©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Poliovirus Protein 2C Contains Two Regions Involved in RNA Binding Activity (*)

Pedro L. Rodrguez (§) , Luis Carrasco (¶)

From the (1) Centro de Biologia Molecular, Universidad Autónoma de Madrid-Consejo Superior de Investigaciones Cientficas, Universidad Autónoma de Madrid, Cantoblanco 28049, Madrid

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Poliovirus protein 2C is involved in poliovirus RNA replication, although the exact function of 2C is still unknown. Recently, it was shown that 2C can be purified to high levels when expressed as a fusion protein with maltose-binding protein (MBP). Evidence was presented that 2C has ATPase and GTPase activities; preliminary results also indicated that 2C interacts with RNA (Rodrguez, P. L., and Carrasco, L. (1993) J. Biol. Chem. 268, 8105-8110). In the present study, 20 variants of 2C have been generated, and their NTPase and RNA binding activities were analyzed. Moreover, an easy procedure to obtain genuine 2C after factor Xa cleavage of an MBP-2C fusion protein is described. This work has determined that 2C has two regions involved in RNA binding: a NH-terminal region located between amino acids 21 and 45 and a COOH-terminal region involving an Arg-rich region located between amino acids 312 and 319. Deletion of either the NH- or COOH-terminal RNA-binding region abolishes RNA binding. Deletion of an internal region of protein 2C that includes the nucleotide-binding motif does not affect RNA binding, whereas this deletion destroys ATPase and GTPase activities. Therefore, the NTPase activity and the RNA binding capacity of protein 2C are located in different regions of the molecule.


INTRODUCTION

The 11 mature proteins that are encoded by the RNA poliovirus genome arise by cleavage of a polyprotein that is initially processed at the polysomal level to generate three polypeptides, P1-P3 (1, 2) . Further cleavage of P1 produces the four structural proteins that form part of mature virus particles (3) , whereas hydrolysis of P2 and P3 generates seven polypeptides that are involved in poliovirus vegetative functions (4, 5) . The first cleavage of P2 gives rise to 2Aand the precursor 2BC, and the latter is then further hydrolyzed to produce 2B and 2C (2) . In addition to forming the mature products 2B and 2C, the precursor 2BC may itself participate in certain processes in the poliovirus replication cycle (5) . Support for this idea comes from experiments that show that the insertion of the encephalomyocarditis virus 5`-untranslated region between 2B and 2C is lethal for poliovirus. In contrast, if the same region is inserted between 2Aand 2B, a viable poliovirus-encephalomyocarditis hybrid is formed (5, 6) . The precise functions of 2B and 2BC in the poliovirus replication cycle remain to be defined, although it is known that poliovirus with mutations in the 2B gene is defective in its ability to replicate the poliovirus genome (7, 8) . Some 2B mutants cannot be complemented in trans and even interfere with wild-type poliovirus, thus suggesting that the mutated 2B protein can override the normal functioning of 2B (8) .

Poliovirus protein 2C is a 329-amino acid polypeptide that contains a typical NTP-binding domain (9) . The sequence GSPGTGKS (amino acids 129-136) forms the A site that is involved in the interaction of the protein with phosphate. The B site, comprising the DD motif at amino acids 176-177, could interact with magnesium. Point mutations expressed in these two sites of protein 2C lead to an impairment of viral RNA replication and are thus lethal for poliovirus (10, 11) . Biochemical studies using isolated poliovirus protein 2C have shown that this protein possesses NTPase activity (10, 12) . Moreover, there is some evidence that suggests that 2C is an RNA-binding protein since gel retardation experiments indicate that it interacts with a partial double-stranded RNA molecule (12) . The exact function that 2C plays in the poliovirus replication cycle remains to be determined. Two lines of evidence suggest that the activity of 2C is necessary for viral genome replication. Mutant forms of this protein are unable to synthesize viral RNA (11, 13, 14) . In addition, guanidine, a compound that selectively blocks poliovirus RNA synthesis, interferes with the mode of action of 2C (15) , although the mechanism(s) underlying this effect have not been elucidated. It has been suggested, however, that guanidine hinders the conformational change that normally follows the NTP binding and/or hydrolysis carried out by 2C (16) . Alternatively, the compound may block the interaction of 2C with vesicular membranes (17) . In accord with this idea, there are indications that 2C might attach the poliovirus replication complexes to membranous vesicles that proliferate during poliovirus infection (17, 18) . For example, isolation of poliovirus RNA replication complexes from infected cells provides a membranous system that actively synthesizes viral RNA (4) . Detergent treatment of this complex reduces its RNA synthetic activity although 2C-related proteins still remain attached to the RNA (18) , thus suggesting that 2C or 2BC can interact directly with the viral genome and that membranes play an important part in poliovirus genome replication. In fact, inhibition of lipid synthesis or interference with the vesicular system profoundly depresses poliovirus RNA synthesis (19, 20, 21) . The addition of cerulenin, an inhibitor of phospholipid synthesis, or brefeldin A, a macrolide antibiotic that interferes with the vesicular system, immediately arrests poliovirus genome replication in infected cells (19, 20, 22) . Therefore, membrane proliferation and vesicular traffic are both necessary for the replication of poliovirus nucleic acids. We have already suggested that 2C could mediate the traffic of viral RNA through the vesicular system (12) , and we now provide evidence that two specific regions within the protein control its interaction with viral RNA.


