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J. Biol. Chem., Vol. 277, Issue 20, 17789-17796, May 17, 2002

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Modification of Late Membrane Permeability in Avian Reovirus-infected Cells

VIROPORIN ACTIVITY OF THE S1-ENCODED NONSTRUCTURAL p10 PROTEIN^*

Gustavo Bodelón, Lucía Labrada, José Martínez-Costas, and Javier Benavente^§

From the Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain

Received for publication, February 28, 2002, and in revised form, March 13, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Infection of chicken embryo fibroblasts by avian reovirus induces an increase in the permeability of the host plasma membrane at late, but not early, infection times. The absence of permeability changes at early infection times, as well as the dependence of late membrane modification on both viral protein synthesis and an active exocytic route, suggest that a virus-encoded membrane protein is required for avian reovirus to permeabilize cells. Further studies revealed that expression of nonstructural p10 protein in bacterial cells arrested cell growth and enhanced membrane permeability. Membrane leakiness was also observed following transient expression of p10 in BSC-40 monkey cells. Both its permeabilizing effect and the fact that p10 shares several structural and physical characteristics with other membrane-active viral proteins indicate that p10 is an avian reovirus viroporin. Furthermore, the fusogenic extracellular NH₂-terminal domain of p10 appears to be dispensable for permeabilizing activity, because its deletion entirely abolished the fusogenic activity of p10, without affecting its ability to associate with cell membranes and to enhance membrane permeability. Similar properties have reported previously for immunodeficiency virus type I transmembrane glycoprotein gp41. Thus, like gp41, p10 appears to be a multifunctional protein that plays key roles in virus-host interaction.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cytolytic viruses induce profound injurious effects in susceptible host cells, resulting in morphological, structural, and biochemical changes and in cell degeneration. A widespread phenomenon during viral infection is the alteration of the permeability of the host plasma membrane (reviewed in Refs. 1-3). Viruses can modify the membranes of their host cells in at least two ways: (i) by promoting membrane fusion between virus and cell and/or between cell and cell and (ii) by altering the permeability of the plasma membrane (reviewed in Ref. 4). The latter can occur at two different stages of infection: either during virus entry or at late infection times. Early membrane permeabilization is induced by input virus particles during the process of virus entry and uncoating, and causes structural and functional changes in the membrane. The extent and degree of these modifications vary with the virus-cell system and with the multiplicity of infection. On the other hand, enhanced permeability at late infection times requires virus gene expression and is manifested as a general increase in permeability to ions and small molecules, but not to macromolecules, suggesting the formation of hydrophilic pores in the plasma membrane. Recent evidence suggests that these pores are formed by specific viral proteins, whose ultimate function is to disorganize the membrane and kill the infected cell. These membrane-perturbing viral proteins, termed viroporins, share several physical and structural characteristics (reviewed in Ref. 1).

Avian reoviruses are members of the Orthoreovirus genus, one of the nine genera of the Reoviridae family. These agents replicate in the cytoplasm and contain a genome formed by 10 segments of double-stranded RNA enclosed within a double protein capsid shell, but lack a lipid envelope (5, 6). The infection of cultured cells by avian reoviruses causes strong cytopathic effects, manifested by shrinkage, rounding, and detachment from the plate. The cytopathic effects of avian reovirus infection are rather complex, involving syncytia formation and apoptosis (7, 8).¹ Previous reports have revealed that the syncytia formation activity of avian reoviruses is associated with genome segment S1 (10), a gene that contains three partially overlapping, out-of-phase, open reading frames (ORFs),² which are highly conserved in all avian reovirus strains examined to date (Fig. 1). All three S1 ORFs are expressed in infected cells (11, 12); ORFs 1 and 2 direct the synthesis of the nonstructural proteins p10 and p17, which associate with cell membranes, whereas distal ORF3 expresses protein C, a minor trimeric structural protein that is involved in virus attachment to cell receptors (13, 14). Recent evidence indicates that there is a close association between nonstructural p10 protein and the syncytial phenotype displayed by avian reoviruses (11, 15). This protein is a small transmembrane type-I (N-out) protein, and mutagenic analysis revealed that a region of its extracellular NH₂-terminal domain displays a sequence-dependent effect on the fusogenic activity of p10 (Ref. 15 and Fig. 1).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Structure of the S1 genome segment and of the p10 protein of avian reovirus S1133. A, schematic representation of the ORF distribution of the tricistronic S1133 S1 gene. The positions of the start and stop codons are indicated at the ends of the corresponding ORFs. The name, number of amino acid residues, and molecular mass of the proteins encoded by each ORF are indicated within boxes. B, amino acid sequences of p10 protein and of a deleted version of p10 lacking its first 22 NH2-terminal amino acid residues (p10*). The predicted transmembrane, extracellular, and intracellular domains of these proteins are indicated within boxes in the middle of the two sequences. Basic amino acids located in the intracellular region adjacent to the transmembrane domain are dotted and cysteine residues marked with an asterisk.

