Poliovirus binding to its receptor in lipid bilayers results in particle-specific, temperature-sensitive channels

Magdalena T. Tosteson1, Hong Wang2,{dagger}, Anatoli Naumov2,{ddagger} and Marie Chow2

1 Department of Cell Biology, Laboratory for Membrane Transport, Harvard Medical School, One Kendall Square, Building 600, Third Floor, Cambridge, MA 02139, USA
2 Department of Microbiology and Immunology, University of Arkansas, Medical School, Little Rock, AR 72205, USA

Correspondence
Magdalena T. Tosteson
mtosteson{at}hms.harvard.edu


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Poliovirus (PV) infection starts with binding to its receptor (PVR), followed by a receptor-aided, temperature-sensitive conformational change of the infectious particle (sedimenting at 160S) to a particle which sediments at 135S. Reported in this communication is the successful incorporation into lipid bilayers of two forms of the receptor: the full-length human receptor and a modified clone in which the extracellular domains of the receptor were fused to a glycosylphosphatidylinositol tail. Addition of virus (160S) to receptor-containing bilayers leads to channel formation, whereas no channels were observed when the receptor-modified viral particle (135S) was added. Increasing the temperature from 21 to 31 °C led to a 10-fold increase in the magnitude of the single channel conductance, which can be interpreted as a conformational change in the channel structure. A mutant PV with an amino acid change in VP4 (one of the coat proteins) which is defective in genome uncoating failed to produce channels, suggesting that VP4 might be involved in the channel architecture. These studies provide the first electrophysiological characterization of the interactions between poliovirus and its receptor incorporated into a lipid bilayer membrane. Furthermore, they form the foundation for future studies aiming at defining the molecular architecture of the virus–receptor complex.

{dagger}Present address: Department of Medicine, Louisiana State University, Shreveport, LA, USA.

{ddagger}Present address: Center for Biofilm Engineering, Montana State University-Bozeman, Bozeman, MT, USA.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
To initiate viral infection, poliovirus (PV) binds to a cellular receptor present only in cells of some primates (Mendelsohn et al., 1989) and undergoes a receptor-mediated conformational change that gives rise to an altered particle (135S or ‘A’ particle) (de Sena & Mandel, 1977; Joklik & Darnell, 1961). Analysis of the human poliovirus receptor (hPVR) structure reveals that the receptor consists of three extracellular immunoglobulin-like domains, a transmembrane domain and a cytoplasmic domain (Koike et al., 1991; Mendelsohn et al., 1989). The PV binding site residues reside in the extracellular N-terminal Ig-like domain 1, whereas the cytoplasmic domain is completely devoid of virus binding and/or recognition activity (Koike et al., 1991; Zibert et al., 1992). In vitro binding of PV to a secreted form of PVR lacking the cytoplasmic domain produces the conversion of the 160S to the 135S particle, which lacks the majority of the VP4 protein and in which the N terminus of VP1 is irreversibly externalized (Gomez Yafal et al., 1993; Kaplan et al., 1990). The 135S particle does not bind to any form of the receptor (Fenwick & Cooper, 1962; Joklik & Darnell, 1961; Lonberg-Holm et al., 1975).

To understand the molecular mechanisms underlying the post-receptor-binding steps of virus entry we decided to work with model systems that allow for precise control of the membrane composition as well as of the ionic composition of the aqueous environment on both sides of the bilayer.

Our results show the incorporation into lipid bilayer membranes of the full-length poliovirus receptor and the incorporation of a modified form of the receptor, in which the transmembrane domain was replaced by a glycosylphosphatidylinositol (GPI) tail (PVR–GPI). The electrical properties of the membranes containing either one of the PVR forms change in response to the addition of the native 160S virus particle and in response to temperature changes, but remain unchanged upon addition of the receptor-induced 135S particle, indicating specific recognition and binding of the 160S particle. This successful incorporation of the full-length poliovirus receptor and of the PVR–GPI into lipid bilayers establishes the model system as a new tool to study the membrane events that occur when PV and other viruses bind to their receptors to initiate virus entry during infection.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cells and media.
Methods for HeLa cell propagation, propagation and purification of [35S]methionine-radiolabelled wild-type poliovirus (serotype 1, Mahoney strain) and 4028T.G mutant virus stocks, determination of virus titres, concentrations of virus particles and virus growth curves were as previously described (Moscufo & Chow, 1992; Rueckert, 1990; Simons et al., 1993). Monolayers of L929 and NIH3T3 cells were grown at 37 °C in 5 % CO2 in Dulbecco's modified Eagle's medium supplemented with 10 % foetal calf serum (FCS), 50 U penicillin/streptomycin ml–1, (DMEM/10 % FCS). Spodoptera frugiperda Sf21 cells were routinely maintained in suspension (between 1–2x105 to 2x106 cells ml–1) at 27 °C in Excel 420 medium (JRH-Biosciences) supplemented with 3 % FCS (BacPAKTM Baculovirus Expression System User Manual, Clontech, www.clontech.com 1999).

