The Yeast Saccharomyces cerevisiae as a Genetic System for Obtaining Variants of Poliovirus Protease 2A*

(Received for publication, November 27, 1996, and in revised form, March 3, 1997)

Angel Barco Dagger §, Ivan Ventoso and Luis Carrasco

From the Centro de Biología Molecular, Consejo Superior de Investigaciones Científicas-UAM, Universidad Autónoma de Madrid, Canto Blanco, 28049 Madrid, Spain

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

The inducible expression of poliovirus protease 2A (2Apro) blocks the growth of Saccharomyces cerevisiae. A number of yeast colonies that grow after 2Apro induction have been isolated. The majority of these clones express 2Apro to control levels, suggesting that their ability to divide is not due to the loss of 2Apro gene inducibility. The sequences of the 2Apro genes isolated from 22 clones were determined. Most of the 2Apro sequences from these colonies contain point mutations in the poliovirus protease. The different variant protease sequences were transferred to an infectious poliovirus cDNA clone. Translation of genomic RNA obtained from these poliovirus mutants in cell-free systems revealed that some of them had defects in their ability to cleave P1-2A in cis. In addition, several of these variants cleaved the translation initiation factor eIF-4G inefficiently. Transfection of the RNA generated from the full-length poliovirus genomes mutated in 2Apro yielded five viable polioviruses with a small plaque phenotype. These five polioviruses efficiently cleaved p220 but showed defects in viral protein synthesis, transactivation of a leader-luciferase mRNA, and 3CD cleavage to 3C' and 3D'. All 2Apro mutant sequences, including those that did not yield viable viruses, were cloned in pTM1 vector under a T7 promoter. Only the 2Apro variants that have activity to cleave 3CD produced viable poliovirus. Our findings indicate that S. cerevisiae represents a useful system for obtaining poliovirus 2Apro variants that may provide further insight into the role of this protease during the poliovirus replication cycle.


INTRODUCTION

Poliovirus gene expression relies on the synthesis of a large polypeptide precursor that is proteolytically cleaved to generate the mature viral proteins. Polyprotein processing is accomplished by two virus-encoded proteases: 2Apro1 and 3Cpro (1, 2). The poliovirus endopeptidase 2Apro is a polypeptide of 149 amino acid residues in which the catalytic triad is formed by His20, Asp38, and Cys109 (3, 4). The fact that cysteine instead of serine forms part of the active site of 2Apro has been taken as evidence that this protease is an evolutionary intermediate between serine and cysteine proteases (5). However, sequence similarities between 2Apro and trypsin-like serine proteases have conclusively classified 2Apro as belonging to the serine protease group (6, 7).

Both proteases 2Apro and 3Cpro cleave substrates in cis and trans (1, 2). cis cleavage by 2Apro occurs once the protease has been synthesized as a precursor on the nascent polypeptide chain still bound to ribosomes (8). Thus, 2Apro activity cleaves the Tyr-Gly peptide bond between P1 and 2A, releasing P1, the precursor of the capsid proteins, from the rest of the nascent polyprotein (1). The second proteolytic cleavage effected by 2Apro on a viral protein precursor lies in 3CD, where a Tyr-Gly dipeptide is hydrolyzed, giving rise to 3C' and 3D', two products of unknown function (9). Apparently, the generation of 3C' and 3D' is not necessary for poliovirus growth in culture cells, since a poliovirus mutant unable to produce 3C' and 3D' grows as well as wild type poliovirus does (10). In addition to the two cleavages performed by 2Apro on poliovirus protein precursors, this protease also cleaves cellular proteins (2). A number of cellular polypeptides are degraded after poliovirus infection, although the proteases responsible for these cleavages have not yet been identified (11). The best documented cleavage of a cellular protein by 2Apro is that of eIF-4G (also known as p220), a component of the translation initiation factor eIF-4F (12). It has not been formally proven that poliovirus 2A directly cleaves p220, but the fact that coxsackievirus 2A and rhinovirus 2A directly degrade p220 (13-15) makes it unlikely that a cellular protease activated by 2Apro is responsible for p220 cleavage (16, 17). The proteolytic cleavage of the dipeptide present in the initiation factor eIF-4G generates the N-terminal eIF-4G moiety involved in eIF-4E interaction, whereas the C-terminal eIF-4G moiety, which binds eIF-4A, participates in the translation of picornavirus RNA (18). Translation of newly synthesized mRNAs is halted by p220 cleavage (19), but protein synthesis on mRNAs already engaged in the protein-synthesizing machinery continues (20, 21). On the other hand, uncapped picornavirus RNA does not require the cap binding factor eIF-4E but does depend on the C-terminal eIF-4G moiety and eIF-4A to become engaged in translation (18).

Poliovirus 2Apro is a multifunctional enzyme. Apart from proteolytic cleavage of viral protein precursors and eIF-4G, 2Apro can also enhance the translation of poliovirus RNA (22). The activation of protein synthesis by 2Apro requires only the poliovirus internal ribosomal entry site (IRES) sequences, since translation of reporter genes placed under the IRES is stimulated by this protease (22-24). The exact molecular basis by which 2Apro stimulates translation remains unknown, but the simple generation of eIF-4G cleavage products is not sufficient for this enhancement to take place (24). The finding that poliovirus mutants in the IRES region compensate for this defect with mutations in the 2Apro sequence (25) suggested an interaction between 2Apro and the IRES, but direct evidence for such an interaction is still lacking. 2Apro not only enhances poliovirus RNA translation but also participates in viral RNA replication by a still unknown mechanism (26, 27). Unlike aphtoviruses and hepatitis A virus, in which the L and 2Apro genes, respectively, are not necessary for viral infectivity (28, 29), the poliovirus 2Apro is required for virus RNA replication and viability (30, 31). Certainly, the generation and analysis of additional poliovirus 2Apro variants should provide more insights into the different activities of this multifunctional protease that regulates viral and cellular gene expression.