In this study we have investigated the capability of avian reovirus to alter the permeability of the host plasma membrane. We found that infection of chicken embryo fibroblasts (CEF) by avian reovirus S1133 causes membrane leakiness at late infection times, and that this alteration is dependent on both viral protein synthesis and protein translocation through the vesicular system. We also found that expression of avian reovirus nonstructural p10 protein in both prokaryotic and eukaryotic cells induces destabilization of the cell membrane, and that this function is not abolished by deletion of its extracellular fusogenic domain.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cells, Viruses, Antibodies, and Cloning-- Primary cultures of CEF were prepared from 9-10-day-old chicken embryos and grown in medium 199 supplemented with 10% tryptose phosphate broth and 5% calf serum. Monkey BSC-40 cells were grown in monolayers in medium 199 supplemented with 10% fetal bovine serum. Strain S1133 of avian reovirus was grown in semiconfluent monolayers of primary CEF, as described previously (16).

Preparation of polyclonal antibodies against the three S1-encoded avian reovirus proteins has been described previously (11). Rabbit polyclonal antibodies against the purified nonstructural protein µNS were raised in our laboratory.

Cloning of ORF1 into prokaryotic and eukaryotic expression vectors has also been described (11). To generate ORF1*, ORF2, and ORF3 DNA inserts, the recombinant plasmid pBsct-S1 (11) was subjected to PCR amplification with the following primers. For ORF1*, the forward primer was 5'-CGGAATTCACGATGGCTCAGAACACGGCAGGT-3' (EcoRI site underlined) and the reverse primer was 5'-CCCGTCGACTCAAACGTCGTATGGCGGAG-3' (SalI site underlined). For ORF2, the forward primer was 5'-CGGAATTCGTAAGCGCAATGGAATGGCTC-3' (EcoRI site underlined) and the reverse primer was 5'-CCCGTCGACTCATAGATCGGCGTCAAATCG-3' (SalI site underlined). For ORF3, the forward primer was 5'-CGGAATTCTTCATTGGGATGGCGGG-3' (EcoRI site underlined) and the reverse primer was 5'-CCCGTCGACTTAGGTGTCGATGCCGGTAC-3' (SalI site underlined). Forward primers were designed to contain an initiation AUG codon with a strong context for translation initiation (17). The resulting amplification products were digested and cloned into EcoRI and SalI sites of expression vectors pMalC and pCIneo. The correct orientation of the inserts was confirmed by nucleotide sequencing of the recombinant plasmids.

Induction of Recombinant Protein Expression in Escherichia coli-- Overnight cultures of either BL21(DE3) or BL21(DE3)pLysE cells containing the indicated plasmid, grown at 37 °C in LB medium, supplemented either with 0.2% glucose and 100 µg/ml ampicillin for BL21(DE3) cells or with 0.2% glucose, 100 µg/ml ampicillin, and 34 µg/ml chloramphenicol for BL21(DE3)pLysE cells, were diluted 100-fold in the same supplemented medium. When an absorbance of 0.6 at 600 nm was reached, cultures were induced by the addition of 1 mM isopropyl-1-thio--D-galactopyranoside (IPTG).

Viral Infections, Transfections, Metabolic Radiolabeling, and Electrophoretic Analysis of Proteins-- Infection of CEF by avian reovirus S1133 and transient expression of viral proteins in BSC-40 cells have been described previously (11). For assessing permeabilization of eukaryotic cells to hygromycin B (HB) (Calbiochem, San Diego, CA), the inhibitor was added, at a final concentration of 1.5 mM, to the cultured medium 45 min before the start of radioactive labeling. For protein radiolabeling, the cultured medium was supplemented with 100 µCi/ml [³⁵S]methionine-cysteine (1.45 Ci/mmol, Amersham Biosciences) and incubated at 37 °C for 45 min. Cells were washed with phosphate-buffered saline and then lysed in 3× Laemmli sample buffer (18), and proteins were resolved on a 10% SDS-PAGE gel and visualized by autoradiography. For metabolic radiolabeling in bacteria, cells were incubated for 20 min at 37 °C in the presence of 0.4 mM HB and 70 µCi/ml [³⁵S]methionine-cysteine. Labeled cells were then collected and lysed in 3× Laemmli sample buffer.