MATERIALS AND METHODS

General Recombinant DNA Protocols Plasmids encoding the different fusion proteins between MBP() and 2C or its variants were constructed by standard molecular cloning procedures (23) . The methods used for the generation and purification of polymerase chain reaction products were performed as described previously (12) , and the template pT7XLD was used throughout, except where otherwise indicated. The 5`-primers contain different coding sequences for 2C (sense primers). The different sequences of 2C encoded by the 3`-primers are complementary to the coding sequence (antisense primers). The 3`-primers add two stop codons (boldface (see sequences below)) and a HindIII site (underlined) to the sequence that ends at the indicated nucleotide of the 2C gene. The regions of 2C amplified by PCR were sequenced by the dideoxynucleotide chain termination method (23) . Construction of pMal-c.2C Plasmids for the Expression of Poliovirus Protein 2C Deletion Mutants in the Form of MBP-2C Fusion Proteins Plasmids encoding the different fusion proteins were constructed using the pMal-c vector. The construction of pMal-c.2C has been described previously (12) . Construction of 2C Variants with Carboxyl-terminal Deletions MBP-2C-(1-161)-A DNA fragment encompassing nucleotides 4124-4606 of the 2C gene was generated by PCR. The 5`-primer used, with the SmaI site underlined, was 5`-2C.B2 (5`-GGC CGG CCCGGG GAC AGT TGG TTG AAG AAG). The 3`-primer used,with the HindIII site underlined and stop codons in boldface, was 3`-2C.4606 (5`-GGG CCC AAGCTT ACTATGGA TCC GGG GGTAGC GAG TAC). The PCR product was subjected to SmaI- HindIII double digestion and ligated to StuI- HindIII-digested pMal-c. MBP-2C-(1-255)-This construction has been described previously (12) . Briefly, pMal-c.2C was digested with XbaI and treated with Klenow enzyme. This generates a deletion mutant of MBP-2C with the relevant gene containing a serine codon and a nonsense codon added after that coding for amino acid 255 of 2C. MBP-2C-(1-297)-A DNA fragment encompassing nucleotides 4424-5014 of the 2C gene was generated by PCR. The 5`-primer used was 5`-2C.4424 (5`-ATACAGAAACTAGAGCATACT). The 3`-primer used was 3`-2C.5014 (5`-GGGCCCAAGCTTACTAGGAAGATTTGTCCATTAATTG). The PCR product was subjected to XbaI- HindIII double digestion and ligated to XbaI- HindIII-digested pMal-c.2C. MBP-2C-(1-319)-A DNA fragment encompassing nucleotides 4424-5080 of the 2C gene was generated by PCR. The 5`-primer used was 5`-2C.4424. The 3`-primer used, with the HindIII site underlined, was 3`-2C.5080 (5`-GGG CCC AAG CTT ACT AGTTG GATCTT CTG TTT CTC TCA TT). The PCR product was subjected to XbaI- HindIII double digestion and ligated to XbaI- HindIII-digested pMal-c.2C Construction of 2C Variants with Amino-terminal Deletions MBP-2C-(17-329) and MBP-2C-(21-329)-These mutants were constructed by digesting pMal-c.2C with SphI followed by treatment for different periods of time (30, 60, and 90 s) with Bal-31 nuclease (47) . Deletions with 60 nucleotides removed were selected after analysis with restriction enzymes. After transformation of Escherichia coli DH5 cells, clones that express proteins with a slight but appreciable difference in mobility with respect to that of MBP-2C were isolated. After sequencing of such clones, two mutants were selected with 16 and 20 amino acids, respectively, deleted from the NHterminus. MBP-2C-(42-329)-A DNA fragment encompassing nucleotides 4246-4606 of the 2C gene was generated by PCR. The 5`-primer used, with the SacI site underlined, was 5`-2C.4246 (5`-GGC CGG GAG-CTC AGAT AAG TTG GAA TTC GTA A CA). The boldface C at position 4261 is a silent change from T to C that introduces a new EcoRI site. The 3`-primer used was 3`-2C.4606. The PCR product was subjected to SacI- BamHI double digestion and ligated to SacI- BamHI-digested pMal-c.2C. MBP-2C-(101-329)-A DNA fragment encompassing nucleotides 4424-5110 of the 2C gene was generated by PCR. The 5`-primer used was 5`-2C.4424. The 3`-primer used was 3`-2C.B2 (5`-GGG CCCAAG CTT ACT ATTGA AAC AAA GCC TCC ATA C). The PCR product was blunt-ended with Klenow enzyme, further digested with HindIII, and ligated to StuI- HindIII-digested pMal-c. MBP-2C-(173-329)-A DNA fragment encompassing nucleotides 4640-5110 of the 2C gene was generated by PCR. The 5`-primer used was 5`-2C.4640 (5`-GTGATT ATGGTCGACCTGAAT). The boldface T at position 4650 is a change from A to T that introduces a new SalI site and generates the mutation D176V. The 3`-primer used was 3`-2C.B2. The PCR product was blunt-ended with Klenow enzyme, further digested with HindIII, and ligated to StuI- HindIII-digested pMal-c. MBP-2C-(201-329)-A DNA fragment encompassing nucleotides 4724-5110 of the 2C gene was generated by PCR. The 5`-primer used was 5`-2C.4724 (5`-CCACCCATGGCATCCCTGGAGGAGAAAG).The 3`-primer used was 3`-2C.B2. The PCR product was blunt-ended with Klenow enzyme, further digested with HindIII, and ligated to StuI- HindIII-digested pMal-c. MBP-2C-(234-329)-A DNA fragment encompassing nucleotides 4823-5110 of the 2C gene was