Inducible expression of poliovirus nonstructural proteins in the yeast Saccharomyces cerevisiae showed that two of them, namely 2Apro and 2BC, inhibited yeast growth (32-34). Recently, a similar effect was found for rhinovirus 2Apro (35). This finding allows the use of yeast cells as a genetic system in which to select 2Apro variants that permit S. cerevisiae growth. We now report the generation and characterization of several 2Apro variants selected by this method. In principle, this approach can be applied to analyze the structure-function relationship of any other viral or nonviral protein that interferes with yeast cell division.


EXPERIMENTAL PROCEDURES

Yeast Growth, Transformation, and Induction

Transformation of yeast by the lithium acetate procedure was performed as described previously (36). Yeast growth and induction of UASGAL-CYC promoter was performed as described (34). The different media used in this work (YNB. Glu, YNB. Gal, and YNB. LGal) have been defined previously (34).

Hydroxylamine Mutagenesis and Genetic Assay

The plasmid pEMBL.2A (32) was randomly mutated using hydroxylamine as described (36). This mutated DNA was used to transform the S. cerevisiae strain W303-1B (Mat alpha , Ade2, His3, Leu2, Trp1, Ura3) to obtain 100-200 colonies in each YNB.Glu plate. These plates were replicated in YNB.Gal medium and incubated for 48 h either at 30 or 37 °C. Colonies able to growth in YNB.Gal medium were considered potential mutants in the inhibitory activity of 2A. These clones were amplified, and their capacity to grow at different temperatures and to express the 2Apro poliovirus protein in liquid medium was tested. Mutant plasmids were isolated from yeast cells using standard procedures (36), and Escherichia coli DH5 cells were transformed with these lysates. The locations of the different mutations were determined by DNA sequencing following the dideoxy method (37).

General Recombinant DNA Protocols

E. coli DH5 (37) was used for the construction of all expression plasmids described. To introduce the different mutations in plasmid pT7XLD (38) containing the infective clone of poliovirus, a subcassette with the 3235-3953 sequence of poliovirus was constructed (pSub.2A). The mutants 2, 3, 4, 5, 8, and 9 were cloned in this plasmid using the PstI and Asp718 sites; the mutants 6, 7, 10, 12, 13, 15, and 21 were cloned using the NcoI sites. Mutant 17 was cloned using the PstI and NcoI (partial digestion) sites. The various pSub.2A* vectors were digested with BstEI and cloned in pT7XLD digested with this same enzyme to obtain the different pT7XLD(2A*) mutants. The sequence of each plasmid was verified by partial DNA sequencing of the 2A region by the dideoxy method (37). The different pTM1-2A* plasmids were constructed by digestion of pTM1-2A (WT) (39) and all the pEMBL-2A* plasmids with PstI and SalI.

Western Blot Analysis

The alpha -2A, alpha -2C, and alpha -3C sera were obtained by inoculation of the fusion proteins MBP.2A, MBP.2C, or MBP.3C in rabbits. The antiserum against p220 (eIF-4G) protein has been previously described (39). Yeast extracts for Western blot analysis were prepared as indicated (40). For standard immunoreactions, sera dilutions of 1:1000 in phosphate-buffered saline-Tween 20 (0.05%) were used. Immunoblots were carried out as described elsewhere (34).

In Vitro Transcription and Translation

All plasmids were linearized and transcribed with phage T7 RNA polymerase as described (24). HeLa S10 cell extract treated with micrococcal nuclease was used for in vitro translation as described previously (41)

Transfections and Electroporations

For DNA and RNA transfections we used the protocol described by Aldabe et al. (42) and Ventoso and Carrasco (24), respectively. To obtain poliovirus after RNA electroporations, about 1 × 107 cells were transferred to a 0.4-cm cuvette and 5 µg of in vitro synthesized full-length poliovirus RNA was added. Electroporation was performed by a single pulse at 250 V and 960 microfarads using a Bio-Rad Gene Pulser apparatus with the pulse-controller unit set at maximum resistance. Half of these cells were transferred to a 6-cm culture dish, and 4 h later the cells were overlaid with 0.45% agarose and incubated at 32.5, 37, or 39.5 °C for several days before staining with crystal violet. The remaining cells were diluted with nonelectroporated HeLa cells and were overlaid or not to pick plaques or recover the supernatant. WT and viable mutant polioviruses were isolated by picking several plaques visualized after staining with neutral red. To verify the presence of the mutated 2Apro, the sequence was amplified from the extracted viral RNA by reverse transcriptase polymerase chain reaction and sequenced by the dideoxy method (37).

Virus Infection of Cell Monolayers

Maintenance of HeLa and infection with WT or 2A mutant polioviruses, analysis of viral proteins, transactivation assay, measurement of viral RNA synthesis, and dot blot analysis were carried out as described previously (24).