Estimation of Uridine Release-- Uridine preloading of cultured cells and subsequent estimation of release were done essentially as described previously (19, 20). For eukaryotic cells, 14 h before infection or transfection, semiconfluent cell monolayers were incubated for 14 h at 37 °C in medium containing 2 µCi/ml [5,6-³H]uridine (32 Ci/mmol, Amersham Biosciences). The cells were then washed with phosphate-buffered saline and subjected to either viral infection or lipofection. At the indicated times, the culture medium was collected, centrifuged at 8,000 × g for 5 min, and the supernatant was mixed with scintillation mixture (989, PerkinElmer Life Sciences). The radioactivity was then quantified in a liquid scintillation counter. For bacterial cells, 90 min before IPTG induction, cultures were incubated for 1 h in medium supplemented with 4 µCi/ml [5,6-³H]uridine. The cells were then pelleted and washed twice with prewarmed isotope-free culture medium, resuspended in the initial volume of growth medium, and incubated at 37 °C. After IPTG induction, 0.3-ml aliquots of the culture were collected and cells were pelleted. Quantification of the radioactivity in the supernatant was done as above.

Subcellular Fractionation and Immunoblot Analysis-- Fractionation of transfected BSC-40 cells and Western blotting analysis were done as described previously (11). Isolation of bacterial membranes was essentially done as described by Lama and Carrasco (21) with minor modifications. Briefly, 30 min after IPTG induction, bacteria were pelleted and washed twice with a buffer containing 50 mM Tris-HCl, pH 7.6, and 100 mM NaCl. Cells were finally resuspended in buffer A (50 mM Tris-HCl, pH 7.6, 100 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, and 5% glycerol) and lysed by sonication. The resulting extracts were clarified by low speed centrifugation (2,500 × g for 10 min), and the supernatant was centrifuged again (100,000 × g for 30 min). The pelleted membrane fraction was resuspended in buffer A, and proteins in this fraction and in the supernatant fraction were resolved by SDS-PAGE and subsequently visualized by Coomassie Blue staining.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Avian Reovirus Causes Membrane Leakiness at Late Infection Times-- To analyze membrane permeability changes in response to avian reovirus infection, several assays were performed. First, the ability of HB to cross the membrane of avian reovirus-infected cells was investigated at different infection times. HB, which does not normally trespass the membrane barrier of intact cells, does penetrate cells with permeabilized membranes, causing strong inhibition of intracellular protein synthesis; it is therefore widely used to study changes in membrane permeability (1). The results shown in Fig. 2A indicate that, although HB was unable to cross the plasma membrane of both uninfected CEF (Fig. 2A, compare lanes 1 and 2) and early-infected CEF (Fig. 2A, lanes 3-6), it readily penetrated S1133-infected CEF at late infection times, blocking protein synthesis (Fig. 2A, compare lanes 7 and 8). These results indicate that viral infection induces enhanced membrane permeability at late, but not early, infection times. To determine whether the perturbed plasma membrane also allows increased efflux of compounds, we next examined the leakage of radioactivity from [³H]uridine-preloaded cells. As can be observed in Fig. 2B, a substantially greater amount of radioactivity was released from infected cells at 9 h after infection than at 0 or 2 h after infection. These data clearly illustrate that the plasma membrane of avian reovirus-infected cells is permeabilized bidirectionally at late infection times, allowing increased entry and exit of metabolites.

View larger version (31K): [in this window] [in a new window]   Fig. 2.   Membrane permeability assays. A, mock-infected (CEF) and avian reovirus-infected cells (50 pfu/cell; S1133-CEF) were incubated, at the indicated times after infection, for 45 min in the presence (even lanes) or absence (odd lanes) of 1.5 mM HB. Cells were then labeled with [35S]methionine-cysteine for another 45 min in the presence of HB. Cell extracts were prepared and analyzed by SDS-PAGE and autoradiography. Positions of molecular size markers are indicated on the left, and positions of the three size classes of avian reovirus polypeptides are indicated on the right. B, cells were pre-incubated in medium containing 2 µCi/ml [5,6-3H]uridine for 14 h and then infected. At the indicated infection times, the radioactivity released to the medium was measured. The values shown are means of four independent experiments, and error bars indicate standard deviations of the mean.

Membrane Permeabilization by Avian Reovirus Requires Viral Protein Synthesis and Protein Translocation through the Vesicular System-- We next performed experiments to assess whether newly synthesized viral proteins are required for the late permeability changes induced by avian reovirus. First of all, we checked whether the onset of viral protein synthesis and membrane modification are concurrent, by performing infections at two different multiplicities of infection (Fig. 3). When cells were infected with a multiplicity of 5 pfu/cell, viral protein synthesis and enhanced permeability to HB were both first detected at 9 h after infection, as shown by ³⁵S-metabolic labeling and immunoblot analysis of the nonstructural viral protein µNS in HB-treated and untreated cells (Fig. 3, A and B). Increasing the multiplicity of infection up to 100 pfu/cell accelerated both processes; onset of both viral protein synthesis and membrane leakiness was now detectable at 6 h after infection (Fig. 3, C and D). The fact that the onsets of these two events are concurrent suggests a cause-effect relationship.

