INTRODUCTION

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Poliovirus, a representative member of the Picornaviridae family, profoundly modifies cellular architecture and metabolism after infection. These alterations include enhanced membrane permeability (14, 17); inhibition of DNA, RNA, and protein syntheses (16, 44); blockade of membrane traffic (22); increased intracellular calcium levels (29); and modifications in phospholipase activities and lipid turnover (28, 30). There are also dramatic morphological alterations in chromatin structure and in the cytoskeleton (32). Moreover, the Golgi apparatus is not recognizable as such in poliovirus-infected cells (20, 42), and numerous membranous vesicles (50 to 400 nm) fill most of the cytoplasm at late times of infection (11, 21). All of these changes are referred to as the cytopathic effect (13, 15).

Membrane proliferation is essential for poliovirus genome replication, and viral RNA synthesis is associated with the newly generated vesicles (10, 28). For some time it was considered that these cytoplasmic vesicles originated by budding from the endoplasmic reticulum (ER) (9). However, more recent evidence suggests that the ER constitutes a significant but not exclusive source for the intracellular membranes induced by poliovirus infection (43). Thus, the cellular origin of these membranous vesicles, the exact mechanism used by poliovirus to induce them, and the mode by which the replicative complexes associate with these vesicles are not yet clear. It is clear, however, that protein 2BC and its proteolytic products, 2B and 2C, play an important role in these processes (2, 6, 11, 19, 22, 47). Understanding the functions of proteins 2B, 2C, and 2BC would help to elucidate the mechanisms of picornavirus genome replication and cytopathogenicity at the molecular level.

Processing of 2BC by 3C^pro gives rise to the mature products 2B and 2C, although a large fraction of 2BC remains uncleaved in picornavirus-infected cells. Genetic studies have suggested putative roles of 2B and 2C in RNA replication (4, 8, 31, 33, 36, 37, 45, 46). More recently, 2B, 2C, and 2BC were also implicated in the induction of diverse alterations in poliovirus-infected cells. Notably, proteins 2B and 2BC enhance membrane permeability and block the exocytic pathway when they are expressed individually in cultured cells and have been implicated in virus release (1, 22, 47, 48). The expression of proteins 2C and 2BC in mammalian cells with recombinant vaccinia viruses promotes membrane proliferation in the cytoplasm of transfected cells. Vesicle proliferation induced by 2BC is similar to that observed in poliovirus-infected cells, while 2C induces the formation of tubular membrane structures with a myelin-like arrangement (2, 20). In addition, the RNA replicative complexes contain a number of poliovirus proteins, including 2C (12). Three functions have been ascribed to 2C: ATPase and GTPase enzymatic activities (35, 38), interaction with viral RNA, and RNA binding by two regions of 2C (39) and interaction of 2C with membranes by means of an amphipathic helix at the N terminus (23). Based on these results, it was hypothesized that 2C or its precursor, 2BC, is responsible for poliovirus RNA binding to the induced cytoplasmic vesicles and participates in the spatial organization of the replicative complexes (39).

Inducible expression of poliovirus 2BC in yeast cells inhibits growth and provokes a number of morphological and metabolic modifications in these cells similar to those observed in mammalian cells (6). Thus, the synthesis of 2BC interferes with the exocytic pathway and promotes the formation of cytoplasmic vesicles in yeast cells. We now report the generation and characterization of a large collection of 2BC variants obtained by site-directed mutagenesis and random mutagenesis. Our results allow the dissection of the different activities assigned to the 2BC protein.

	MATERIALS AND METHODS

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Microbial strains. Escherichia coli DH5 (41) was used to obtain all expression plasmids described. The Saccharomyces cerevisiae strain used was W303-1B (MAT ade2 his3, leu2 trp1 ura3).

General recombinant DNA protocols. Construction of vectors was carried out by standard procedures (41). The yeast expression plasmid used was the yeast-E. coli shuttle vector pEMBLyex4 (18), a 2µm plasmid derivative. Poliovirus sequences were amplified by PCR from pT7XLD (a plasmid including the cDNA of poliovirus type 1, generously provided by E. Wimmer, Stony Brook, N.Y.).