RESULTS

Generation and Isolation of Poliovirus 2Apro Variants

The finding that expression of 2Apro was inhibitory to S. cerevisiae cell growth (32) opened the possibility of isolating 2Apro variants devoid of this inhibitory capacity. To this end, plasmid pEMBL containing the poliovirus 2Apro sequences was mutagenized with hydroxylamine. Yeast cells were transformed with the mutated pEMBL-2A, and colonies that grew in glucose agar were replicated in plates containing galactose to induce the synthesis of 2Apro. To assay for thermosensitivity of the yeast clones obtained, the galactose plates were incubated at two different temperatures, 30 and 37 °C. Upon galactose induction 22 colonies of yeast cells that grew at 37 °C were obtained, indicating that poliovirus 2Apro was not induced or was noninhibitory for these yeast clones (Table I). Six of these clones presented a ts phenotype, since their growth in liquid (results not shown) and solid media was selectively arrested at 30 °C (Fig. 1A). This result gives the percentage of yeast clones that grow in galactose after mutagenesis of 1.2% (Table I). Plasmid DNA was obtained from these clones, and the 2A sequences were determined. These results are summarized in Tables I and II. Thus, 15 different point mutations in the 2Apro sequence have been identified. In addition, two nonsense mutants at codon 24 arose. Finally, in two other clones (numbers 1 and 11) the mutation that conferred the ability of yeast cells to grow in galactose could not be identified. Notably, none of these 2Apro variants contained a mutation in the catalytic triad of the protease.

Table I. Hydroxylamine mutagenesis of pEMBL.2A


Transformant colonies in Glu plates/Gal replica plates
  pEMBLyex4 + hydroxylamine 5000/5000
  pEMBL.2A 5000/0
  pEMBL.2A + hydroxylamine 1800/22 right-arrow 1.2%
Thermosensitivity
  Colonies on replica plates at 30 °C: 16
  Colonies on replica plates at 37 °C: 22
  Clones with temperature-sensitive   phenotype 27%
Summary of the mutations
  Undetermined mutations: 2 (mutants 1 and 11)
  Nonsense mutations: 1 (mutants 14 and 16)
  Point mutations: 15 (3 of them appear twice)
  Transitions G to A: 8
  Transitions C to T: 6
  Others
    1 change C to A
    1 double change dimer GG to AA


Fig. 1. Growth of yeast colonies upon induction of 2Apro synthesis. A, growth of the different clones of yeast cells obtained in the genetic analysis. Cells expressing the different 2Apro mutants were streaked on YNB.Glu (left plate) or YNB.Gal (middle and right plates). The cells were grown at the temperatures indicated. No significant differences in YNB.Glu plates were observed at 30 and 37 °C. B, immunoblot analysis of 2Apro expression in the mutant clones. Yeast cells were grown in YNB.LGal medium, and crude extracts were obtained 8 h after induction, separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and immunoreacted with alpha -2A rabbit antiserum. Yeast cells bearing the plasmids pEMBLyex4 (V) or pEMBL.2Awt (2A), HeLa cells (-), or poliovirus-infected HeLa cell (+) extracts were used as controls. The position of the recombinant poliovirus 2Apro is indicated by an arrow.
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Table II. Poliovirus 2Apro mutants obtained in S. cerevisiae

pb, mutated nucleotide; aas, amino acid change; ts, thermosensitivity; 2A, 2A expression in yeasts. pb, mutated nucleotide; aas, amino acid change; ts, thermosensitivity; 2A, 2A expression in yeasts.
Mutant pb aas ts 2A

1 ND*a ND  - +
2 right-arrow A G60R  - +
3 right-arrow T S66F + +
4 right-arrow T L39F  - +
5 right-arrow T N18K  - +
6 right-arrow A D135N + +
7 right-arrow A V119M + +
8 right-arrow A A22T  - +
9 right-arrow A C55Y  - +
10 right-arrow T S134L  - +
11 *2b - +  -
12 right-arrow A G110S  - +
13 right-arrow A G126D + +
14 right-arrow T Q24Stop  -  -
15 right-arrow A G121E  - +
16 right-arrow T Q24Stop  -  -
17 GG  right-arrow AA G100N  - +
18 right-arrow T S134L  - +
19 right-arrow Ac V9M  - +
20 right-arrow A G60R  - +
21 right-arrow T A125V + +
22 right-arrow A G121E  - +
2A WT - -  - +

a The plasmid pEMBL-2A* was not recovered in bacteria.
b Recombinant plasmid, 1 kilobase bigger than WT. No substitutions in 2A sequence.
c Substitution close to N terminus of the protein; mutant not subcloned.

To test if the ability of the yeast clones to grow in galactose medium was due to the absence of 2Apro synthesis, cell extracts were obtained, separated by SDS-polyacrylamide gel electrophoresis, and immunoblotted with anti-2A polyclonal antibodies (Fig. 1B). The majority of the clones expressed 2Apro upon galactose induction at higher levels than in yeast cells bearing the control plasmid pEMBL-2A. Only three clones (numbers 11, 14, and 16) did not synthesize any polypeptide that reacted with anti-2A antibodies. This is logical in the case of clones 14 and 16, which encode a truncated 2A protein of only 23 amino acids, whereas clone 11 still contains the 2Apro sequence but has lost its inducibility. This finding suggests that the various mutated 2Apro enzymes that are expressed in these clones have lost their capacity to inhibit yeast growth. Therefore, yeast cells represent an amenable genetic system in which to easily obtain poliovirus 2Apro variants.