View larger version (50K): [in this window] [in a new window]   Fig. 3.   Effects of viral multiplicity on viral protein synthesis and membrane permeability. Cells were infected with either 5 pfu/ml (panels A and B) or 100 pfu/ml (panels C and D) of avian reovirus S1133 and incubated with (even lanes) or without (odd lanes) 1.5 mM HB for 45 min, at the times indicated on top. A and C, cells were then labeled with [35S]methionine-cysteine for another 45 min in the presence of the inhibitor, and then lysed. Total cell extracts were analyzed by SDS-PAGE, with visualization of proteins by autoradiography. Positions of molecular mass markers are indicated on the left of panel A, and positions of the three size classes of avian reovirus polypeptides are indicated between the two panels. B and D, nonradiolabeled cell extracts were subjected to immunoblot analysis with anti-µNS rabbit polyclonal antibodies.

To confirm this, membrane permeabilization to HB was analyzed in infected cells cultured in the presence of ribavirin. Ribavirin is a guanosine nucleoside analog that has been shown to inhibit avian and mammalian reovirus gene expression, at the level of virus transcription, without affecting the expression of cellular genes (22).¹ We have shown previously that the presence of ribavirin in the culture medium of uninfected CEF cells, at concentrations up to 400 µM, does not affect either the viability of the cells for at least 3 days or their protein synthesis capacity, analyzed 16 h after addition of the inhibitor. In the same study we also showed that ribavirin, when added to avian reovirus-infected cells at the start of the infection, does not prevent viral uncoating, indicating that the drug does not affect the capacity of the avian reovirus to penetrate and uncoat within endosomes. The results shown in Fig. 4 (A and B) reveal that ribavirin does not inhibit protein synthesis in uninfected cells (compare lanes 1 and 2), but induces a drastic reduction in the synthesis of viral polypeptides, but not cell polypeptides, in avian reovirus-infected cells (compare lanes 5 and 6). This result demonstrates that ribavirin is a specific inhibitor of reoviral, but not cellular, gene expression. Analysis of HB internalization in ribavirin-treated cells showed that, although ribavirin did not promote the entry of HB into uninfected cells (Fig. 4A, compare lanes 3 and 4), its presence prevented HB internalization in late-stage infected cells (Fig. 4A, compare lanes 7 and 8). Likewise, HB internalization did not take place in cells infected with UV-treated, replication-incompetent reovirions (data not shown). Together, these results demonstrate that viral gene expression is required for avian reovirus to permeabilize the host plasma membrane and also suggest that late membrane leakiness is induced by a viroporin.

View larger version (39K): [in this window] [in a new window]   Fig. 4.   Effects of ribavirin and brefeldin A on membrane permeability. Mock-infected cells (CEF) and avian reovirus-infected cells (S1133-CEF) were incubated for 9 h in the presence (even lanes) or absence (odd lanes) of 200 µM ribavirin (Rib.; panels A and B) or 5 µg/ml brefeldin A (BA; panel C). The culture medium was then supplemented (lanes +) or not (lanes ) with 1.5 mM HB and incubated for another 45 min. A and C, cells were subsequently labeled with [35S]methionine-cysteine for 45 min and then lysed, and total cell extracts were analyzed by SDS-PAGE and autoradiography. Positions of molecular mass markers are indicated on the left of panel A, and positions of the three size classes of avian reovirus polypeptides are indicated between the two panels. B, nonradiolabeled cells were lysed, and the resulting cell extracts were subjected to immunoblot analysis with rabbit anti-µNS antiserum.

Because viroporins are transmembrane proteins that exert their permeabilizing effect when inserted into the cell membrane, we next investigated whether an active exocytic route is required for membrane permeabilization in avian reovirus-infected cells. With this end, we investigated the effect of brefeldin A on the capacity of HB to penetrate late-stage infected cells (Fig. 4C). Brefeldin A is a macrolide antibiotic that has been shown to inhibit vesicle transport to the cell surface, by causing the resorption of the Golgi complex into the endoplasmic reticulum (23-25). At the concentration of 5 µg/ml, brefeldin A did not inhibit protein synthesis in uninfected CEF (compare lanes 1 and 2) and caused only a minor reduction in protein synthesis in avian reovirus-infected cells (compare lanes 5 and 6). Furthermore, the presence of brefeldin A only induced a slight decrease in the intracellular production of infectious viral particles (data not shown), indicating that this antibiotic hardly affects virus replication and reovirion assembly. Similar results regarding the effect of brefeldin A have been reported previously for the replication of avian reovirus 176 in the continuous quail fibrosarcoma cell line QT6 (26). However, the presence of brefeldin A abrogated the ability of the virus to induce both membrane permeabilization to HB (Fig. 4C, compare lanes 7 and 8) and syncytia formation (data not shown), indicating that an active exocytic system is required for avian reovirus to induce these alterations.