Construction of plasmids encoding 2BC mutant proteins. Plasmids pEMBL. 2B(B), pEMBL.2C, pEMBL.2BC, pEMBL.2BcXbaI, pEMBL.2BcSphI, pEMBL.2BcGKS, pEMBL.2BcEcoRI, pEMBL.2BcSalI, pEMBL.2bC-S, pEMBL.2bC-D, and pEMBL.2bC30N have been described elsewhere (6).

Yeast growth, transformation, and induction. Transformation of yeast by the lithium acetate procedure was done as previously described (40). Yeast growth and induction of the UAS_GAL-CYC promoter (18) was carried out as described previously (6). The different media used in this work (YNB.Glu, YNB.Gal, and YNB.LGal) have been defined previously (6).

Hydroxylamine mutagenesis and genetic assay. Plasmid pEMBL.2Bc128 or pEMBL.2bcSphI(N/B) was randomly mutated with hydroxylamine as described previously (7, 40). The mutated DNA was used to transform S. cerevisiae W303-1B to obtain 100 to 200 colonies on each YNB.Glu plate. These colonies were replicated with YNB.Gal medium and incubated for 72 h. Colonies able to grow in YNB.Gal medium were considered potential 2BC variants. These clones were amplified, and their capacity to express poliovirus 2BC variant proteins was tested. Mutant plasmids were isolated from yeast cells by standard procedures (40). E. coli DH5 cells were transformed with these lysates. The location of the different mutations was determined by DNA sequencing by the dideoxy method (41).

Permeability changes tested with HB sensitivity. The permeability changes in the plasma membrane were detected by testing the entry of the aminoglycoside antibiotic hygromycin B (HB) in yeast cells and the consequent inhibition of protein synthesis. The cultures were preincubated with or without 1 mM HB for 10 min before labeling of cells with 7.5 µCi of [³⁵S]methionine per ml for 45 min. Finally, the samples were processed as described previously (51).

Other techniques. Most of the other techniques used in this work have been described elsewhere (6). The isolation of antisera against poliovirus 2B and 2C was previously described (6). The antiserum against invertase was a generous gift from R. Schekman (University of California, Berkeley). The monoclonal antibodies against carboxypeptidase Y (CPY) were purchased from Molecular Probes, Inc. Cell labeling and immunoprecipitations were carried out as described previously (27). Yeast extracts for Western blot analysis were prepared as described previously (51). Immunoblot analyses were carried out as described elsewhere (6). The protocol for electron microscopy was described previously (6).

	RESULTS

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Site-directed mutagenesis of poliovirus 2BC protein. Poliovirus 2BC is a multifunctional protein that blocks cell growth and the exocytic pathway in yeast. In addition, 2BC induces the proliferation of intracellular cisternae that fill most of the cytoplasm. To define the 2BC regions involved in these activities, a variety of point and deletion mutations in 2BC were generated as described in Materials and Methods. The schematic representation and the nomenclature used for the 2BC mutants described in this work and in our previous work (6) are shown in Fig. 1. A summary of the results obtained with all of the 2BC mutants for yeast growth, inhibition of the exocytic pathway, and enhancement of membrane permeability is also shown in Fig. 1. The effects of several mutations in conserved regions of 2BC on cytotoxicity for yeast cells were reported previously (6). Now, the collection of 2BC mutants has been expanded with the aim of determining the minimal portion of 2BC able to block yeast growth, to permeabilize cells, and to inhibit glycoprotein processing.