Reconstitution of Poliovirus Genomes Containing the Mutated 2A Sequences

The next step in our studies was to reconstitute the entire poliovirus genomes bearing the different 2Apro variants. To this end, a subcassette containing the 3235-3953 poliovirus sequence was engineered. The 2Apro sequences obtained from the variant pEMBL-2A* isolates were initially cloned in this subcassette, and finally the different sequences were passed to plasmid pT7XLD containing the full-length poliovirus genome. Thus, 14 poliovirus genomes bearing different mutations in 2Apro were reconstructed. Poliovirus RNA was obtained from these mutants by in vitro transcription with T7 RNA polymerase. These RNAs were translated in a HeLa cell-free system, and the proteins synthesized were analyzed by SDS-polyacrylamide gel electrophoresis. Fig. 2A reveals that some of the mutants, namely numbers 3, 6, 7, 8, and, to a lesser extent, 12, show almost no P1-P2 precursor but produce significant amounts of 2A. The other mutants show various amounts of P1-P2 precursor and mature 2Apro. No P1 is detected in some of the mutants, such as 2, 5, 9, 13, and 15, but instead a protein band of higher molecular weight is detected that could correspond to P1-2A. Therefore, mutants in which P1 is absent should have a defect in the ability of 2Apro to cleave in cis the dipeptide between P1 and 2Apro.


Fig. 2. A, in vitro translation of poliovirus mRNA and proteolytic processing of the mutant poliovirus polyproteins. RNA transcripts prepared from pT7XLD reconstructed (WTr) and mutant derivatives of this plasmid were translated in HeLa cell-free lysates for 16 h at 30 °C. Translation reactions were also programmed with poliovirus virion RNA isolated from infected cells (V). Poliovirus-infected cells were used as control (P). The positions of poliovirus proteins are indicated. B, p220 cleavage by pT7XLD(2A*) transfection. HeLa cells were infected with VT7 (5 plaque-forming units/cell) and transfected with pT7XLD reconstructed (WTr) and mutant derivatives of this plasmid. C-, HeLa cells infected with VT7 and nontransfected. HeLa cells (-) or poliovirus-infected HeLa cell (+) extracts were used as controls. The positions of eIF-4G (p220) protein and cleavage products (CP) are indicated. C, genomic structure of poliovirus showing the 5' leader noncoding region and the coding regions for each protein.
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To further characterize the enzymatic capacities of the different mutated 2Apro enzymes, eIF-4G cleavage was examined by transfection of the different pT7XLD(2A*) plasmids and infection with a recombinant vaccinia virus that expresses the T7 RNA polymerase in HeLa cells. Fig. 2B shows that almost no cleavage of eIF-4G is detected with mutants 5 and 15, little cleavage of this initiation factor occurred with mutants 2 and 17, and the rest of the mutants clearly accomplished eIF-4G cleavage. Since mutants 5 and 15 do not show any sign of synthesis of mature 2Apro, it is possible that the 2Apro present in the P1-2A precursor is very inefficient to cleave eIF-4G. Alternatively, it is possible that 2Apro as such is devoid of eIF-4G cleavage activity. To investigate which of these two possibilities is correct, all the individual 2Apro variants were cloned in pTM1 and their activities analyzed (shown below).

The formation of lytic plaques upon transfection of HeLa cells with the different poliovirus RNA variants was examined (Fig. 3). Poliovirus plaques were obtained with mutants 3, 6, 7, 8, and 12 after 3 days of incubation, although these plaques have a reduced size compared with WT poliovirus RNA. Minute plaques arise with mutant 10 and extra-minute plaques with mutant 4 after 5 days of incubation, whereas no recovery of virus was possible from the rest of the mutants. Production of batches of mutants 4 and 10 without loss of their phenotype has not been possible. The attempts to recover reconstituted virus by electroporation of pT7XLD(2A*) plasmids have been done at least twice, with the plates incubated at 32.5, 37, and 39 °C. We were also unable to recover reconstituted virus from cells transfected by the Lipofectin procedure, although we successfully recovered mutants 3, 6, 7, 8, and 12 by this approach.


Fig. 3. Isolation of mutant polioviruses by electroporation of mutant viral RNAs. HeLa cells electroporated with the indicated viral RNAs were overlaid with 0.7% agar and incubated for the times indicated at 37 °C. Plaques were visualized by crystal violet staining. Similar results were obtained at 32.5 and 39 °C.
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Characterization of the Reconstituted Viable Poliovirus Mutants in 2Apro

To determine at the molecular level the defect that prevents poliovirus mutants 3, 6, 7, 8, and 12 from forming large plaques, several analyses were carried out. Initially, the capacity of these variant polioviruses to synthesize proteins was investigated. Fig. 4A shows that all five poliovirus mutants examined show defects in their capacity to synthesize proteins at control levels, although they all shut down host translation efficiently. To further characterize the level of poliovirus protein synthesis in cells infected with these polioviruses, cell extracts corresponding to 2 and 6 hpi were immunoblotted with an antibody against poliovirus protein 2C (Fig. 4B). Clearly, all five poliovirus variants synthesize lesser amounts of proteins 2BC and 2C compared with cells infected with control poliovirus. The reduced levels of poliovirus proteins produced by these five mutants may be due to synthesis of less viral RNA or to a lower capacity of the viral RNA to be translated. A poliovirus 2Apro mutant was described recently that showed defects in translation of the viral RNA, whereas genome replication was much less affected (24). Therefore, the amount of poliovirus RNA was examined at various times after infection with the five poliovirus mutants (Fig. 4C). Less viral RNA was present at 5 hpi in all five variants compared with WT poliovirus, whereas this amount was similar in all of them by 8 hpi. However, the cytopathic effect of WT poliovirus at this late time was much higher than with the rest of the mutated polioviruses (results not shown). These results indicate that viral genome replication is slower in the mutant polioviruses but reaches levels at 8 hpi similar to those present in WT-infected cells at 5 hpi. Despite this similarity, viral protein synthesis by the variant poliovirus is much lower than that observed with WT poliovirus.


