contributed equally to this work.