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
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ABSTRACT |
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Present address: Department of Medicine, Louisiana State University, Shreveport, LA, USA.
Present address: Center for Biofilm Engineering, Montana State University-Bozeman, Bozeman, MT, USA.
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INTRODUCTION |
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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 (PVRGPI). 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 PVRGPI 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.
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METHODS |
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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 1342) 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/PVRGPI, was used to generate baculovirus expressing the PVRGPI recombinant shown in Fig. 1
. pCI-PVRGPI and pCI-PVR contain the PVRGPI recombinant and the full-length PVR sequences, respectively, were inserted into pCI-neo expression vector (Promega) under the control of the cytomegalovirus (CMV) immediateearly and T7 promoters.
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Receptor.
Full-length PVR was expressed by in vitro transcriptiontranslation 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 (1025 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.
PVRGPI 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/PVRGPI (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 PVRGPI-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 PVRGPI, 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 PVRGPI 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 PVRGPI 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 PVRGPI 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 PVRGPI.
[35S]Methionine-labelled poliovirus (107 p.f.u. per 300 µl, final concentration) was incubated overnight (4 °C) with various concentrations of the purified PVRGPI or parallel BacVec fractions in PBS containing 10 mM OG and 1 % BSA. The resultant virusreceptor 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 PVRGPI 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 PVRGPI was assessed by infection with PVM1EGFP and detection of GFP expression by fluorescent microscopy.
Direct incorporation of protein.
(i) Purified PVRGPI fractions (1 µg ml1 final concentration) were incubated with confluent cell monolayers at 37 °C for 1 h. The cells were then washed twice with DMEM, infected with PVM1EGFP (m.o.i. 2) and observed under a fluorescence microscope 58 h post-infection (p.i.). (ii) Protein transfections: the proteinDOTAP complex (35 µl) containing 5 µl DOTAP, 1 µg PVRGPI 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 PVRDOTAP mixture was added directly to the cell monolayers in 12-well dishes and incubated for 24 h at 37 °C. The media was replaced with fresh DMEM/10 % FCS to remove the unincorporated receptor. The cells were infected with PVM1EGFP 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 103 Hz) for 3060 min to check for stability and absence of channels. After each new experimental condition, the current in response to a small voltage (2040 mV) was monitored until it became constant (2040 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 (0·52 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 2025 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 (5x103105 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 conductancevoltage curve determined (as described above). The new electrical parameters of the bilayer became stable, on average, 25 min after PV addition.
Incorporation of PVRGPI was accomplished as described for a GPI-linked form of CD4, the receptor for HIV in Tosteson et al. (1991). Briefly, PVRGPI 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 2030 min after bilayer formation, the time necessary to check for stability and absence of a change in conductance.
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RESULTS |
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We determined that expression of the PVRGPI construct could function as a virus receptor by transiently transfecting NIH3T3 cells with the PVRGPI 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 PVRGPI 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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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 transcriptiontranslation 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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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 PVRGPI 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 receptorvirus 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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To test for the specificity of the PVRGPI bilayer system for virus particles, 135S particles were added to PVRGPI-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 PVRGPI 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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DISCUSSION |
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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 Q102 (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 PVRPV 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 PVRPV 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) mol1, which is comparable with, but somewhat lower than the activation energy for the transition from 160S to 135S,
100 kcal (
420 J) mol1 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 PVRlipid 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 (PVRPV) 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 virusreceptor 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.
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ACKNOWLEDGEMENTS |
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Received 21 October 2003;
accepted 11 February 2004.