generated by PCR. The 5`-primer used was 5`-2C.4823 (5`-CACAGTGATGCATTAGCCAGGCGCTTTGCGTTCG). The 3`-primer used was 3`-2C.B2. The PCR product was blunt-ended with Klenow enzyme, further digested with HindIII, and ligated to StuI- HindIII-digested pMal-c. Construction of NH -terminal Deletions from MBP-2C-(1-161) MBP-2C-(21-161)-PCR was carried out with malE primer (New England Biolabs Inc.) and 3`-2C.4606 using pMBP-2C-(21-329) as template. The PCR product was digested with BamHI and ligated to pMBP-2C-(1-161) previously digested with BamHI and treated with calf intestinal alkaline phosphatase. MBP-2C-(45-161)-PCR was carried out with malE primer and 3`-2C.4606 using pMBP-2C-(42-329) as template. The PCR product was EcoRI- HindIII-digested and ligated to pMal-c previously digested with the same enzymes. MBP-2C-(101-161)-PCR was carried out with malE primer and 3`-2C.4606 using pMBP-2C-(101-329) as template. The PCR product was digested with BamHI and ligated to pMBP-2C-(1-161) previously digested with BamHI and treated with calf intestinal alkaline phosphatase. Construction of pMal-c2.2C pMal-c2.2C is an expression plasmid encoding an MBP-2C fusion protein that, upon cleavage with factor Xa, renders 2C with five extra glycines at the NHterminus. The pMal-c2 vector was purchased from New England Biolabs Inc. The 2C coding region was amplified by PCR. The 5`-primer used was 5`-2C.B3 (5`-GGTGGTGGTGGTGGTGGTGACAGTTGGTTGAAGAAG). This primer contains five GGT codons, encoding five Gly residues, placed in frame with the first codon of 2C. The 3`-primer used was 3`-2C.B2. The PCR product was blunt-ended with Klenow enzyme, further digested with HindIII, and finally cloned into XmnI- HindIII-digested pMal-c2. Construction of Internal Deletions from pMal-c2.2C MBP -2C-(161-188)-pMal-c2.2C was digested with XmnI and BamHI, blunt-ended with Klenow, and self-ligated. MBP -2C-(129-172)-This plasmid was constructed using the methodology of overlap extension described by Higuchi et al. (48) . Three different PCRs were carried out. PCR1 was carried out with primers 5`-2C.B2 and 3`-2C.D129-172 (5`-CAGGTCGACCATAATCAC-ATGTACTAGCAAACATAC). PCR2 was carried out with primers 5`-2C.4640 and 3`-2C.B2. The overlap created by PCR1 + PCR2 was extended in a PCR3 carried out with primers 5`-2C.B2 and 3`-2C.B2. This PCR3 product was SphI- XbaI-digested and ligated to pMal-c2.2C previously digested with the same enzymes. Construction of pMal-c2.2C Plasmids for the Expression of MBP -2C-(1-313), MBP -2C-(1-315), and MBP -2C-(1-319) Fusion Proteins A DNA fragment encompassing nucleotides 4424-5080 of the 2C gene was generated by PCR. The 5`-primer used was 5`-2C.4424. The 3`-primer used was 3`-2C.5080Stop (5`-GGGCCCAAGCTTACTAGTTGGATC(A/T)TC(A/T)GTTTC(A/T)CTCATT). This primer is an oligonucleotide mixture that introduces stop codons in place of arginines at residues 314, 316, and 317 of 2C. The PCR product was submitted to XbaI- HindIII double digestion and ligated to XbaI- HindIII-digested pMal-c2.2C Purification of the Fusion Proteins and Cleavage with Factor Xa E. coli DH5 cells, transformed with the plasmids encoding the different deletion mutants, were grown in 20 ml of LB medium containing 0.2% glucose and 100 mg/ml ampicillin to an absorbance at 600 nm of 0.6. Induction and purification of the fusion proteins were as described (12, 24) . MBP-2C and its derivatives were cleaved by following two approaches. After elution, the purified proteins were cleaved in the elution buffer containing 1 mM CaClat a factor Xa/fusion protein ratio of 1:100. Alternatively, MBP-2C was cleaved while it was attached to the amylose resin. For this purpose, instead of eluting the fusion protein with maltose, the 1.5 ml of amylose resin (to which the fusion protein had been bound as described above) was resuspended in factor Xa cleavage buffer containing 1 mM CaCland 5 µg of factor Xa. The reaction mixture was incubated by rocking in an Eppendorf tube at 4 °C for 2 h. Then it was transferred to the column. The amylose resin was washed with factor Xa cleavage buffer and with factor Xa cleavage buffer containing 10 mM maltose. Although this protocol might have provided a single-step purification of 2C, after its cleavage, the protein eluted with factor Xa cleavage buffer containing 10 mM maltose, thereby indicating that MBP and uncleaved MBP-2C were also present. The noncanonical products resulting from cleavage with factor Xa were also present in this eluate. Nonradioactive Northwestern Assay The purified proteins were submitted to SDS-PAGE under standard conditions (25) . Electrophoresis was performed on 15% acrylamide gels at a constant current of 30 mA applied for 6 h. The proteins were transferred overnight from the gel to a nitrocellulose membrane using the wet electrotransfer protocol as described (25) . The details of the nonradioactive Northwestern and Western immunoblot assays have been described recently (24) . ATPase and GTPase Activity Assays Assays were performed as described previously (12) using 0.3 µg of the different purified fusion proteins.