View larger version (32K): [in this window] [in a new window]   FIG. 1.   Schematic representation of 2BC mutants and summary of the effects of their expression in yeast cells. The amino acids of each protein are indicated. Black bars indicate sequences unrelated to 2BC. Growth agar (HB/+HB) indicates growth on YNB.Gal plates supplied (+HB) or not supplied (HB) with 50 µg of HB per ml; +++, growth rate similar to that of control cells; ++ growth at a rate lower than that of control cells; +, growth resulting in individual colony formation; , no growth. The third value shown for 2B(1-97) corresponds to cells streaked for a second time on YNB.Gal plates supplemented with HB. Growth liquid indicates that exponentially growing yeast cultures were diluted in YNB.Lac medium, and galactose (2%) was added (initial A660, 0.150). Cells were harvested at 2-h intervals, and the A660 was measured to quantitate cell density: the optical density of the culture after 24 h of incubation increased more than 10-fold (+++), between 7- and 10-fold (++), between 5- and 7-fold (+), between 3- and 5-fold (), or less than 3-fold (). pCPY arrest indicates that the ratio of precursor form of CPY (pCPY) to mature CPY in wild-type 2BC-expressing cells was calculated by densitometric analysis, and this value was taken as 100%. The relative proportion of pCPY was calculated for the rest of the mutants: +++, 100 to 70% wild-type 2BC activity; ++, 70 to 40% wild-type 2BC activity; +, 40 to 10% wild-type 2BC activity; , no pCPY accumulation. HB sensitivity indicates that the ratio of protein synthesis in yeast cells expressing 2BC in the absence or in the presence of HB (1 mM) was calculated by densitometric analysis, and this value was taken as 100%. Therefore, 100% represents full permeabilization to HB. The relative inhibition of protein synthesis by HB was calculated for the rest of the mutants: +++, 100 to 70% wild-type 2BC activity; ++, 70 to 40% wild-type 2BC activity; +, 40 to 10% wild-type 2BC activity; , no HB permeabilization.

Effects of poliovirus 2BC and variants on yeast growth. Poliovirus 2BC potently blocks yeast growth. The action of different 2BC variants on yeast growth both on solid and in liquid media was tested (Fig. 1 and 2). Two different types of solid media, supplemented or not supplemented with 50 µg of HB per ml, were used in order to test the effect of long-term exposure to this antibiotic.

View larger version (66K): [in this window] [in a new window]   FIG. 2.   Growth of yeast cells expressing different 2BC mutants. (A) Yeast cells expressing different 2BC variants were streaked on agar plates with the indicated composition and incubated for 3 days at 30°C. HigB, hygromycin B. (B) Extracts of yeast cells expressing the different 2BC variants indicated or bearing plasmid pEMBLyex4 (V) were obtained after 6 h of induction and assayed with antibodies against 2B or 2C proteins. Immunoblot analysis of these samples was performed as described in Materials and Methods.

Enhanced membrane permeability induced by poliovirus 2BC in yeast cells. Poliovirus proteins 2B and 2BC enhance membrane permeability in mammalian cells, as demonstrated by the entry of HB, a nonpermeating inhibitor of translation. First, we analyzed membrane permeabilization induced by poliovirus proteins 2B, 2C, and 2BC using the HB test with yeast cells. 2BC strongly promoted the entry of HB into yeast cells soon after its induction (Fig. 3A). In contrast to mammalian cells, yeast cells were not permeabilized by 2B (Fig. 3B). This result is in agreement with the finding that 2BC enhances membrane permeability more strongly than 2B in mammalian cells. Other poliovirus proteins, such as 2C (Fig. 3B), 2A, which is strongly cytotoxic for yeast cells (7), 3A, and 3AB, had no effect on the entry of HB into yeast cells (results not shown for 2A, 3A, and 3AB).

View larger version (76K): [in this window] [in a new window]   FIG. 3.   Action of 2BC variants on HB permeabilization. (A) 2BC induces permeability changes. Protein synthesis in yeast cells bearing plasmids pEMBLyex4 (V) and pEMBL.2BC (2BC) in the presence (+) or absence () of HB (1 mM) was assayed. The samples were obtained as described in Materials and Methods at the postinduction times indicated in hours. (B) Effects of 2BC variants on membrane permeability. Protein synthesis in yeast cells expressing the 2BC variants indicated or bearing plasmid pEMBLyex4 (V) in the presence (+) or absence () of HB (1 mM) was assayed. The samples were obtained as described in Materials and Methods at 5 h postinduction. This experiment was performed with all of the mutants obtained, and the results of the densitometric analysis of the corresponding sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiograms are included in Fig. 1.