Plasmids.
pSVL20A containing a full-length PVR clone was provided by Marian Freistadt (Freistadt et al., 1990) and a syndecan-1 clone containing the rat glypican-1 GPI-linkage (329S/G) was provided by Ralph D. Sanderson (Wei Liu et al., 1998). The cDNA encoding extracellular domain (aa 1–342) of PVR was fused in-frame to the 3'-terminal sequence encoding the rat glypican GPI-linkage through a linker that encodes a Myc epitope and six-histidine tags (5'-GAACAAAAACTCATCTCAGAAGAGGATCTGAATAGCGCCGTCGACCATCATCATCATCATCAT-3') and placed in the pBacPAK6 transfer vector (BD Biosciences Clontech). The resultant construct, BacPAK6/PVR–GPI, was used to generate baculovirus expressing the PVR–GPI recombinant shown in Fig. 1. pCI-PVR–GPI and pCI-PVR contain the PVR–GPI recombinant and the full-length PVR sequences, respectively, were inserted into pCI-neo expression vector (Promega) under the control of the cytomegalovirus (CMV) immediate–early and T7 promoters.



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Fig. 1. (A) Schematic diagram of a construct encoding PVR with a GPI modification (PVR–GPI). The extracellular domain of the wild-type hPVR was fused to the glypican GPI addition sequence via a Myc epitope–His tag linker. TM, transmembrane domain; Cyt, cytoplasmic domain. (B) Flow diagram of the purification scheme of PVR–GPI; the details are in Methods. (C) Sf21 cells were infected either with the parental BacPAK6 vector (BacVec) or with BacPAK6/PVR–GPI baculovirus and the extent of purification was followed by Western blot analyses. Lane 1, lysate from cells infected with BacPAK6; lane 2, lysate from cells infected with BacPAK6/PVR–GPI. All other lanes correspond to fractions at the indicated purification stages in the flow diagram. Upper panel, silver-stained 10 % SDS-PAGE gel; lower panel, Western blots probed with anti-Myc antibodies. * PVR–GPI without GPI modification.

 
Viruses.
A recombinant poliovirus (serotype 1, Mahoney strain) expressing the enhanced form of the green fluorescent protein (PVM1–EGFP) was constructed using a strategy similar to that developed by Andino et al. (1994) and Mueller & Wimmer (1998). Briefly, a PV 3C protease cleavage site was introduced by changing the N-terminal methionine of the VP4 capsid protein sequence to a glutamine via PCR-mediated mutagenesis (Ex-Site PCR based mutagenesis, Stratagene). The EGFP sequence was fused in-frame to the VP4 sequence and the genome organization of the resultant virus is shown in Fig. 3(B). Seed stocks of the PVM1–EGFP virus were generated using coupled vaccinia virus infection–PV cDNA transfections (Simons et al., 1993) and partially purified on 15–30 % sucrose density gradients. The working stocks used for all experiments described were generated from the seed stocks by a single low multiplicity passage in HeLa cells and the virus was released from the cells by three cycles of freeze-thaw. The titres of PVM1–EGFP in these lysates were typically ~2x108 p.f.u. ml–1.



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Fig. 3. Incorporation of PVR–GPI onto cell surfaces. (A) L929 cells were incubated in the absence (a and b) or in the presence of purified PVR–GPI (c and d) or of PVR–GPI/DOTAP (e and f) as described in Methods. The cells were then infected with PVM1–EGFP (m.o.i. 2) and the expression of GFP assessed by visual examination of fluorescence. Left panels (a, c and e) show the phase-contrast view. Right panels (b, d and f) show the fluorescence view of the same fields. (B) Schematic diagram of the recombinant PV expressing the enhanced green fluorescence protein (PVM1–EGFP) (details in text). P1, PV capsid proteins precursor; P2 and P3, non-structural PV proteins; IRES, internal ribosome entry site.