Fig. 4. A, time course of protein synthesis. Autoradiogram of proteins synthesized in the 2Apro mutant-, WTR-, and WT (our laboratory stock)-infected cells (20 plaque-forming units/cell) during 1-h pulses with [35S]methionine. At the indicated times postinfection, the cells were recovered in sample buffer and processed as described under "Experimental Procedures." B, immunoblot against 2C protein of the same samples used in A. C, estimation of poliovirus positive strand RNA. Total RNA was extracted from WT (square )-, mutant 3 (black-square)-, mutant 6 (open circle )-, mutant 7 (bullet )-, mutant 8 (triangle )-, and mutant 12 (black-triangle)-infected cells (20 plaque-forming units/cell) at the indicated times postinfection. Dot blot analysis was performed with a biotinylated riboprobe to detect viral RNA as described under "Experimental Procedures." Only one of the four serial dilutions in the linear range of the signal is shown; the densitometric quantification from these blots is plotted in the graph.
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Poliovirus 2Apro has the capacity to activate the translation of mRNAs that contain the poliovirus 5' untranslated region (22). Therefore, we next examined the ability of the different poliovirus variants to enhance the translation of a reporter mRNA that bears the poliovirus 5' untranslated region before the luciferase sequence. Fig. 5A shows that transfection of poliovirus leader-luciferase mRNA in poliovirus-infected HeLa cells results in almost a 6-fold stimulation of luciferase synthesis compared with mock-infected cells. This activation is observed even when the infected cells are treated with guanidine, indicating that poliovirus genome replication is not necessary for this phenomenon to occur. The five poliovirus mutants assayed are defective to varying degrees at enhancing the translation of the leader-luciferase mRNA. Thus, transactivation by mutant 3 is about 1.2 times that of control uninfected cells, whereas mutant 6 enhances leader-luciferase translation by about 2.5 times. Our conclusion from these results is that all five poliovirus mutants are hampered in their ability to enhance leader-luciferase mRNA translation.


Fig. 5. Transactivation capacities of WT and 2A mutant viruses. HeLa cells were infected with WTR (WT) or 2Apro mutant viruses (50 plaque-forming units/cell), and 2 h later they were transfected with 2 µg of leader-luciferase RNA (Ventoso and Carrasco, 1995). Incubation was continued for 3 h (5 hpi), and the cells were recovered in lysis buffer. 3 mM guanidine (GUA) was added at 1 hpi as indicated. A, one-tenth of each sample was used to measure luciferase activity. The data shown are normalized to the protein content in each sample. B, immunoblot analysis of p220 of the same samples used in A. The positions of eIF-4G (p220) protein and cleavage products (CP) are indicated.
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Importantly, analysis of p220 cleavage in HeLa cells infected with the different poliovirus variants shows that all of them cleave p220 as efficiently as control poliovirus (Fig. 5B). This finding supports the idea that p220 cleavage is not sufficient to transactivate the poliovirus leader region. In addition, the presence of a WT poliovirus 2Apro is necessary for this phenomenon to take place.

The capacity of the different viable mutants to generate 3C' was also evaluated. The poliovirus precursor 3CD undergoes alternative cleavages: either it is processed by 3Cpro to yield 3Cpro plus 3D, or 3CD is cleaved by 2Apro to produce 3C' and 3D'. The function, if any, of these alternative cleavage products of 3CD remains obscure, since a poliovirus unable to generate 3C' and 3D' replicates well in culture cells (10). Fig. 6A shows that generation of 3C' is greatly diminished in cells infected with any of the five poliovirus variants assayed. Some 3C' is clearly visible, particularly in mutants 7 and 8, but the proportion of 3C' to 3CD or to 3Cpro in the five mutants is very small compared with that in HeLa cells infected with WT poliovirus.


Fig. 6. Thermosensitivity of the 2Apro mutant viruses. A, all the 2Apro mutants failed to mediate alternative cleavage of 3CD polypeptide. This defect is detected at three temperatures: 32.5 °C (a), 37 °C (b), and 39.5 °C (c). Extracts from WTR- and 2Apro mutant-infected cells (25 plaque-forming units/cell), 6 h postinfection, were analyzed by Western blotting with anti-3C serum. The positions of the poliovirus proteins are indicated by arrows. B, plaque morphology of WTR and mutant 6 poliovirus in HeLa cells. Plaques were visualized after staining with crystal violet 48 h (37 °C incubation) or 72 h (32.5 °C incubation) after infection. Similar results were obtained with mutants 3, 7, 8, and 12.
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Some of these 2Apro variants, such as 3, 6, and 7, showed a temperature-sensitive phenotype to induce the inhibition of yeast growth (Fig. 1A). Therefore, it was of interest to determine if this characteristic was preserved in the reconstituted polioviruses. For this purpose, eIF-4G cleavage (results not shown), generation of 3C' (Fig. 6A), and virus plaque formation (Fig. 6B) were assayed at different temperatures. None of the poliovirus variants studied showed a ts phenotype in any of these assays compared with the WT poliovirus.

Therefore, the five polioviruses mutated in 2Apro showed defects in viral protein synthesis, transactivation of leader-luciferase translation, and 3CD cleavage to generate 3C'. However, these poliovirus variants retained their capacity to cleave eIF-4G. A summary of these results is shown in Table III.