Modification of Membrane Permeability by Avian Reovirus p10 Protein-- We have recently shown that the two nonstructural proteins, p10 and p17, encoded by the tricistronic avian reovirus S1 gene become associated with cell membranes in both prokaryotic and eukaryotic cells (11). A priori, then, these two proteins appear to be good candidates for involvement in membrane permeabilization. To assess the permeabilizing activity of the S1-encoded proteins, we first used an inducible Escherichia coli expression system that has been shown to be suitable for the characterization of other membrane-active viral proteins. In this study we used E. coli BL21(DE3)pLysE cells, which contain an IPTG-inducible T7 RNA polymerase gene within the chromosome as well as the pLysE plasmid that constitutively expresses high levels of the T7 phage lysozyme (27). This system, therefore, allows us to test for permeabilization of the inner bacterial membrane, because, when intact, intracellular lysozyme cannot reach the cell wall, whereas a perturbed membrane will allow lysozyme to gain acces to and degrade the cell wall peptidoglycan, giving rise to rapid cell lysis.

As a first approach to analyzing the effect of the three S1-encoded proteins on bacterial membranes, their open reading frames were individually cloned into the maltose-binding protein (MBP) gene fusion vector, pMalC, and the resulting recombinant plasmids were used to transform BL21(DE3)pLysE E. coli cells. Electrophoretic analysis of extracts from recombinant bacteria showed that MBP and all three MBP-fused polypeptides were expressed in IPTG-induced bacteria (Fig. 5C, odd lanes), but not in uninduced bacteria (data not shown). The growth rates of the transformed bacterial cells were analyzed spectrophotometrically by measurement of optical density at 600 nm. The results shown in Fig. 5A revealed that, whereas bacteria transformed with pMalC, pMalC-ORF3, or pMalC-ORF2 grew exponentially during the 180-min period following IPTG induction, a drastic arrest of cell growth followed IPTG induction of pMalC-ORF1-transformed bacteria. This finding indicates that expression of p10 enhances the permeability of the inner membrane to intracellular lysozyme, giving rise to rapid cell lysis. On the other hand, the expression of MBP-p10, but not of the other two MBP-fused proteins, promoted the release of substantial amounts of radioactivity from [³H]uridine-preloaded cells (Fig. 5B) and rendered cells susceptible to HB inhibition (Fig. 5C, compare odd lanes with even lanes).

View larger version (34K): [in this window] [in a new window]   Fig. 5.   Effects of the three S1-encoded proteins on bacterial membrane permeability. E. coli BL21(DE3)pLysE cells carrying the plasmids indicated within the box were induced with 1 mM IPTG. A, the optical densities of cell cultures at 600 nm were measured at the indicated times after induction. B, cells that had been preloaded with [5,6-3H]uridine were IPTG-induced and, at the indicated times after induction, the radioactivity released to the medium was measured. The values shown are means of four independent experiments. C, 1 h after induction, cells transformed with pMalC (lanes 1 and 2) or with one of the pMalC recombinant plasmids containing the inserts shown on top (lanes 3-8) were incubated for 20 min with [35S]methionine-cysteine in the absence (odd lanes) or presence (even lanes) of 0.4 mM HB. Cells were then lysed, and the extracts analyzed by SDS-PAGE and autoradiography. Positions of MBP and of MBP-fused proteins are indicated on the sides of the figure.

To gather more evidence on the permeabilization activity of p10, we next compared the capacity of p10 and p17 to induce membrane leakiness in eukaryotic cells. First of all, the p17- and p10-encoding ORFs were individually cloned into the eukaryotic expression vector pCIneo, and the resulting recombinant plasmids were lipofected into monolayers of BSC-40 monkey cells. The Western blot analysis shown in Fig. 6 (A and B) demonstrates that the two proteins were synthesized in transfected cells, as previously reported (11). A subsequent electrophoretic analysis of ³⁵S-radiolabeled cell extracts revealed that, although HB could readily penetrate cells transformed with pCIneo-ORF1 (Fig. 6C, lane 3), this inhibitor was unable to cross the plasma membrane of cells lipofected with either the empty vector or the pCIneo-ORF2 recombinant vector (Fig. 6C, lanes 1 and 2). Furthermore, the amount of radioactivity released from [³H]uridine-preloaded cells was much greater in pCIneo-ORF1-transformed cells than in cells lipofected with either pCineo or pCIneo-ORF2 (Fig. 6D). These data demonstrate that p10, but not p17, possesses permeabilizing activity in both eukaryotic and prokaryotic cells.