 
Recombinant baculovirus expressing PVR–GPI was recovered from Sf21 cells after co-transfection of pBacPAK6/PVR–GPI with BacPAK6 viral DNA that was digested with Bsu36I (BD Biosciences Clontech). The recombinant baculovirus clones were amplified in Sf21 monolayers for three or four passages. Viral stocks were generated by pelleting the virus from the cell supernatant (45 min at 15 000 g) after removal of cell debris (10 min at 1000 g). The resultant virus pellet was resuspended in PBS and virus titres were determined by plaque assay on Sf21 cells (O'Reilly et al., 1992).

Receptor.
Full-length PVR was expressed by in vitro transcription–translation reactions in the presence of trace amounts of [35S]methionine, by using the TNT-T7 coupled reticulocyte lysate system (Promega) and was used without further purification. The yield of receptor (estimated from the radioactivity of the expressed protein) was variable (10–25 ng) and degraded within 2 weeks of storage at –80 °C. For this reason, each preparation was only used during the first week after synthesis.

PVR–GPI was isolated and purified according to the following protocol. Confluent Sf21 cells in 150 mm dishes (5x106 cells per dish) were infected with either the parental baculovirus BacPAK6 or with BacPAK6/PVR–GPI (m.o.i. 0·3). After 96 h incubation at 27 °C, cells were harvested by scraping and pelleted by centrifugation. Cells infected with the PVR–GPI-expressing or BacPAK6 parental vector virus were processed in parallel to isolate a detergent-insoluble lipid fraction, using the method described by Brown & Rose (1992). Based on the location of the PVR–GPI, the identical fractions from the BacPAK6 virus lysate were pooled to produce the BacVec fraction. The low-density insoluble lipid fraction was concentrated by pelleting in an SW28 rotor (27 000 r.p.m., 2 h) and solubilized by incubating in PBS with 300 mM NaCl, 60 mM octylglucoside (OG), PIC (EDTA-free protease inhibitor cocktail, Roche Applied Science) for 1 h at 20 °C. The PVR–GPI protein from this fraction was purified to homogeneity by batch loading onto regular Talon Co2+-resin (5 ml, BD Biosciences Clontech) and binding for 1 h at 4 °C. The resin was transferred to a column at room temperature and the flow-through recycled twice onto the column. All wash and elution buffers contained 60 mM OG. The PVR–GPI was eluted from the column with extraction/wash buffer (50 mM imidazole pH 7·0) in 3 ml step fractions and immediately concentrated using a microconcentrator (Centricon YM-30, Millipore) to exchange the imidazole buffer for PBS. The final yield of protein was about 120 µg of PVR–GPI from 6x108 cells. The extent of purification was followed by SDS-PAGE and Western blot analyses using anti-Myc-antibody (Oncogene Research) or anti-His antibody (BD Biosciences Clontech) at 1 : 10 000 dilution and subsequently, peroxidase-conjugated goat anti-mouse secondary antibody (Accurate Chemical & Scientific Corp.).

Biological assays
In vitro conversion of PV particles by PVR–GPI.
[35S]Methionine-labelled poliovirus (107 p.f.u. per 300 µl, final concentration) was incubated overnight (4 °C) with various concentrations of the purified PVR–GPI or parallel BacVec fractions in PBS containing 10 mM OG and 1 % BSA. The resultant virus–receptor complexes were incubated at 37 °C for 20 min to catalyse the conversion of 160S to 135S, and subsequently analysed by sedimentation in sucrose density gradients (Moscufo & Chow, 1992). Native 160S and heat-converted 135S particles were run in parallel gradients as sedimentation markers.

Incorporation of purified PVR–GPI into cell membranes.
NIH3T3 or L929 cell monolayers were used to incorporate PVR on the surface of non-permissive cells by either adding the protein directly to cell monolayers or as a protein transfection complex with DOTAP (liposome transfection reagent). In both procedures, successful incorporation of PVR–GPI was assessed by infection with PVM1–EGFP and detection of GFP expression by fluorescent microscopy.