RESULTS

Expression of Poliovirus Protein 2C and 2C Deletion Mutants in the Form of MBP-2C Fusion Proteins

The DNA sequence encoding poliovirus protein 2C was cloned into the pMal-c vector in order to generate a fusion protein between 2C and maltose-binding protein (MBP-2C) as described (12) . A number of poliovirus protein 2C deletion mutants were generated as depicted in Fig. 6A. Most of the mutants were made by employing PCR and using the primers and procedures detailed under ``Materials and Methods.'' Two mutants, MBP-2C-(17-329) and MBP-2C-(21-329), were obtained by Bal-31 nuclease digestion. One mutant, MBP-2C-(1-255), was obtained from pMal-c.2C as detailed under ``Materials and Methods.''


Figure 6: A, schematic diagram of the poliovirus protein 2C deletion mutants expressed in E. coli as MBP-2C fusion proteins: summary of their ability to bind RNA and NTPase activity. ND, not determined. B, mutants expressed as MBP-2C fusion proteins: summary of their ability to bind RNA. C, linear map of poliovirus protein 2C. Putative amphipathic regions (41), the NTP-binding motif ( NTPBM), the position of guanidine mutants with a change at Asn( Gua), and the Arg-rich region at the COOH terminus are indicated. The Arg-rich regions located at the COOH termini of the following picornaviral 2C proteins are indicated: bovine enterovirus ( BEV); rhinovirus ( HRV) types 89, 2, and 1B; encephalomyocarditis virus type b ( EMCVb); and coxsackie b virus type 1 ( COXb1).