View larger version (34K): [in this window] [in a new window]   Fig. 6.   Permeabilization activity of p10 and p17 expressed in eukaryotic cells. Semiconfluent monolayers of BSC-40 cells were lipofected with the recombinant expression vectors pCIneo, pCIneo-ORF1, or pCIneo-ORF2 and then incubated for 20 h. A and B, cells were then lysed and subjected to immunoblot analysis with polyclonal antibodies raised against the recombinant proteins MBP-p10 (panel A) and His-p17 (panel B). C, cells were then incubated for 45 min in the presence of 1.5 mM HB, and for another 45 min in the presence [35S]methionine-cysteine. Total cell extracts were then prepared and analyzed by SDS-PAGE and autoradiography. Positions of molecular mass markers are indicated on the left. D, the radioactivity released to the medium from [5,6-3H]uridine-preloaded cells was measured. The values shown are means of four independent experiments, and error bars indicate standard deviations of the mean.

The Fusogenic Domain of p10 Is Dispensable for Its Membrane Permeabilizing Activity-- It has been recently shown that p10 is a type I transmembrane protein that causes cell-cell fusion when transiently expressed in mammalian cells, and that deletion of a region of its extracellular NH₂-terminal domain abrogates the fusogenic activity of p10, without affecting its ability to associate with cell membranes (15). As a first approach to assessing whether this "fusogenic sequence" is also involved in membrane permeabilization, a 5'-truncated version of the ORF1 (ORF1*) was cloned into the prokaryotic expression vector pMalC or the eukaryotic vector pCIneo. The resulting recombinant vectors pMalC-ORF1* and pCIneo-ORF1*, as well as their corresponding empty vectors and the recombinant vectors containing nontruncated ORF1 inserts, were then introduced into prokaryotic or eukaryotic cells.

The experiments carried out with bacterial cells are shown in Fig. 7. Both electrophoretic and immunoblot analysis of cell extracts demonstrated that MBP, MBP-p10, and MBP-p10* were all expressed in the corresponding transformed bacteria after IPTG induction (Fig. 7, A and B, lanes 2, 6, and 10), but not in uninduced bacteria (lanes 1, 5, and 9). Fractionation of the extracts from induced bacteria (I) into supernatant (S) and pellet (P) fractions revealed that, whereas most MBP is associated with the soluble fraction (Fig. 7, A and B, compare lanes 3 and 4), both MBP-p10 and MBP-p10* segregated almost exclusively with the pelleted fraction (Fig. 7, A and B, compare lanes 7 and 11 with lanes 8 and 12), suggesting that they become associated with the bacterial membrane. Furthermore, IPTG induction of bacteria transformed with pMalC-ORF1 and with pMalC-ORF1*, but not with the empty plasmid pMalC, caused arrest of cell growth (Fig. 7C), suggesting that both p10 and p10* facilitate access of the intracellular lysozyme to the cell wall, by permeabilizing the inner membrane. Finally, both HB internalization and leakage of radioactivity from [³H]uridine-preloaded bacteria were observed in cells transformed with the two recombinant plasmids, but not in cells transformed with the empty plasmid pMalC (Fig. 7, D and E). From these results, we conclude that both p10 and p10* have the ability to permeabilize bacterial membranes.

View larger version (50K): [in this window] [in a new window]   Fig. 7.   Permeability changes induced by expression of p10 and p10* in bacterial cells. A and B, overnight cultures of E. coli BL21(DE3) cells carrying the plasmids pMalC (), pMalC-ORF1 (ORF1), or pMalC-ORF1* (ORF1*) were induced with IPTG (lanes I) or not (lanes U), and 30 min later cells were lysed and the extracts fractionated into supernatant (S) and pellet (P) fractions by centrifugation. Samples were then analyzed by SDS-PAGE, and proteins were either visualized by Coomassie Blue staining (panel A) or transferred to a polyvinylidene difluoride membrane and subjected to immunoblot analysis with a polyclonal anti-MBP antiserum (panel B). C, the optical densities at 600 nm of cultures of E. coli BL21(DE3)pLysE transformed with the plasmids indicated within the box were measured at the indicated times after induction. Values shown are means of three independent experiments. D, 1 h after induction, transformed E. coli BL21(DE3)pLysE cells were incubated for 20 min with [35S]methionine-cysteine in the presence (even lanes) or absence (odd lanes) of 0.4 mM HB. Cells were then lysed, and the extracts analyzed by SDS-PAGE and autoradiography. Positions of MBP-p10 and of MBP-p10* are indicated on the right. E, transformed E. coli BL21(DE3)pLysE cells that had been preloaded with [5,6-3H]uridine were induced with IPTG and the radioactivity released to the medium was measured at the indicated times after induction. Values shown are means of three independent experiments.

A similar conclusion was reached from the results of the experiments performed with eukaryotic cells (Fig. 8). Thus, whereas p17, p10, and p10*, but not C, segregate exclusively with the membrane fraction when transiently expressed in BSC-40 monkey cells (Fig. 8A), only p10 and p10* are able to promote both HB internalization and uridine release (Fig. 8, B and C). Taken together, these results demonstrate that the fusogenic amino-terminal fragment of p10 is dispensable for membrane association and for membrane permeabilization. However, this fragment is indeed required for the fusogenic activity of p10, because its deletion abrogated the syncytia formation capacity of p10 (Fig. 8D), as reported previously (15).