Direct incorporation of protein.
(i) Purified PVR–GPI fractions (~1 µg ml–1 final concentration) were incubated with confluent cell monolayers at 37 °C for 1 h. The cells were then washed twice with DMEM, infected with PVM1–EGFP (m.o.i. 2) and observed under a fluorescence microscope 5–8 h post-infection (p.i.). (ii) Protein transfections: the protein–DOTAP complex (35 µl) containing 5 µl DOTAP, 1 µg PVR–GPI in PBS with 60 mM OG (5 µl), was formed according to manufacturer's recommendations (Roche Molecular Biochemicals), and subsequently mixed with 1 ml DMEM/10 % FCS by gentle pipetting. The PVR–DOTAP mixture was added directly to the cell monolayers in 12-well dishes and incubated for 2–4 h at 37 °C. The media was replaced with fresh DMEM/10 % FCS to remove the unincorporated receptor. The cells were infected with PVM1–EGFP and assessed for GFP expression for up to 19 h p.i.

Lipid bilayers and receptor incorporation.
The lipid bilayers were formed as described by Montal & Mueller (1997) by apposition of two lipid monolayers onto a Teflon partition separating the two aqueous compartments of the chamber. The chambers, method of membrane formation and data acquisition and analysis were as described in Tosteson & Chow (1997). The temperature in the chambers was clamped using a dual temperature controller (Warner Instruments). Lipids used to form the bilayers are indicated in the figures. The value of the applied potential is given with the trans-chamber as the reference and zero current corresponds to the level when the voltage is zero. The aqueous solution used throughout was: 300 mM NaCl, 10 mM Tris/HEPES pH 7·4. The conductance of the bilayer was determined (21 °C) before any additions, applying a 75 mV pulse of 2 min duration (rate of 10–3 Hz) for 30–60 min to check for stability and absence of channels. After each new experimental condition, the current in response to a small voltage (20–40 mV) was monitored until it became constant (20–40 min). This was followed by determination of the current vs time in response to at least three different voltages for each different experimental condition. The averaged results reported in the text were always obtained in at least three different bilayers. The data were analysed using pClamp 6.0 (Axon Instruments).

hPVR was incorporated directly by the addition of an aliquot (0·5–2 µl) of primed TNT-T7 lysate to one of the aqueous solutions bathing a preformed lipid bilayer (cis-side), following the protocol described in Tosteson et al. (2001). Each new receptor preparation was calibrated so that an addition of a fixed volume of the primed TNT-T7 resulted in a bilayer conductance (in the absence of PV) of 20–25 pS. Preliminary results indicated that this amount of hPVR in the membrane was enough to obtain successful interaction with PV in a reproducible manner. The concentration of each receptor preparation was subsequently adjusted so that this conductance would be obtained upon addition of 0·5 µl of the TNT-T7-primed lysate.

Binding of PV (5x103–105 virus particles) to hPVR-bilayers was accomplished by direct addition (21 °C) of an aliquot of a virus preparation to the cis-compartment, after the membrane conductance had stabilized, and the conductance–voltage curve determined (as described above). The new electrical parameters of the bilayer became stable, on average, 25 min after PV addition.

Incorporation of PVR–GPI was accomplished as described for a GPI-linked form of CD4, the receptor for HIV in Tosteson et al. (1991). Briefly, PVR–GPI was added to the subphase of one of the aqueous solutions (cis-side) and the lipid monolayer preincubated for 20 min before a bilayer was formed. PV was added 20–30 min after bilayer formation, the time necessary to check for stability and absence of a change in conductance.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Purification and characterization of PVR–GPI
The cloned receptor was purified according to a scheme that we developed based on the observation that most GPI-anchored proteins are associated with low temperature- and detergent-resistant membrane microdomains (‘lipid rafts’) (Brown & Rose, 1992). The two-step purification procedure is schematically shown in Fig. 1(B) and the results of the purification are shown Fig. 1(C). Consistent with the sequestration of GPI-anchored proteins into detergent-resistant lipid microdomains, the 65–70 kDa PVR–GPI form is greatly enriched in the Triton-resistant lipid fraction and is the major protein species in the fraction (Fig. 1C). Lane 7 shows that using the six-histidine tag, the PVR–GPI protein was purified to homogeneity from the OG-solubilized lipid fraction by chromatography on Ni2+- or Co2+-resins. Anti-Myc antibodies recognized two major species (approximately 40–45 and 60–65 kDa) of the Myc-tagged PVR–GPI construct in crude cell lysates of BacPAK6/PVR–GPI-infected cells. The heterogeneous migration of both the 45 and 65 kDa forms is due to glycosylation as assessed in tunicamycin-treated cells (data not shown). The size of the smaller protein species corresponds to the predicted size (40 kDa) of the PVR-extracellular domain. The larger 65 kDa protein species corresponds to the GPI-modified PVR protein.