The different MBP-2C mutants were expressed in E. coli, and the corresponding fusion proteins were purified as described under ``Materials and Methods.'' Fig. 1( A and B) illustrates Ponceau S staining (after SDS-PAGE and transfer to nitrocellulose) of the different MBP-2C fusion proteins. A major protein band is observed in each case that has a calculated molecular weight that corresponds to that of MBP-2C or its variants. The major bands react with an antiserum prepared against 2C, indicating that they contain 2C-related sequences that are immunologically recognized within the fusion protein (data not shown).


Figure 1: RNA binding activity of poliovirus protein 2C deletion mutants expressed in E. coli as fusion proteins with MBP. A, Ponceau S staining after wet electrotransfer of proteins to nitrocellulose sheets; B, same procedure described in A carried out with NH-terminal truncations; C, Northwestern binding assay using biotin-labeled poliovirus (amino acids 2099-4600) RNA as the probe; D, same assay described in C carried out with NH-terminal truncations.



RNA Binding Activity of 2C Mutants

When 2C is expressed as a fusion protein with maltose-binding protein, it gives rise to a retardation complex after association with a partially double-stranded RNA substrate (12) . Although the fusion protein MBP-2C, which contains genuine poliovirus protein 2C, can be shown by this assay to bind RNA, MBP alone is devoid of this activity. Furthermore, the deletion mutant MBP-2C-(1-255), lacking the COOH-terminal region between amino acids 256 and 329, also loses the capacity to interact with RNA (12) . Since these results indicate that protein 2C has at least one region that is involved in RNA binding, we have extended this analysis in order to define more precisely where this region(s) is located by employing a Northwestern assay (see ``Materials and Methods'') recently developed in our laboratory (24) . Tests on the RNA binding capacities of the different 2C deletion mutants generated (see Fig. 1, C and D) revealed that deletions of 32 or 74 amino acids (mutants 1-297 and 1-255, respectively) at the carboxyl terminus rendered 2C variants incapable of binding RNA. Therefore, it appears that the last 32 amino acids of the carboxyl terminus control the interaction of 2C with RNA. A smaller deletion of 10 amino acids has no influence on the RNA binding capacity of 2C, thereby locating the RNA binding activity to residues 298-319. A larger deletion at the COOH terminus (mutant 1-161) restores the capacity of protein 2C to bind RNA. This finding agrees well with the RNA binding properties of other proteins of the RNA-binding family, where small deletions abrogate RNA binding, but larger deletions restore this activity (26, 27, 28) . On the other hand, deletion of the first 16 or 20 residues from the amino terminus of 2C does not abolish RNA binding; longer deletions of 41, 100, or 172 residues at this terminus completely destroy this capacity. These findings suggest that two regions in 2C participate in RNA binding: one located at the amino terminus and the other located at the carboxyl terminus. Additional evidence for the existence of two regions within 2C that have RNA binding activity comes from results obtained with MBP-2C-(21-161), MBP-2C-(1-161), MBP-2C-(201-329), and MBP-2C-(234-329). The first 161 amino acids of 2C confer RNA binding ability upon the fusion protein MBP-2C-(1-161), showing that this amino-terminal segment of 2C participates in RNA binding. Moreover, deletion of the first 20 amino acids (MBP-2C-(21-161)) does not affect this property. The carboxyl terminus of 2C is also involved in the phenomenon since the last 129 and 96 amino acids of 2C also confer RNA binding activity to the fusion proteins MBP-2C-(201-329) and MBP-2C-(234-329), respectively. These results are in agreement with other findings on RNA-binding proteins. MBP alone does not bind RNA, but it acquires this ability if it is fused with the heterogeneous nuclear RNP U protein or with a smaller fragment of this protein containing only 110 amino acids located at the carboxyl terminus (27) . Eukaryotic initiation factor 4B, expressed as a fusion protein with glutathione S-transferase, also binds RNA (29) , as does delta hepatitis antigen fused with TrpE (30) .

Since 2C is also endowed with ATPase and GTPase activities (12) , the different protein variants used in this work were tested for these activities. Fig. 2shows that 2C mutants lacking 32 or 74 amino acids from the carboxyl terminus still retain NTPase activity. A similar effect is observed by deleting the first 16 or 20 amino acids located at the amino terminus, whereas longer deletions of 41 or 100 residues in this region abolish the NTPase activities of 2C. Therefore, sequences located between residues 21 and 255 of 2C are essential for NTPase activity, and these results agree well with the fact that the GKS motif is located at positions 134-136 of 2C, whereas the DD motif is located at positions 176-177.