Table III. Summary of the activities of the different poliovirus 2Apro variants

2A*, different 2A variants; aas, amino acid change; PP, plaque phenotype (wt, wild type plaque size; sp, small plaque; mp, minute plaque; emp, extra-minute plaque; nv, nonviable); ts, thermosensitivity; cis, cis cleavage in cell-free system after translation; p220, nu  p220 cleavage; 3CD, 3CD cleavage; R, viral replication; Ta, transactivation activity; -, no activity; ++, maximal activity; ND, not determined. 2A*, different 2A variants; aas, amino acid change; PP, plaque phenotype (wt, wild type plaque size; sp, small plaque; mp, minute plaque; emp, extra-minute plaque; nv, nonviable); ts, thermosensitivity; cis, cis cleavage in cell-free system after translation; p220, nu  p220 cleavage; 3CD, 3CD cleavage; R, viral replication; Ta, transactivation activity; -, no activity; ++, maximal activity; ND, not determined.
Mutant aas Viruses/pT7XLD(2A*)
pTM1-2A*
PP ts cis p220 3CD R Ta p220 3CD Ta

2 G60R nv  -  - + ND ND ND +  - +
3 S66F sp  - ++ ++ +/- + +/- ++ +/- +
4 L39F emp  - + ++ ND ND ND ++  - +/-
5 N18K nv  -  -  - ND ND ND +/-  - +
6 D135N sp  - ++ ++ +/- + + ++ +/- +
7 V119M sp  - ++ ++ +/- + +/- ++ +/- +
8 A22T sp  - ++ ++ +/- + +/- ++ +/- +
9 C55Y nv  -  - ++ ND ND ND +  - +
10 S134L mp  - + ++ ND ND ND ++ +/- +/-
12 G110S sp  - ++ ++ +/- + + ++ +/- +
13 G126D nv  -  - + ND ND ND ++  - +/-
15 G121E nv  -  -  - ND ND ND +/-  - +/-
17 G100N nv  - +/- + ND ND ND +  - +/-
21 A125V nv  - +/- ++ ND ND ND +  - +/-
2A WT  - wt  - ++ ++ ++ ++ ++ ++ ++ ++

Activities of the 2Apro Variants Using the pTM1/VT7 System

To extend the analysis of the 2Apro activities not only to the mutated 2Apro that rendered infectious polioviruses but also to the rest of the 2Apro variants obtained in this work, the 14 2Apro sequences were cloned in plasmid pTM1 bearing a phage T7 promoter. Synthesis of 2Apro is achieved upon transfection and infection with a recombinant vaccinia virus that synthesizes the T7 RNA polymerase. Expression of the 2Apro gene occurs even when viral replication is inhibited by Ara-C (39). [35S]Methionine labeling under these conditions detected no vaccinia virus proteins, whereas 2Apro was synthesized at high levels (Fig. 7A). In fact, all 14 2Apro variants were synthesized to higher levels than WT poliovirus 2Apro in this system. The various degrees of 2Apro synthesis may correlate, at least in part, with their toxicity for the transfected cells. Thus, some mutated 2Apro variants, such as 2, 4, 5, 9, and 15, are synthesized to very high levels compared with those that yield viable polioviruses (3, 6, 7, 8, and 12). Analysis of eIF-4G cleavage in cells transfected by the 14 2Apro variants shows that all of them possess the ability to cleave eIF-4G, although some of them still leave significant amounts of intact eIF-4G (Fig. 7B). Densitometric analyses to quantitate the proportion of cleavage products versus intact eIF-4G indicate that mutants 2, 5, and 15 are the least active (<5% of the activity of WT 2Apro).


Fig. 7. Activities of the 2Apro variants in the pTM1/VT7 system. A, metabolic labeling with [35S]methionine of COS cells infected with VT7 (5 plaque-forming units/cell) and transfected with the pTM1-2A* plasmids in the presence of ara C (40 µg/ml) (39). After 16 h of infection cells were labeled and collected. C1, pTM1-transfected cells. B, immunoblot analysis with anti-eIF-4G antiserum of the same samples used in A. The positions of eIF-4G (p220) protein and cleavage products (CP) are indicated. C, immunoblot analysis with anti-3C antiserum showing the failure of the mutant 2A proteins to mediate alternative cleavage of the 3CD polypeptide. HeLa cells were co-transfected with pTM1.3CD and pTM1.2A* plasmids, and extracts were obtained 16 h posttransfection. C1, HeLa cells co-transfected with pTM1 and pTM1.3CD; C2, HeLa cells co-transfected with pTM1 and pTM1.2Awt; P, poliovirus-infected HeLa cells. The positions of the poliovirus proteins are indicated by arrows. D, transactivation capacities of mutant 2Apro proteins. HeLa cells were co-transfected with 1 µg of pT75'NCLUC RNA and 5 µg of pTM1.2C RNA (CONTROL) or 5 µg of each pTM1-2A* RNA as described previously (24). The data were corrected for the protein content of each sample and for the specific luciferase RNA transfected in each sample as estimated by dot blot analysis.
[View Larger Version of this Image (40K GIF file)]

The capacity of 2Apro to cleave 3CD in trans was assayed by double transfection of cells with pTM1 bearing the different 2Apro- and pTM1-expressing poliovirus 3CD. The proteolytic activity of 2Apro on 3CD becomes apparent by the generation of 3C' that is detected by immunoblot assays with a polyclonal anti-3C antiserum (Fig. 7C). Notably, only the 2Apro variants able to reconstitute viable poliovirus generate 3C', albeit at lower levels than WT 2Apro. None of the other 2Apro mutants were able to produce even minimal amounts of 3C' that could be detected after longer exposures of the x-ray film.