We determined that expression of the PVR–GPI construct could function as a virus receptor by transiently transfecting NIH3T3 cells with the PVR–GPI clone and measuring virus titres after infecting these transfectants with PV. The single-cycle growth curves (Fig. 2A) demonstrate that PV infects and replicates in the PVR–GPI transfectants with similar kinetics as transfectants expressing the wild-type PVR (WT-PVR), whereas NIH3T3 cells transfected with the vector alone (pCI-neo) were not permissive for viral infection (Fig. 2A).



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Fig. 2. (A) Transfected PVR–GPI is a functional PVR. NIH3T3 cells were transiently transfected with pCI vectors expressing WT-PVR ({bullet}), PVR–GPI ({blacktriangleup}) or with the pCI vector alone ({circ}). After 24 h, the cells were infected with PV (m.o.i. 10). At the indicated times p.i., cells were harvested and the titres of cell-associated virus were determined by plaque assay. (B and C) PVR–GPI induces the 160S to 135S transition in vitro. The BacVec fraction (B) or the fractions of PVR–GPI (C) were incubated in parallel with radiolabelled PV particles at 4 °C. The resultant complexes were either kept at 4 °C ({circ}) or incubated at 37 °C ({bullet}) for 20 min and analysed by sedimentation in sucrose density gradients. The molar ratio of receptor to virus in (C), was approximately 100.

 
Previous studies have shown that the receptor-mediated conformational transition, which forms 135S particles during infection, can be achieved in vitro by incubating 160S particles with soluble forms of hPVR (Arita et al., 1998; Gomez Yafal et al., 1993). We tested whether the purified PVR–GPI was a functional receptor according to this in vitro assay and the results are shown in Fig. 2(B, C). Fig. 2(B) shows that when radiolabelled virus particles were incubated at 4 °C with the fraction (BacVec) purified from cells infected with the baculovirus vector alone, no conversion of the 160S particles (PV) was observed either at the low temperature or after increasing the temperature to 37 °C. However, when the labelled virus particles were incubated at 4 °C with the purified Myc-tagged receptor, subsequent increase of the temperature of the virus–receptor complexes to 37 °C led to conversion of the 160S native form of the particle to the 135S receptor-altered form, consistent with the temperature-dependence of this conversion during infection (cf. Fig. 2C). We also confirmed the formation of receptor–virus complexes present in the peak sedimenting at ~100S by the simultaneous detection of the radiolabelled viral proteins and of the Myc-tagged receptor by Western blot analyses (data not shown). We further established that the purified PVR–GPI protein functions as a PVR in vivo, by incorporating it onto the surface of non-permissive cells, following procedures to incorporate proteins containing a GPI tail into cellular membranes (Acosta et al., 2000). Fig. 3 shows that after incorporation of the purified PVR–GPI into mouse L929 cells, it is possible to observe GFP fluorescence in infected cells (details in figure legend). These data taken together validate the use of PVR–GPI as a bona fide receptor, both for further studies in cells as well as in the model membrane system.

Incorporation of the poliovirus receptor into lipid bilayers
Since the identification, cloning and expression of the hPVR (Mendelsohn et al., 1989), there have been numerous studies on the interaction of virus with the extracellular domains of the purified receptor in solution. These studies, however, cannot address the events that follow the binding of PV to its receptor embedded in the membrane. This can be accomplished through functional incorporation of the receptor in a model membrane system. To this end, the full-length hPVR was expressed using a coupled in vitro transcription–translation system, shown to be effective in the incorporation of other membrane proteins and transcription factors (Tosteson et al., 2001). The results of the addition of hPVR to lipid bilayers, shown in Fig. 4, demonstrate that hPVR incorporates into planar lipid membranes, as indicated by the changes in the bilayer conductance as a function of the receptor concentration (Fig. 4A).