Figure 2: NTPase activity of poliovirus protein 2C deletion mutants expressed in E. coli as fusion proteins with MBP. A, ATPase activity; B, GTPase activity. Lane 1, MBP-2C-(1-255); lane 2, MBP-2C-(1-297); lane 3, MBP-2C-(1-319); lane 4, MBP-2C; lane 5, MBP-2C-(17-329); lane 6, MBP-2C-(21-329); lane 7, MBP-2C-(42-329); lane 8, MBP-2C-(101-329); lane 9, MBP-2C-(173-329); lane 10, MBP-2C-(201-329); lane 11, MBP-2C-(234-329).



Cloning and Expression of an MBP -2C Variant Fusion Protein That Is Cleaved to Generate Genuine 2C

We previously reported that cleavage of MBP-2C by protease Xa gives rise to a cleaved product of 2C since factor Xa recognizes, with high efficiency, an internal region of 2C, rather than the MBP-2C junction (12, 49) , despite the fact that 2C does not contain the IEGR tetrapeptide that is normally required for protease Xa activity (31) . We reasoned that the addition of a less structured sequence adjacent to the correct recognition site would increase the accessibility of the protease to this position. For example, improved thrombin cleavage occurs after the addition of five glycines after the cleavage site (32) . Similarly, specific cleavage of MBP-2C by factor Xa might be enhanced by connecting the two proteins MBP and 2C by a glycine-rich arm. To test if such an MBP-2C fusion protein would liberate 2C upon Xa cleavage, the construction indicated in Fig. 3was made. Cleavage of MBP-2C produces a significant amount of 2C (Fig. 3 B) that migrates electrophoretically in the same way as genuine 2C made in poliovirus-infected cells and is recognized by specific antiserum against 2C (Fig. 3 C). This new cloning strategy allows a rapid assay for the RNA binding capacity of the proteins expressed because in the RNA binding assay described in this work, the proteins are separated by SDS-PAGE. This enables rapid analysis of the different mutated 2C proteins not only in fusion with other proteins, but also as isolated individual components.


Figure 3: A, construction of pMal-c2.2C; B, MBP-2C liberates 2C upon factor Xa cleavage. The MBP-2C fusion protein was cleaved with factor Xa while it was attached to the amylose resin as described under ``Materials and Methods.'' Lanes 1-3 contain 1, 0.5, and 0.25 µg of MBP, respectively. Lanes 4-6 are individual fractions eluted from the amylose resin column after factor Xa cleavage. Broken and unbroken arrows indicate the products obtained after canonical and noncanonical cleavage of factor Xa, respectively. C, Western immunoblot assay carried out with anti-2C antiserum (kindly provided by Dr. Wimmer). Lane 1, analysis of fraction 5 from B; lane M, biotinylated standard (45 kDa); lane PV, proteins from poliovirus-infected cells.



Internal Deletions of MBP -2C

Since poliovirus protein 2C is an NTPase containing the A and B sites that are involved in nucleotide interaction, two internal deletions of MBP-2C were generated that lacked these motifs (see Fig. 6 B). Both constructions rendered fusion proteins that were efficiently cleaved by factor Xa (Fig. 4, A and B). Cleavage of the fusion protein MBP-2C or its variants is achieved after a 1-h incubation, and longer incubation times do not increase the amount of 2C generated (Fig. 4 A). Factor Xa digestion of MBP-2C liberates 2C, and several minor components are formed as a result of internal cleavages (Fig. 4, A and B). Genuine 2C (but containing five extra glycines at the amino terminus) possesses RNA binding capacity (Fig. 4 C). Both of the 2C deletion mutants, one lacking amino acids 129-172 and the other lacking amino acids 161-188, were similar to 2C with respect to their interaction with RNA (Fig. 4 C). However, the 2C mutant that lacks amino acids 129-172 has background ATPase activity only, whereas this activity in the variant that lacks amino acids 161-188 is residual but reproducible (Fig. 4 D). Further experiments are required to define with more precision the role that the DD motif plays in ATPase activity. Nevertheless, the results obtained with the 2C mutant lacking amino acids 129-172 indicate that 2C can bind RNA in the absence of NTPase activity.


Figure 4: Internal deletions of MBP-2C. A, Western immunoblot assay carried out with anti-2C antiserum. MBP-2C, MBP-2C-(161-188), and MBP-2C-(129-172) were submitted to digestion with factor Xa for different time periods (1, 3, or 8 h) as described under ``Material and Methods.'' Lane PV, proteins from poliovirus-infected cells. B, Coomassie Blue staining of the proteins obtained after an 8-h digestion with factor Xa of MBP-2C, MBP-2C-(161-188), and MBP-2C-(129-172) ( lanes 1-3, respectively). Lane M, molecular weight standards. C, Northwestern assay; D, ATPase assay.



