1 Institut für Virologie der Veterinärmedizin and
2 Rudolf-Buchheim-Institut für Pharmakologie, Justus-Liebig-Universität, D-35392 Giessen, Germany
Correspondence
Gerd Wengler
gerd.wengler{at}gmx.de
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ABSTRACT |
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Introduction |
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The core of alphaviruses is an icosahedral complex of 240 molecules of core protein and 42S viral genome RNA. During virus multiplication, newly synthesized cores accumulate in the cytoplasm prior to budding. A special process must therefore account for the disassembly of cores that are released into the cellular cytoplasm after fusion of the viral into the endosomal membrane. Core disassembly involves an interaction of cores with 60S ribosomal subunits (Wengler & Wengler, 1984; Singh & Helenius, 1992
). Experiments have been reported indicating that this interaction is regulated by an unidentified process (Singh et al., 1997
). Recently, we have analysed the disassembly of alphavirus cores in vitro in order to identify this process (Wengler & Wengler, 2002)
. In these studies, we discovered that a low-pH environment strongly stimulated the disassembly. At first sight this situation seemed to be unphysiological but it has been shown that the accumulation of viral structural proteins in the cell membrane that occurs during virus multiplication alters the permeability of the membrane at low pH late in infection (Lanzrein et al., 1993
; Dick et al., 1996
). These data led us to propose that the viral surface proteins that are transferred to the endosomal membrane after fusion of the viral and the endosomal membranes in the early stage of virus infection form a pore (Wengler & Wengler, 2002)
. The resulting flow of protons from the endosome into the cytoplasm through this pore would lead to a region of low pH at the correct time and place to allow the efficient disassembly of alphavirus cores. The molecules and processes involved in the fusion of the viral and the cellular membranes have been studied intensively [for reviews see Garoff et al. (1994)
; Kielian (1995)
]. It is important to note that the data presented below did not analyse the fusion process or the formation or structure of the fusion pore, which is rather large and allows the passage of the core into the cytoplasm. The aim of the experiments was to analyse whether the membrane patch, generated after insertion of the viral membrane into the target membrane, contained ion-permeable pores, which might support the above-mentioned proton flow.
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Methods |
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Preparation of [3H]choline-loaded liposomes.
Liposomes were prepared from phosphatidylethanolamine (PE), phosphatidylcholine (PC), sphingomyelin (SPM) and cholesterol. Thirty-five mg of a dry mixture of these lipids in the proportions indicated in the individual experiments was solubilized in 1 ml 1-propanol at 50 °C and injected rapidly through a 0·45x12 mm needle into 20 ml 50 mM NaCl, 20 mM Tris/HCl, pH 8·1, prewarmed to 50 °C under strong stirring. After a 2 min incubation at 50 °C, the opalescent solution was cooled to 0 °C, filtered through a 0·45 µm Millex (Durapore) membrane and liposomes were pelleted by centrifugation of the filtrate. Liposome pellets were stored at 0 °C. For loading with [3H]choline, liposomes containing 5 mg lipid were suspended in 300 µl water, and 25 µCi [3H]choline in 12·5 µl ethanol was added. The opalescent solution was sonicated at room temperature 20 times for 0·1 s each and incubated at 37 °C for 30 min to allow resealing. The material was then adjusted to 50 mM NaCl, 10 mM Tris/HCl, pH 8·1, and subjected to gel filtration on a Superose 6 HR 10/30 column in 50 mM NaCl, 10 mM Tris/HCl, pH 8·1. Approximately 1 % of the radioactivity was eluted in the excluded volume together with the liposomes. The liposomes were stored on ice and used within 5 days.
Assay of [3H]choline release from liposomes.