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Fig. 4. Incorporation of the hPVR into lipid bilayer membranes. Lipid membranes were formed and the hPVR added to one of the aqueous solutions bathing this membrane as detailed in Methods. (A) Conductance (peak amplitude/voltage) as a function of [hPVR]. (B) Effect of PV addition to hPVR-bilayers. Determination of the electrical parameters of the hPVR-bilayer, WT-PV particles (Mahoney) were added. The panel shows 30 s of a continuous recording of the current vs time response to applied voltages (as shown in the figure) of an hPVR-bilayer before (first trace) and after addition of a total of 15 000 PV particles to 1·5 ml (last three traces). The amplitude markers are in conductance units, to emphasize the independence on voltage, the 20 pS marker corresponds to the first trace only. (C) Current amplitude distribution curves for the complete traces (120 s each) of the bilayer after addition of PV. The numbers to the left of each current amplitude distribution correspond to the applied voltage. Lipids, 20 mg asolectin ml–1 in pentane. Temperature, 21 °C.

 
Addition of PV particles (160S, Mahoney Type 1) to receptor-containing membranes led to significant increases in the bilayer conductance, as shown by the representative traces of the time-course of the current in response to applied voltages, after addition of virus particles (Fig. 4B). These results clearly indicate that the electrical properties of the channels are different from the ones observed for hPVR-membranes in the absence of PV (compare the first trace of Fig. 4B, the current response in the absence of virus, with traces 2–4 after PV addition, shown with different conductance scales). Thus, the single channel conductance, calculated from the amplitude distribution histograms (Fig. 4C) increases from a value ~10 pS in the absence of PV (histogram not shown) to a conductance (65±15) pS after addition of virus. The mean open time is also markedly changed, from ~2–5 ms to 100–300 ms, indicating that the lifetime of the channel formed by the complex of PV with its receptor is relatively long-lived.

Addition of the 135S particles to an hPVR-bilayer produced no change in the electrical properties of the receptor-containing bilayer (data not shown). We can conclude tentatively that it is highly probable that these particles do not bind to the receptor in lipid bilayers, just as the 135S particles do not bind to receptors present on the plasma membranes of susceptible cells.

The results shown in Fig. 4 demonstrate that the full-length PVR incorporates into lipid bilayers and that once the receptor is integrated into the membrane, addition of PV changes the conductance of the membrane further, in a particle-specific manner. We speculate that this new conductance is due to the formation of a new channel, which arises as a consequence of the binding of PV to hPVR. Thus, we expect that similar results would be obtained if the GPI-linked receptor were to be incorporated into lipid bilayers. This expectation is born out by the data presented in Fig. 5. First we found, as expected for a GPI-linked protein, that incorporation of the PVR–GPI into lipid bilayers did not alter the electrical properties of the membranes, independent of the lipid used to form the membrane (first trace of Fig. 5A). However, as shown in the second trace in Fig. 5(A) and first trace of 5(C), there is a dramatic change in the conductance of the membrane following the addition of PV to the cis-aqueous phase, as with the full-length receptor (e.g. Fig. 4B). Thus, these data, together with the data presented above, show that binding of PV to its receptor in a membrane leads to the formation of ion-permeable channels, independent of the source of receptor and of the type of lipid used to incorporate the receptor. As with other receptors however, the absolute value of the single channel conductance is a function of the lipid used, but not of the type of the receptor (cf. Table 1). To validate further the usefulness of the model system, studies were done to determine the effects of a temperature increase on the conductance of the receptor–virus complex. An increase of 10 °C in the temperature of the aqueous media (from 21 to 31 °C) results in a marked increase in the bilayer conductance (Fig. 5A, C) as well as in the conductance of the single channel (Fig. 5B, D).