Involvement of the NERNRR Motif in the Binding of RNA by Poliovirus Protein 2C

The results described above identify a region at the carboxyl terminus of 2C, located between residues 297 and 319, that is involved in RNA binding. There is a sequence in this region that is rich in arginines and resembles, in this respect, the RNA-binding domains described for other proteins that are endowed with this activity (27, 30, 33, 34, 35) . Therefore, we decided to mutate the sequence NERNRR (amino acids 312-317) in order to determine its importance for RNA binding. To this end, an oligonucleotide mixture was synthesized that contained several stop codons in place of arginines (Fig. 5 A). After PCR amplification with this oligonucleotide mixture followed by cloning and sequencing of the different clones, three variants of 2C were obtained: one without arginines in this motif (mutant 1-313), one with a single arginine (mutant 1-315), and one with three arginines (mutant 1-319). The corresponding fusion proteins of these mutants were obtained and purified as described above. The purified fusion proteins were digested with factor Xa, separated by SDS-PAGE, and assayed for their RNA binding capacity and, subsequently, their reactivity with anti-2C antibodies. Fig. 5F shows that all these fusion proteins are efficiently cleaved by factor Xa to yield significant amounts of the corresponding 2C variants. The mutant that contains no arginines in the COOH-terminal region is devoid of RNA binding capacity; some binding is detected with mutant 1-315, which contains one arginine; and full RNA binding capacity is observed with mutant 1-319, which contains three arginines (Fig. 5, C and E). These findings suggest that the region between residues 313 and 319 of 2C is essential for RNA binding. The motif NERNRR of 2C is similar in sequence to those described in other proteins with RNA binding properties (see below). Moreover, analogous sequences rich in basic amino acids are not only located in this region of poliovirus protein 2C, but are found in other picornaviruses, suggesting, again, an involvement of such sequences in RNA binding activity (Fig. 6 C).


Figure 5: Schematic diagram and RNA binding activity of poliovirus protein 2C deletion mutants from the Arg-rich COOH-terminal region. A, schematic diagram of the constructions ( nt, nucleotides; aa, amino acid); B, Ponceau S staining after wet electrotransfer of proteins to nitrocellulose sheets. Lane 1, MBP-2C-(1-313); lane 2, MBP-2C-(1-315); lane 3, MBP-2C-(1-319); lane 4, MBP-2C-(1-329). C, Northwestern assay of the fusion proteins indicated in B; D, Western immunoblot assay carried out with anti-2C antiserum. The nitrocellulose membrane containing the MBP-2C fusion proteins, which were analyzed in the Northwestern RNA binding assay shown in C, was subsequently analyzed in the Western immunoblot assay. E, Northwestern assay carried out as described for C. The fusion proteins were partially cleaved with factor Xa as described under ``Materials and Methods.'' The positions of MBP-2C and 2C are indicated ( arrows). F, Western immunoblot assay carried out as described for D.




DISCUSSION

Proteins endowed with RNA binding activity are involved in different biological functions including the metabolism of nucleic acids and the regulation of gene expression (36, 37) . This diverse group of proteins comprises at least nine families that have been distinguished on the basis of their RNA recognition motifs (38) . Most of the RNA-binding proteins thus far identified contain more than one binding domain (37) . Some of them, including the small nuclear RNP U1A protein, have two domains located one at each end of the molecule. Functional and structural aspects of the RNA-protein interaction are exemplified by protein U1A, which contains an RNA-binding motif variously termed the ribonucleoprotein consensus sequence, the RNA-binding domain, or the RNA recognition motif. This RNA-binding domain is 90 amino acids long and contains two conserved sequences (RNP1 and RNP2) that are separated by 30 amino acids (39) . Both basic and aromatic amino acids are present in RNP1 and RNP2, and they may form a complementary surface to allow interaction with RNA (37, 38, 39) . The arginine-rich sequence is another well established RNA-binding motif that is found in several viral, bacterial, and ribosomal RNA-binding proteins (33) . Two viral members of this family are the human immunodeficiency virus Tat and Rev proteins. A short cluster of arginine residues within the human immunodeficiency virus type 1 Tat protein can directly bind a specific RNA sequence, denominated TAR, and a single arginine in that cluster is responsible for direct interaction of the protein with the phosphate backbone of TAR RNA (34) . Another viral protein that contains arginine-rich motifs is typified by delta hepatitis antigen (30) . Arginine residues are also present in the RGG box, and it has been suggested that these are responsible for RNA binding activity (27) . These RGG boxes have a strong positive charge, but the RNA binding capacity is lost if the arginines are replaced by lysines, so this property is not acquired simply by the presence of a positively charged region. Aromatic residues are also present in RGG motifs, and these could contribute to hydrophobic stacking interactions with RNA bases (27, 38) .