Finally, the transactivation capacity of the 2Apro variants on a leader-luciferase mRNA were assayed. To this end, both the RNA encoding 2Apro and the leader-luciferase mRNA were transfected into HeLa cells by the Lipofectin method (24). As occurred with the 3CD cleavage activity, all 14 2Apro variants showed defects in transactivating the synthesis of luciferase (Fig. 7D). Two of them, showing a higher transactivation capacity (mutants 3 and 6), were also able to produce small plaque polioviruses. However, the rest of the 2Apro mutants able to reconstitute polioviruses (mutants 7, 8, and 12) had transactivation activities similar to another 2Apro variant incapable of producing viable polioviruses (mutant 2). Our conclusion from these experiments is that the capacity of the various 2Apro to cleave eIF-4G or 3CD differs. Moreover, some 2Apro variants produce significant amounts of eIF-4G cleavage products but are devoid of transactivation activity. This result agrees with the idea that the simple cleavage of eIF-4G is not sufficient to enhance the translation of a mRNA bearing the poliovirus leader sequence. Finally, there is a good correlation between the ability of 2Apro to generate 3C' (and 3D') and its capacity to produce viable polioviruses. A summary of these results is shown in Table III.


DISCUSSION

S. cerevisiae as a System for Obtaining Poliovirus 2Apro Variants

The isolation and characterization of gene variants is of primary importance for understanding poliovirus molecular biology (31). Three main strategies have been used to isolate poliovirus variants: selection of spontaneous mutants with a given phenotype (plaque size, drug resistance, etc.), random mutagenesis followed by selection, and site-directed mutagenesis (31). Random mutagenesis of a given poliovirus gene combined with a system for assaying a particular function facilitates the isolation of mutant viruses with phenotypes of interest. Some systems have been described to detect rhinovirus 2Apro and coxsackievirus 3Cpro variants using E. coli and S. cerevisiae, respectively (43, 44). Both assays are based on the so-called "proteinase-trapping" technique in which hybrid variants between the picornavirus protease and B-galactosidase devoid of cis cleavage activity are selected. However, the mutated proteins thus selected have not yet been analyzed in mammalian cells or in reconstituted viruses. Other functional analyses of human viral proteins in yeast cells have been reported recently (34, 35, 45-49), but again, those studies were restricted to yeast cells.

The versatile and simple genetic system described in this work for selecting poliovirus 2Apro mutants unable to block yeast growth will provide new 2Apro variants that will offer further insight into the structure-activity relationship of this protease and provide more details about its functioning during the poliovirus lytic cycle. The random mutagenesis method of the 2Apro gene used in this work yields approximately 1% of 2Apro mutants; about 25% of those have a ts phenotype in yeast cells. Notably, most of the 2Apro variants obtained contain point mutations, allowing the characterization of new regions in this poliovirus protease involved in substrate recognition. The isolation of many more 2Apro variants using this approach would provide not only a more detailed characterization of the domains in 2Apro but also the possibility of reconstituting ts poliovirus mutants. However, thus far, our attempts in this direction have been unsuccessful. Another still unexplored possibility for this approach is to screen for second site revertants of some of the 2Apro variants. This would provide additional data on the interactions of different regions of the 2Apro.

Although poliovirus 2Apro blocks S. cerevisiae growth strongly, the exact target of its activity remains unknown (32). Although gene expression in yeast cells is clearly affected, the target recognized by 2Apro is probably unrelated to mammalian eIF-4G (35). It is possible that eIF-4G and the yeast target of 2Apro share common structural motifs that make them both substrates for 2Apro. Certainly, the 2Apro variants isolated from yeast cells show defects in substrate recognition and cleavage when tested in mammalian cells. This finding validates the use of yeast cells as an assay to isolate 2Apro variants with defects in poliovirus growth and 2Apro activity in human cells. One limitation of the system described for generating different 2Apro variants is that it relies on the same selective pressure: the toxicity of 2Apro for yeast cells. However, the different 2Apro mutants obtained can be classified in different groups: some generate viable polioviruses, others still retain full eIF-4G activity, and a few are deficient in all the activities assayed. In particular, all the mutant viruses obtained are deficient in 3CD cleavage and transactivation of the poliovirus IRES region. Most likely, the basis of the genetic selection in yeast is related to these phenomena in molecular terms. In conclusion, these findings show that the use of this selection system would provide a wide range of 2Apro mutants. Moreover, it is possible that varying the conditions of yeast growth and the composition of the medium would also influence the selective pressure and the types of 2Apro variants isolated.

Poliovirus 2Apro as a Multifunctional Enzyme

Poliovirus 2Apro is not only involved in cis and trans cleavage of poliovirus protein precursors but also degrades cellular substrates (2). In addition, 2Apro is a translational activator of mRNAs bearing the poliovirus IRES sequence (22) and is involved in viral RNA replication (26, 27). Some of these 2Apro activities appear to be associated in the protease variants analyzed. Thus, the five viable poliovirus mutants analyzed in this work (namely 3, 6, 7, 8, and 12) show a small plaque phenotype, defects in the transactivation activity, and reduced viral protein synthesis. The 2Apro variants 4 and 10 produce viruses with the minute plaque phenotype, show defects in cis cleavage, and accumulate P1-P2 and P1-2A precursors (see Fig. 2A); however, they cleave eIF-4G as efficiently as the other five viable polioviruses. All viable polioviruses are able to cleave P1-2A efficiently but cleave 3CD very ineffectively. Curiously, all of the 2Apro variants that did not give rise to viable viruses were unable to cleave 3CD. This finding is puzzling, since no role has yet been assigned to 3C' and 3D' in the poliovirus replication cycle. Moreover, the mutation of this alternative cleavage site in 3CD has no consequences for virus growth (10). Our finding, however, could suggest either that the 3CD cleavage products or the 2Apro proteolytic capacity responsible for this cleavage is essential for virus viability. The fact that the 3CD cleavage site recognized by 2Apro is maintained after passage in cultured cells, coupled with our present findings, could foster additional work on the potential function of 3C' and 3D'.