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Fig. 5. Incorporation of PVR–GPI into bilayers of different lipid composition. The PVR construct, PVR–GPI (600 ng ml–1 final concentration) was added to one subphase and after bilayer formation, 6500 (160S particles) ml–1 were added. The temperature of the bilayer, as indicated in the figure, was changed and the records shown are taken from the same membrane at each of the different lipids tested. (A) Current vs time traces in response to +20 mV voltage pulse for a lipid bilayer made from asolectin. Top trace, before addition of virus, temperature 21 °C; second and third traces, after PV added. (B) Single channel conductance vs voltage, at both temperatures. The solid lines correspond to the mean value of the conductance at each temperature and the error bars at 0 mV correspond to the SD of the mean (values obtained in eight different membranes). (C) Current vs time traces in response to +20 mV voltage pulse for a lipid bilayer made from phosphatidyl-ethanolamine : phosphatidyl-serine (1 : 1 molar ratio) after addition of PVR–GPI at the temperatures indicated in the figure. (D) Single channel conductance vs voltage, at both temperatures. The solid lines correspond to the mean value of the conductance at each temperature and the error bars at 0 mV correspond to the SD of the mean (values obtained in four different membranes).

 

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Table 1. Effect of temperature on the conductance of lipid PVR-bilayers in the presence of PV

 
Further shown in Fig. 5(B), (D) is that the single channel conductance is independent of voltage both at 21 and at 31 °C. The results of experiments like those shown in Fig. 5, as well as results obtained with hPVR, are summarized in Table 1. Of particular interest in the data shown is the fact that the ratio of the conductance increases 6–8 times upon increasing the temperature to 31 °C. These values, which are much larger than the expected values for ion diffusion in a channel (Q10~1·6–2), suggest that there is a conformational change of the receptor–PV complex at the bilayer surface upon an increase in temperature. Experiments where the temperature of the same bilayer was decreased from 31 to 21 °C resulted in a Q10=2 (three different bilayers, one made from PE : PS and two from asolectin, PVR–GPI), a value expected for diffusion of ions in the aqueous environment provided by a channel (Hille, 2001). This suggests that the change in the channel conformation produced by the increase in temperature (with a resulting Q10~7) is irreversible and that this conformation is different from the one formed initially at 21 °C, given that the single channel conductances are different.

To test for the specificity of the PVR–GPI bilayer system for virus particles, 135S particles were added to PVR–GPI-containing bilayers. As when hPVR was incorporated into the bilayers, it was found that 135S particles failed to produce channels in the bilayers, independent of the temperature at which it was tested (data not shown).

VP4 sequences are important in channel formation
In order to validate the model system presented in this report, we took advantage of a site-specific mutant with a glycine substitution at the conserved threonine-28 of VP4. This virus, 4028T.G, is the only known mutant defective in entry, with a defect in genome uncoating at a step after 135S formation (Danthi et al., 2003; Moscufo et al., 1993). Addition of 4028T.G 160S particles to asolectin membranes containing PVR–GPI did not produce changes in the current vs time traces, as shown in Fig. 6, either at 21 or 31 °C. These results suggest that VP4 might be important for the formation and/or the structure of the channels that we are studying. More importantly, they suggest that the channels that are seen in the model system when wild-type PV is added to receptor-containing bilayers are structures relevant to the biology of virus entry.



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Fig. 6. 4028T.G mutant fails to produce channels in PVR–GPI bilayers. Experimental details as in legend to Fig. 5, except that virus was the mutant 4028T.G. Lipid, 20 mg asolectin ml–1 in pentane; virus, 52 000 particles ml–1; V, +60 mV.

 