View larger version (58K): [in this window] [in a new window]   Fig. 8.   Permeabilization and fusogenic activities of p10 and p10* expressed in eukaryotic cells. A, immunoblot analysis of soluble (S) and membrane (M) fractions prepared from extracts of BSC-40 cells lipofected with pCIneo-ORF3 (C), pCIneo-ORF2 (p17), pCIneo-ORF1 (p10), or pCIneo-ORF1* (p10*). Protein C was detected with anti-C, protein p17 with anti-His-p17, and proteins p10 and p10* with anti-MBP-p10 polyclonal antiserum. B, at 20 h after transfection, lipofected cells were incubated for 45 min with 1.5 mM HB, and for another 45 min in the presence of [35S]methionine-cysteine. Cells were then lysed, and total cell extracts were analyzed by SDS-PAGE and autoradiography. Positions of molecular mass markers are indicated on the left. C, cells that had been lipofected with the plasmids shown on top were visualized at 36 h after transfection by microscopy under visible light after staining with Giemsa. Lanes , cells were mock-transfected with Lipofectin.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The alteration of membrane permeability is a common feature of infection by cytolytic animal viruses, and such alterations not only exert profound effects on the metabolism of the infected cell, they also contribute to the development of the cytopathic effect. Early alterations of the membrane allow low molecular weight compounds as well as macromolecules to enter cells together with viral particles. Late membrane leakiness requires virus gene expression and allows the passage of ions and small molecules, but not macromolecules (reviewed in Ref. 1).

In the first part of the present study, we examined host membrane alterations in response to avian reovirus infection. We found that the plasma membrane of S1133-infected CEF becomes permeabilized at late, but not early, infection times, and that the perturbed membrane allows increased influx and efflux of metabolites. Enhanced membrane permeability at late infection times has been also observed in cells infected with flaviviruses (28), herpesviruses, rhabdoviruses (29), paramyxoviruses (29), papovaviruses (30), picornaviruses (31), retroviruses (32, 33), and togaviruses (34).

The absence of increased permeability at early infection times, and the dependence of membrane leakiness on both virus gene expression and intracellular protein trafficking, strongly suggest that viroporins are involved in avian reovirus-induced permeabilization. Accordingly, in the second part of our study, we tried to identify avian reovirus proteins displaying viroporin activity. We focused on the proteins encoded by the tricistronic S1 genome segment because two of them, the nonstructural proteins p10 and p17, have been shown recently to associate with the membranes of both prokaryotic and eukaryotic cells (11), and also because p10 has been shown both to display fusogenic activity (11, 15) and to share structural characteristics with other membrane-active viral proteins (see below). Our results demonstrate that, although both nonstructural proteins p10 and p17, but not C, are entirely associated with prokaryotic and eukaryotic cell membranes, only the expression of p10 is able to induce enhanced membrane permeability to HB and uridine. Furthermore, p10, but not p17 or C, inhibited cell growth in the E. coli system, suggesting that p10 induces disorganization of the inner bacterial membrane and hence allows cytoplasmic lysozyme to reach and digest the peptidoglycan layer of the outer cell wall. Taken together, these results indicate that p10 is an avian reovirus viroporin, and also strongly suggest that p10 is the protein involved in modifying membrane permeability in avian reovirus-infected cells. In support of this possibility, we found that the onsets of membrane permeabilization to HB and p10 synthesis, in cells infected with different avian reovirus multiplicities, were concurrent (data not shown). Several proteins from different viruses have also been reported to possess viroporin activity, including poliovirus 2BC and 3AB (19, 21, 35); adenovirus E3-11.6K (36); togavirus 6K (37); coxsackie B3 virus 2B (38); hepatitis C virus E1 (39); influenza M2 (40); picornavirus 3A and 2B (41); hepatitis A virus 3A, 2B, and 2BC (42, 43); human immunodeficiency virus type-1 gp41 and Vpu (32, 33); rotavirus NSP4 (44); African horse sickness virus NSP3 (45); human respiratory syncytial virus small hydrophobic protein (20); and small hydrophobic nonstructural proteins of Japanese encephalitis virus (28).