Notably, all the 2Apro variants obtained in this work cleaved eIF-4G, although to varying extents. The mutants that affected eIF-4G less were those that processed P1-2A less efficiently. On the other hand, P1-2A and 3CD are left almost intact by some 2Apro mutants, whereas eIF-4G is still hydrolyzed to some extent. These findings indicate that 2Apro has a region involved in substrate recognition manifested not only in yeast cells but also in other substrates of mammalian cells. Mutations in this region do not have exactly the same consequences for recognition and cleavage of different substrates. It would be of interest in the future to isolate 2Apro mutants totally devoid of eIF-4G cleavage activity.

The finding that some 2Apro variants do not transactivate the IRES of leader-luciferase mRNA but still cleave eIF-4G indicates that generation of the C terminus of eIF-4G alone does not suffice for the stimulation of translation of poliovirus mRNA to occur. These results add support to the previous findings from our group that transactivation requires not only eIF-4G cleavage but also intact 2Apro (24). All the 2Apro variants generated are deficient in proteolytic and transactivation capacity. This could suggest that for transactivation to occur the level of 2Apro proteolytic activity needs to pass a threshold to cleave a given cellular substrate. The possibility that this poliovirus protease directly participates in the translation of poliovirus RNA is also an attractive possibility. Further work to elucidate the exact mechanism by which some picornavirus proteases enhance translation is necessary.

2Apro Structure-Activity Relationships

The three-dimensional structure of picornaviral 2Apro has been modeled based on that of alpha -lytic proteinase (3, 6). The three-dimensional structure of 2Apro reported by Yu and Lloyd (3) was based on the structure of smaller bacterial serine proteases that in principle lack some of the functions assigned to picornavirus 2Apro, such as the transactivation activity and participation in RNA replication. Until this theoretical modeling is confirmed by direct determination of the three-dimensional structure, mutational analysis of 2Apro represents a good approach through which to gain insight into its structure-function relationships. Using the three-dimensional model of 2Apro, Macadam et al. (25) localized most of the mutations observed in their study in two clusters near the surface of the protein and away from the active site, suggesting a direct interaction between these regions of 2Apro and the 5' noncoding sequence of poliovirus RNA.

The substitutions of the 2Apro variants that we have found cluster near the catalytic triad. Most of the mutations concentrate in the putative mouth where the substrate binds. The schematic depiction of the different poliovirus 2Apro variants in the three-dimensional model of 2Apro is given in Fig. 8. Four of five 2Apro mutants that show a ts phenotype in yeast cells are grouped in a straight line between amino acids 119 and 136. Conceivably, this region of the protease undergoes conformational changes induced by temperature that influence substrate recognition in yeast cells. The two mutants with stronger defects in cleavage in cis and proteolysis of eIF-4G map close to the catalytic site: mutant 5 in the putative active site and mutant 15 in the middle of the molecule. Both substitutions involve charged amino acids, suggesting that these two residues play a crucial role in the 2Apro activity. The three additional 2Apro mutants lacking cis cleavage capacity but with some activity on eIF-4G are also located in a straight line that overlaps part of the ts region defined above, further strengthening the idea that this region of 2Apro participates in substrate recognition. Therefore, the analysis of multiple random mutants obtained by different genetic assays (2A revertants for mutations in the 5' noncoding region (25) or noncytotoxic 2A mutants in yeast (as shown in this study)) could allow the identification of the different functional domains present in the poliovirus 2Apro.


Fig. 8. Primary and secondary structure of poliovirus protease 2Apro. Sequence and three-dimensional model of the alpha -carbon chain of poliovirus protease 2Apro based on the structure of small bacterial serine proteases (3, 6). The putative catalytic triad (residues 20, 38, and 109, bold type) is shown as well as the different mutations found in our genetic assay (black circles and outline type; ts phenotype, black open circles and italic outline type). The 2Apro sequences of various enteroviruses and rhinoviruses were obtained from the GenBankTM/EMBL data bank and analyzed using the University of Wisconsin Genetics Computer Group programs Pileup and Plotsimilarity. The regions of the protein with a higher degree of similarity between different picornaviruses are shaded. Most of the mutations that we have found in our genetic assay localize to conserved regions of the protein, with only two exceptions: mutant 3, which has a Phe in place of the normal Ser or Tyr (suggesting that the hydroxyl radical at this position plays a crucial role for 2Apro structure or activity), and mutant 17, which has Asn in a position always occupied by an amino acid with a small side chain (Gly or Ala).
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FOOTNOTES

*   This work was supported in part by a grant from the Dirección General de Investigación Científica y Tecnológica (BIO 94-0148) and an institutional grant to the Centro de Biología Molecular from the Fundación Ramón Areces.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.
Dagger    Holder of a Formacion de Personal Investigador fellowship.
   Holder of a Comunidad Autónoma de Madrid fellowship.
§   To whom correspondence should be addressed: Fax: 34-1-3974799.
1   The abbreviations used are: 2Apro, poliovirus protease 2A; IRES, internal ribosomal entry site; WT, wild type; hpi, hours postinfection; ts, temperature-sensitive.

ACKNOWLEDGEMENTS

The expert technical assistance of M. A. Sanz is acknowledged. We thank Eckard Wimmer for generously providing HeLa cell extracts for in vitro translation.


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