Effects of 2BC variants on the exocytic pathway. The action of 2BC and its variants on the exocytic pathway in yeast cells was assayed by analyzing the processing of CPY and invertase (Fig. 4). CPY is a vacuolar enzyme that is synthesized as a proenzyme in the ER, where it is core glycosylated (67 kDa). CPY is further glycosylated in the Golgi apparatus and is cleaved to generate the mature form of the enzyme (61 kDa) within the vacuole (5, 27). Yeast invertase is also synthesized as a precursor that is extensively processed in the ER (79 to 81 kDa) and in the Golgi apparatus (different forms of 100 to 140 kDa) to produce the mature enzyme, which is secreted into the medium (24, 25). In general, there was a good correlation between the enhancement of membrane permeability and the ability to block the exocytic pathway for most of the 2BC variants tested. The majority of the 2BC variants obtained affected CPY processing to different degrees (Fig. 4A). The expression of mutant 2Bc128 resulted in the accumulation of the CPY precursor form to levels similar to those obtained with wild-type 2BC. However, the synthesis of 2Bc108 led to only small amounts of the precursor form of CPY, while mutants 2B-2c40N and 2BX had no effect on CPY processing. No accumulation of the CPY precursor form was seen with 2bcSphI(N/B) or with the 2B deletion variants, while the 2B point variants resulted in CPY precursor form accumulation to different degrees (Fig. 4A and summary in Fig. 1).

View larger version (37K): [in this window] [in a new window]   FIG. 4.   Action of 2BC variants on the exocytic pathway. (A) Extracts of yeast cells expressing the different 2BC variants indicated or bearing plasmid pEMBLyex4 (V) were obtained after 6 h of induction and assayed with antibodies against CPY. mCPY, mature CPY; pCPY, precursor form of CPY. (B) Yeast cells at 4 h postinduction in YNB.LGal medium were labeled for 10 with [35S]methionine and chased for 0 or 30 min. Invertase immunoprecipitation of control cells (bearing pEMBLyex4 [V]) or cells expressing the different 2BC variants indicated was performed as described in Materials and Methods. This pulse-chase experiment was carried out three times for mutant 2Bc128. In all cases, the amount of invertase precipitated was much smaller than that obtained with any other 2BC variant. mInv, mature invertase; pInv, precursor forms of invertase; MW, molecular weight (in thousands).

Modification of the ultrastructure of yeast cells by 2BC variants. The ultrastructure of yeast cells that expressed some of the more interesting 2BC mutants was analyzed by electron microscopy (Fig. 5). The wild-type form of poliovirus 2BC induces the proliferation of a large number of cytoplasmic vesicles, together with the disruption of the yeast vacuole (Fig. 5B). Of the mutants tested, only 2bC-D (Fig. 5D) was able to reproduce the ultrastructural modifications found with 2BC, although it did so at a lower rate. Mutants 2Bc258 (Fig. 5C) and 2Bc128 (Fig. 5E and F), which showed the same behavior as wild-type 2BC in all of the previous assays, induced ultrastructural changes in yeast cells clearly different from those induced by 2BC. Protein 2Bc258 (Fig. 5C) induced the generation of vesicles in the cytoplasm that were larger (±500 nm) than those observed in 2BC-expressing cells (Fig. 5B). A similar phenotype was observed with 2Bc.EcoRI (results not shown). The expression of protein 2Bc128 (Fig. 5E and F) induced membranous structures that probably corresponded to ER swelling. Most yeast cells that expressed 2Bc128 had a cytoplasm occupied by honeycomb-like structures, where ER stacks were piled. This phenomenon has been described for the overexpression of several residential ER proteins, such as the HMG coenzyme A reductase (50) and cytochrome P-450 (49). On the contrary, cells expressing mutants 2Bc108 (Fig. 5G) and 2B-2c40N (Fig. 5I) showed a phenotype more similar to that of control yeast cells. However, cells that synthesized 2Bc108 or 2B-2c40N did not contain the large vacuole typical of control cells (Fig. 5K). Finally, cells expressing 2bC30N (Fig. 5A) and 2B (Fig. 5H) looked like control yeast cells that did not express any poliovirus gene (Fig. 5K). Thus, the region of the 2C protein next to 2B dictates the type of membrane generated in yeast cells, while the N terminus of 2B is essential to induce structural changes.

View larger version (155K): [in this window] [in a new window]   FIG. 5.   Ultrastructure of yeast cells expressing different 2BC variants. Thin-section electron microscopy was carried out for yeast cells expressing 2bC30N (A), 2BC (B), 2Bc258 (C), 2bC-D (D), 2Bc128 (E and F), 2Bc108 (G), 2B (H), 2B-2c40N (I), or 2Bc128*3c (J) or transformed with plasmid pEMBLyex4 (K). Cells were chemically fixed at 20 h postinduction and were processed for electron microscopy as described in Materials and Methods. N, nucleus; V, vacuole; M, mitochondria. Black arrows indicate ER swelling. Bars, 500 nm.