Virus was stored at -70 °C as pellets, each containing 50 µg protein. A fresh pellet was used for each experiment. For a single release reaction, 25 µl 50 mM NaCl, 10 mM Tris/HCl, pH 8·1, containing about 2 µg of the protein or 10 µg of the virus to be assayed and 5 µl [3H]choline-containing liposomes (25 µg lipid) were transferred into a 10 K Nanosep ultrafiltration vial (Pall; 500 µl total volume) at 30 °C. After the addition of 10 µl 300 mM MES buffer of appropriate pH (7·0, 6·6, 6·2, 5·8, 5·4, or 5·0), the sample was incubated at 30 °C for 20 min. After this incubation, 300 µl 50 mM NaCl, 10 mM Tris/HCl, pH 8·1, was added and in order to separate the buffer solution from the liposomes, the vials were subjected to centrifugation at 12 000 g at 6 °C until the buffer solution was present in the filtrate reservoir. The radioactivity present in the filtrate was determined by liquid scintillation counting. Control reactions contained 2 µg BSA.
Analysis of binding of protein to liposomes by flotation.
For a typical binding analysis, a standard release reaction containing liposomes and protein was scaled up by a factor of four, resulting in a final reaction volume of 160 µl. After 20 min incubation at 30 °C, 80 mg sucrose was dissolved in the reaction fluid and the material was loaded into a 0·8 ml cellulose nitrate vial in an SW 55 Beckman rotor. The material was overlaid with approximately 200 µl each of 30% (w/w) and 20 % (w/w) sucrose in 50 mM NaCl, 10 mM Tris/HCl, pH 8·1, and centrifuged at 48 000 r.p.m. at 4 °C for 3 h. The gradients were divided into four fractions by removing the solutions from the top. Fraction 1 contained the top 300 µl of the gradient and fraction 4 contained the bottom 200 µl containing the original reaction. The fractions were transferred into 10 K Nanosep (Pall) concentration vials. The proteins were recovered on the ultrafiltration membrane of the vials by centrifugation, solubilized in sample buffer and subjected to PAGE. Proteins were visualized by Coomassie blue staining.
Patch-clamp analysis of membrane permeability.
Patch-clamp measurements were performed as described by Hamill et al. (1981). HEK 293 cells were grown on 35 mm Petri dishes in growth medium consisting of DMEM and Ham's F12 (1:1) with 10 % (v/v) foetal bovine serum and used at a density of approximately 4x105 cells per dish.
The following solutions were used. Cells were incubated in extracellular solution composed of 140 mM NaCl, 3 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 10 mM glucose and 10 mM HEPES, pH 7·4, adjusted with NaOH. The recording pipette contained an intracellular solution composed of 140 mM potassium glutamate, 10 mM NaCl, 2 mM MgCl2 and 10 mM HEPES, pH 7·3, adjusted with KOH. A free intracellular Ca2+ concentration of 100 nM was obtained using 100 µM of the Ca2+ chelator BAPTA [1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid] and a total Ca2+ concentration of 29·69 µM, assuming an apparent dissociation constant, KD, of 0·24 µM (pH 7·3) for the Ca2+BAPTA complex. Differences in osmolarity between extra- and intracellular solutions were compensated for by adding sorbitol in the range of 1020 mM. Bath and pipette solutions were filtered through 0·2 µm pore filters.
The following materials were used for the recordings. Recording pipettes were pulled from borosilicate glass capillaries (Hilgenberg) with an outer diameter of 1·5 mm and a wall thickness of 0·3 mm. After fire polishing, they had a resistance of 510 M when filled with pipette solution. The currents were recorded and filtered with an EPC-8 patch-clamp amplifier (HEKA GmbH) with sampling frequencies of up to 30 kHz, according to the particular experiment. All signals were filtered at one-third of the sampling frequency. Membrane potentials were measured at zero current in the current-clamp mode of the whole-cell recording configuration. Data acquisition and off-line analysis were performed with Pulse and Pulsefit software (HEKA GmbH). The data were corrected for the liquid-junction potential between the pipette and bath solutions, which was -10 mV for the standard potassium glutamate internal solution. Data were expressed as means±SEM unless stated otherwise.