   DISCUSSION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The idea that virus infection leads to changes in the permeability of the cell membrane has been put forth in several publications (reviewed by Carrasco, 1995) and has been shown to apply for enveloped viruses, through measurements of the formation of fusion pores in the plasma membrane as virus infection starts (Wengler et al., 2003). The goal of the experiments presented in this report was to study the steps that follow the binding of PV to its receptor anchored to a lipid membrane and to identify the molecular determinants of the process. To achieve this goal, we chose to work with two forms of hPVR: the full-length protein and a receptor protein lacking the transmembrane and cytoplasmic domains of hPVR. The data presented show that the PVR–GPI protein, when either expressed or incorporated on the plasma membrane of cells, can function as a bona fide receptor enabling infection of non-permissive cells (Figs 2 and 3). We further show that we have accomplished the incorporation of the full-length hPVR and of the GPI form of the receptor into lipid bilayers, as demonstrated by the formation of channels following the addition of the appropriate ligand, PV. This type of response has been found for other receptors incorporated in lipid bilayers, notably CD4, the receptor for human immunodeficiency virus, as well as for the acetylcholine receptor, the ryanodine receptor, the inositol 1,4,5-triphosphate receptor and the glutamate receptor, for example (Coronado et al., 1994; Mikoshiba et al., 1992; Nelson et al., 1980; Tashmukhamedov et al., 1985; Tosteson et al., 1991). The data in Figs 4 and 5 clearly show that addition of the PV 160S particle induces channels in bilayers in which the receptor (either hPVR or PVR–GPI) has been incorporated. We suggest that these channels are the result of the formation of a binding complex of PVR and PV, particularly since they are particle-dependent and thus might reflect the initial step(s) of virus infection. Further support for the notion that the channels are due to a PVR–PV complex is given by the response to an increase in temperature (cf. Fig. 5 and Table 1). As shown, the conductance of the PVR–PV channels in bilayers was significantly increased following a rise in temperature from 21 to 31 °C, yielding a conductance ratio, Q10~7, which is independent of the receptor incorporated into the bilayer, as well as independent of the lipids used to form the bilayer. This high value of the conductance ratio suggests that the PVR–PV complex undergoes a change in its conformation with the change in temperature (Hille, 2001). We would suggest that these changes in conformation are related to structures in the membrane, and correspond to a change from a complex in which only binding of PV occurs (21 °C) to one at which infection can start (31 °C).

The single channel conductance obtained upon lowering the temperature from 31 to 21 °C is not the same as the one initially obtained at 21 °C following addition of PV to PVR-bilayers. The value of the Q10~2 (conductance at 31 °C/conductance at 21 °C) is the expected ratio of the diffusion rates of the ions in an aqueous environment, such as the one provided by an ion channel. Thus, these results indicate that the change in the conformation of the channel that is created at the higher temperature is irreversible, as is the conformational change of 160S to 135S induced by PV binding to its receptor. We suggest that the PVR–PV complex formed at 31 °C in lipid bilayers corresponds to the complex of PV with its receptor, which participates in the process of uncoating and RNA delivery, when the 160S particles change to the 135S particles. Calculation of the activation energy for the change in conductance of bilayers containing the PVR–PV complex (suggested to represent the uncoating process) using the Arrhenius equation and the value of the experimentally obtained Q10 yields a value of ~60 kcal (~252 J) mol–1, which is comparable with, but somewhat lower than the activation energy for the transition from 160S to 135S, ~100 kcal (~420 J) mol–1 measured in the presence of soluble receptor by Tsang et al. (2001). This reinforces the idea that the receptor-containing lipid bilayers are a useful model to study the very early stages in virus entry.

We have previously found that a mutant (4028T.G) which is defective in genome uncoating, a step after 135S conversion, can go through the 160S to 135S conversion in a temperature-dependent manner upon exposure to a receptor (Moscufo et al., 1993). Interestingly, this mutant does not form channels in lipid bilayers devoid of receptor even at 31 °C and neither does it form channels in lipid bilayers containing receptor, as shown by the data in Fig. 6. Apparently, the ability to make the temperature-dependent, receptor-mediated conversion is not enough to produce channels in the PVR–lipid bilayer membranes, highlighting the importance of the experimental model presented in this report to define defects in virus mutants.

To summarize, our data show that when PV binds to its receptor in the bilayer at room temperature, the (PVR–PV) initial complex embeds in the lipid bilayer and forms an aqueous pathway, which can be used for ions to cross the membrane (Figs 4 and 5). This channel is different from the one previously seen in the absence of receptor in the bilayer (Tosteson & Chow, 1997; Danthi et al., 2003). Increasing the temperature changes the conformation of the channel formed, after tight binding of virus to its receptor in the bilayer, to a new virus–receptor complex that might be connected to uncoating events. Our data also show that VP4 is probably a component of the channel. We believe that this model system will be of help in the detailed characterization of the viral proteins and receptor domains involved in virus entry and uncoating, work which is in progress in our laboratories.


   ACKNOWLEDGEMENTS
 
This work was supported in part by Public Health Service grant AI-42390 from the National Institute of Allergies and Infectious Diseases. The authors wish to thank X. Ning for providing the PVM1–EGFP construct, and Drs H. Aktas, R. Latorre and D. C. Tosteson for reading the manuscript and offering helpful comments and suggestions.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 21 October 2003; accepted 11 February 2004.