Random mutagenesis of 2BC variants. In principle, the inhibition of yeast cell growth induced by 2BC can be used as a genetic assay to obtain 2BC variants deficient in cytotoxicity. This approach has been successfully used in the case of poliovirus 2A^pro (7), and it would be particularly interesting to obtain thermosensitive variants of poliovirus proteins that inhibit yeast cell growth.

Analysis of 2B deletion mutants. The mutants obtained by random mutagenesis of pEMBL.2Bc128, known as 32b and 3c, showed a phenotype that was, in principle, unexpected. Although 2B alone has almost no effect on yeast growth, short deletions at the C terminus give rise to truncated 2B proteins that are cytotoxic (2Bc128*.32b and 2Bc128*.3c). Note that longer deletions of 10 or 26 additional residues generate nontoxic 2B variants (2b67 and 2bSpeI). The sequences of these mutants are shown in Fig. 6.

View larger version (25K): [in this window] [in a new window]   FIG. 6.   Sequences of 2B deletion mutants. Amino acid sequences from position 51 to the C terminus of the indicated mutants (Mut) are shown. Shaded sequences correspond to the wild-type 2B (2Bwt) sequence.

	DISCUSSION

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Activities of 2B, 2C, and 2BC proteins in yeast and human cells. Our knowledge of the mode of action of picornavirus proteins 2B, 2C, and 2BC has benefited from the individual expression of the corresponding genes in both mammalian and yeast cells. These studies have provided evidence that 2B and 2BC block the exocytic pathway and interfere with the correct processing of glycoproteins (6, 22). In addition, 2B and 2BC enhance membrane permeability to low-molecular-weight compounds, such as uridine or HB, and to ions, such as calcium, in mammalian cells (1, 3, 22, 47, 48). These effects are more pronounced with 2BC than with 2B in the case of poliovirus (1, 3). Besides, poliovirus 2B is only slightly toxic for yeast, and only when its expression is prolonged for several generations is there an inhibition of yeast growth. This inhibition is clearly observed when HB is continuously present in the growth medium of 2B-expressing yeast cells. Therefore, yeast cells seem to be more resistant to the effects of 2B than mammalian cells, and only when a more potent protein such as 2BC is expressed are the cytotoxic and permeabilizing activities of the protein clearly observed. In addition, two noncytotoxic 2BC deletion mutants randomly isolated in yeast cells encoded proteins almost identical to 2B (variants 3b and 30b in Table 1).

Structure-activity relationships of 2BC. The ease of manipulation of S. cerevisiae makes this microorganism a useful model system for understanding the mode of action of 2BC and facilitates genetic studies of this protein. The analysis of the effects of the different 2BC variants described in this work leads to a number of conclusions concerning the regions of 2BC involved in the different activities tested.

Functioning of 2B, 2C, and 2BC in the poliovirus replicative cycle. The 2BC precursor is very abundant in cells infected by poliovirus, producing the mature products 2B and 2C after 3C^pro cleavage. It now seems clear that 2BC has activities that are not present or are clearly diminished in the mature products 2B and 2C. This assertion is particularly clear when yeast cells are assayed. It seems logical to propose that the initial generation of 2BC targets this protein to the ER, redirecting membrane trafficking toward the formation of small vesicles. The formation of these membranous vesicles is a requisite for viral RNA synthesis. Therefore, polioviruses mutated in 2B or 2C are deficient in replicating viral RNA. Coxsackievirus 2B has been described as a viroporin, because of its ability to enhance membrane permeability, perhaps by pore formation, thus facilitating the release of progeny virions (47, 48). In addition, coxsackievirus 2B (47) and poliovirus 2BC (3) enhance intracellular calcium levels, a process that could facilitate the assembly of new virions. In conclusion, the release of 2B from the 2BC precursor may locate this protein at the plasma membrane to act as a viroporin. The generation of mature 2C could lead to the transport of mature viral RNA bound to the protein to special locations where the virion assembly process takes place. In fact, some polioviruses mutated in 2C show defects in the assembly of new virions (34).