Whole-cell patch-clamp analysis was performed as follows. Growth medium was removed and cells were incubated for 510 min in 2 ml extracellular solution. Thereafter a tight seal between a cell and the recording pipette was established. Initial seal resistances were usually higher than 5 G. Whole-cell configuration was obtained by applying slight suction to the recording pipette, combined with a 10 ms electrical pulse with an amplitude of -0·9 V. This usually ruptured the membrane, leading to a direct electrical and ionic access to the cytoplasm without loosening the tight seal between the membrane and recording pipette, so that the recording of membrane current could be started. A pellet of UV-inactivated virus particles containing 50 µg viral protein had been suspended in 250 µl extracellular solution containing 3 % (v/v) Ficoll 400, and 50 µl of this material was transferred with a pipette into the immediate surroundings of the cell. This virus suspension contained 10 µg viral protein, which corresponds to about 4 µg E1, 4 µg E2 and 2 µg C protein in the virus particles. After 5 min incubation at 20 °C to allow adsorption of virus, 100 µl low-pH buffer solution consisting of Earle's buffered salt solution containing 20 mM MES buffer adjusted to pH 5·0 with NaOH and containing 5 % Ficoll was applied to the immediate surrounding of the cell. The osmolarity of this solution had been adjusted to the osmolarity of the extracellular solution by the addition of sorbitol. The number of independent experiments (n) that were performed for a specific analysis is indicated in the text.
The outside-out configuration of the patch-clamp technique was performed as described by Hamill et al. (1981). After establishing a whole-cell recording configuration, the pipette was slowly withdrawn from the cell, leading to a long small-diameter sand-glass-shaped tube of cell membrane, connecting the interior of the cell to the interior of the measuring pipette. Merging of the membrane walls of this tube at the isthmus often generates a small bubble of cell membrane of about 4 µm2, the so-called patch. This membrane patch is still tightly sealed with the measuring pipette and has a direct electrical and ionic access to the former cytoplasmic side of the membrane, but has no other connection with the cell. The external side of this membrane patch represents the physiological outside of the former cell membrane and is exposed to the bath solution. Virus adsorption to this membrane and low-pH treatment were performed as described above.
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Results |
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Discussion |
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Our experiments are a continuation of these experiments, but differ from the earlier analyses. In the earlier experiments, the role of low pH on the permeability of cell membranes containing large amounts of virus-specific proteins that had accumulated during virus multiplication or after transfection was analysed. The generation of pores has been postulated, but the formation of individual pores has not been detected, since the time scale of the patch-clamp analysis was not sufficiently rapid. We attempted to identify the formation of individual pores during the early stages of virus infection. Therefore, in our experiments, viral proteins were added to the cell system as part of virus particles, and the proteins were transferred to the cell membrane by low-pH treatment. This meant that permeability changes that accompany the early stages of virus infection were analysed in our experiments. Such experimental settings with appropriate patch-clamp analyses, using a rather short time scale, allowed us to analyse the entry of virus particles and to identify the possible formation of individual pores during this process. The patch-clamp analysis in our experiments is not suitable for detecting membrane currents supported by H+ ions because of the non-selectivity of the pore. The concentration of H+ ions, even at pH 5·0, is very low relative to that of other ions, e.g. Na+ or K+, present in the system, which mask any currents supported by H+ ions. Since the pores allow permeation of larger ions, e.g. glutamic acid and arginine, albeit at a reduced rate (data not shown), the small ions cannot be substituted for by larger impermeable ions in order to detect small H+ currents. These data also indicated that the diameter of the aqueous channel formed is such that the migration of these individual amino acids is significantly restricted.