In contrast to most nonenveloped viruses, including mammalian reoviruses, avian reoviruses induce syncytia formation, an activity related to the nonstructural p10 protein. Furthermore, this fusion activity appears to be located at the extracellular amino terminus of p10, because deletion of its extreme NH₂-terminal fragment abrogates the ability of p10 to cause cell-cell fusion, without affecting its capacity to associate with membranes (15). Therefore, it was important to assess the implication of this fragment in the permeabilizing activity of p10. The results of the present work indicate that the fragment containing the initial NH₂-terminal 22 amino acid residues of p10, although necessary for fusogenic activity, is not required for membrane permeabilization, because its deletion does not affect the capacity of p10 to associate with cell membranes or to trigger permeability changes in either prokaryotic or eukaryotic cells. This finding suggests that syncytium formation and membrane destabilization are associated with different domains of the p10 protein and, therefore, that they are unrelated phenomena. Furthermore, these two events are not concurrent, because membrane permeabilization always precedes syncytia formation in infected and transfected cells (data not shown). This situation resembles that of the immunodeficiency virus type 1 transmembrane glycoprotein gp41, which contains an amino-terminal domain involved in membrane fusion and syncytia formation, whereas both the membrane-spanning region and sequences located at the carboxyl terminus are involved in increasing membrane permeability (32).

In addition to its permeabilizing activity, p10 also shares several structural and physical characteristics with other viroporins. Thus, p10 and all reported viroporins are small hydrophobic integral membrane proteins of 50-120 amino acid residues, containing at least one transmembrane hydrophobic domain of 20-30 residues and an intracellular region rich in basic residues that is adjacent to the transmembrane domain (reviewed in Ref. 1). Furthermore, p10 and some viroporins contain cysteine residues in their cytoplasmic regions, often positioned proximal to their membrane-spanning domains, and several of these cysteine residues have been found to be modified by palmitoylation (46-50). Interestingly, mutagenesis of the two contiguous cysteines 63 and 64 to alanines, and of lysine 69 to methionine, was reported to abolish the capacity of avian reovirus p10 protein to induce syncytia formation but not to associate with membranes (15). Finally, most viroporins have been shown to form hydrophilic pores by oligomerization, thus allowing ions and low molecular weight hydrophilic compounds to diffuse through the membrane (reviewed in Ref. 1). Whether p10 likewise oligomerizes, whether the two contiguous cysteines are palmitoylated, and whether these cysteines and/or the basic residues play a role in the permeabilizing activity of p10 merit further studies.

The hydrophilic channels formed by viroporin activity allow low molecular weight hydrophilic molecules to cross the membrane; hence, membrane potential is disrupted, ionic gradients collapse, and essential compounds are released from the cell (1, 2, 51). The alterations in ion concentration occurring in the cytoplasm of virus-infected cells could favor virus replication, by enhancing the intracellular concentration of sodium ions. This might promote translation of viral versus cellular mRNAs, because translation of mRNAs from many cytolytic animal viruses, including reoviruses, is fairly resistant to high sodium concentrations, whereas high sodium concentrations are inhibitory for the translation of most cellular mRNAs (52-57). On the other hand, progressive membrane damage is thought to promote cell lysis and virus release, facilitating virus spreading to surrounding cells (9, 36). Curiously, our results and those of Duncan et al. (26) demonstrate that membrane permeabilization and syncytia formation are not essential steps in the avian reovirus replication cycle, because their inhibition by brefeldin A was not accompanied by a significant reduction in either viral protein synthesis or infectious progeny virus production. However, the presence of brefeldin A retards and partially inhibits avian reovirus-induced cytopathic effects and virus release (26), suggesting that membrane permeabilization and syncytia formation, although not strictly required, can accelerate the production of a lytic-type infection. This, in addition to the fact that both the p10 ORF and the syncytial phenotype are conserved among all known avian reovirus isolates as well as two atypical mammalian reoviruses, Nelson Bay virus and baboon reovirus, suggests that expression of the p10 protein could confer some selective advantages to the virus, such as enhanced virus spreading in infected animals. Assessing the role that the p10 protein plays in the replication cycle of avian reovirus is problematic, because no method has yet been developed to manipulate the reoviral genome to generate mutant viruses that do not express a particular protein or that express mutated proteins.

	ACKNOWLEDGEMENTS

We are grateful to Laboratorios Intervet (Salamanca, Spain) for providing the specific-pathogen-free embryonated eggs. We thank Luis Carrasco and Rubén Varela for critical reading of the manuscript and José Antonio Trillo for providing technical support and assistance.

	FOOTNOTES

^* This work was supported by Spanish Ministry of Science and Technology Grant PB97-0523.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Dept. of Medicine, Columbia University College of Physicians and Surgeons, New York, NY 10032.

^§ To whom correspondence should be addressed. Tel./Fax: 34-981-599157; E-mail: bnjbena@usc.es.

Published, JBC Papers in Press, March 13, 2002, DOI 10.1074/jbc.M202018200

¹ L. Labrada, G. Bodelón, J. Viñuela, and J. Benavente, submitted for publication.

	ABBREVIATIONS

The abbreviations used are: ORF, open reading frame; CEF, chicken embryo fibroblast; HB, hygromycin B; IPTG, isopropyl-1-thio--D-galactopyranoside; pfu, plaque-forming unit; MBP, maltose-binding protein.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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