We have proposed that, in the case of alphavirus infection, the formation of these pores has a well-defined function: under physiological conditions the low-pH-induced fusion occurs in the endosome. The pores have a permeability for at least Na+, K+ and Ca2+ ions, and it seems reasonable to propose that in the endosome protons will flow into the cytoplasm through these pores, e.g. in exchange for K+ ions. We have recently shown that disassembly of alphavirus cores by 60S ribosomal subunits is strongly stimulated by a pH of about 6·0 (Wengler & Wengler, 2002). Together these results represent a physiological mechanism that can lead to efficient disassembly of alphavirus cores at low pH early in infection and allows the assembly of stable cores during virus multiplication in the cytoplasm at neutral pH. The finding that the alphavirus replicase is located on the cytoplasmic surface of endosomes and lysosomes (Froshauer et al., 1988
) is in accordance with the above interpretation that a low-pH region in the vicinity of the endosomes leads to a localized core disassembly and translation of the viral genome.
After the preparation of this paper, Smit et al. (2002) published a paper stating that the fusion of alphaviruses with liposomes is a non-leaky process. This is in contrast to the experiments described in Fig. 1
. The techniques used in both analyses are quite different. Only a specific analysis of these differences may possibly allow us to identify why the results obtained are so different. For instance, the liposomes used in the studies were prepared by fundamentally different techniques and different labelled molecules were used in the release assays.
Taken together, the results of our experiments involving liposomes and the results obtained in the patch-clamp analysis indicate that the pores are made up of the viral membrane proteins and are not generated by activation of endogenous membrane channels. Further support for this interpretation comes from published data. SF virus particles from which lipid and the E2 protein were removed by protease treatment in the presence of detergent did retain a residual infectivity (Omar & Koblet, 1988). Furthermore, it has been shown that SF virus E1 protein expressed in E. coli is incorporated into the bacterial plasma membrane and that these membranes become permeable to [3H]choline at low pH (Nyfeler et al., 2001
). Comparable results have been obtained in similar experiments using eukaryotic cells (Dick et al., 1996
). These authors concluded that it was highly likely that the E1 protein formed pores in the membrane in which it accumulated. Recently, the atomic structure of the SF virus-derived E1 protein and its organization within the viral membrane have been characterized (Forsell et al., 2000
; Pletnev et al., 2001
; Lescar et al., 2001
). These experiments have shown that the E1 molecules form an icosahedral lattice on the virus surface, which determines the structure of the particle. Proteins homologous to the SF virus E1 protein are present in other viruses. The atomic structure of the E protein of tick-borne encephalitis (TBE) virus (Rey et al., 1995
), which belongs to the Flavivirus genus, is homologous to the structure of the SF virus E1 protein (Lescar et al., 2001
). Furthermore, molecular modelling analysis has indicated that the envelope protein E2 of hepatitis C virus is homologous to the E protein of TBE virus (Yagnik et al., 2000
). The data discussed above indicate that the E1 protein of alphaviruses and the homologous proteins might constitute a family of viral surface proteins that have three properties: (i) they form a continuous icosahedral protein shell on the virus surface at neutral pH (Forsell et al., 2000
; Lescar et al., 2001
; Pletnev et al., 2001
); (ii) they can be converted into a fusion-active state at low pH; and (iii) after fusion they can form an ion-permeable pore in the target membrane. The last point does not imply that the functional role of this pore is identical in the different virus systems. It may be possible that members of this protein family are also present in viruses that do not have a lipid envelope, since alphaviruses from which lipid had been removed do retain a small residual infectivity (Omar & Koblet, 1988
) and the 30S membrane protein complex derived from SF virus led to release of [3H]choline from liposomes at low pH. The fusion of the viral lipid membrane into the target membrane brought about by these proteins could then be regarded as a side-effect of the basic function of these proteins: the ability to form an icosahedral closed surface at neutral pH which, in the presence of an appropriate target membrane, is converted at low pH into a planar assembly of proteins in the target membrane that can form ion-permeable pores. We think that this proposal may lead to further attempts to identify additional proteins belonging to this family and to analyse the possible formation of ion-permeable pores by the corresponding virus.
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ACKNOWLEDGEMENTS |
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Received 8 July 2002;
accepted 23 August 2002.