Our present results indicate that 2C belongs to the group of proteins that bind RNA and that it contains two regions, one NH-terminal and the other COOH-terminal, that have been implicated in this property. Deletion of either 42 amino acids at the NHterminus or 16 amino acids at the COOH terminus abolishes RNA binding. Thus, both RNA-binding regions seem to act in concert to bind RNA, in agreement with other findings on RNA-binding proteins. For example, RNA binding of delta hepatitis antigen involves at least two arginine motifs, and deletion of either results in total loss of RNA binding activity in vitro (30) . Similarly, the protein kinase DAI (double-stranded RNA activated inhibitor) contains two copies of an RNA-binding motif. Deletion of either of the two motifs prevents the binding of RNA (40) . For other RNA-binding proteins, large truncations cause an unpredictable perturbation in the tertiary structure of the RNA-binding site of the protein; thus, even though the primary sequences responsible for RNA binding are still contained in some deletion mutants, they give negative results when assayed for RNA binding (26, 28) . For example, deletion of 174 residues from the 54-kDa protein of the signal recognition particle generates a 330-amino acid protein devoid of RNA binding, but an additional deletion of 122 amino acids renders the resulting 208-amino acid carboxyl-terminal protein capable of interacting with RNA (26) . This might be the case for certain 2C mutants. Thus, a short deletion at the COOH terminus (mutant 1-297) renders a 2C variant devoid of RNA binding activity, but a longer deletion (mutant 1-161) restores the ability of protein 2C to interact with RNA. This finding is rather common among RNA-binding proteins (26, 27, 28) . A short deletion of 16 amino acids at the COOH terminus abolishes RNA binding, but not NTPase activity (data not shown), favoring the hypothesis that the COOH-terminal RNA-binding region acts in concert with the NH-terminal RNA-binding region to bind RNA.

Therefore, poliovirus protein resembles forceps when interacting with RNA in the sense that two RNA-binding domains are located one at each end of the molecule. The NH-terminal RNA-binding region spans amino acids 21-161, with the RNA binding capacity being tentatively assigned to residues 21-45. The amino acid sequence between residues 5 and 19 of 2C contains some homology to a double-stranded RNA-binding consensus sequence (41, 42) . However, the double-stranded RNA-binding consensus sequence comprises 60-70 amino acids, and residues 5-19 of protein 2C only partially match the COOH-terminal part of the consensus sequence. Moreover, our result with mutant 21-161 does not agree with the idea that residues 5-19 are required for RNA binding. To our knowledge, the rest of the 2C sequence (amino acids 20-161) does not contain significant homology to any previously identified RNA-binding motif. The overall region has a basic isoelectric point and is rich in lysine residues. The existence of an amphipathic helix at the amino terminus of 2C comprising residues 10-27 has been predicted (41) . On the basis of similarities between this putative helix of 2C and apolipoprotein C-III, the possibility that this region of 2C is involved in membrane interaction has been advanced (41) . Several poliovirus mutants have been engineered in this region (41) . Two such mutants, designated N2 (I25K) and N3 (K16T, K24T), show diminished 2C and 3AB synthesis due to polyprotein misprocessing. These mutants are nonviable, but it remains uncertain if the lack of viral RNA replication was directly caused by a failure of mutated 2C to participate in RNA synthesis or resulted from a secondary defect involving misprocessing of the viral proteins. Finally, mutations resulting in the conversion of two conserved glutamic acid residues (Gluand Glu) to valines in this region produces a viable mutant with a small plaque phenotype, suggesting that the amino-terminal region plays a part in virus growth.

The second region of 2C that is implicated in RNA binding is located at the COOH terminus and comprises the last COOH-terminal 96 amino acids (234-329). Our data show that this 96-amino acid sequence confers RNA binding capacity to the fusion protein MBP-2C-(234-329). The former sequence has an arginine-rich COOH-terminal region between amino acids 312 and 319 (NERNRRSN). A deletion mutant ending at amino acid 313 does not bind RNA, whereas full binding is observed with protein 2C ending at amino acid 319, indicating an essential requirement for residues 312-319 in this phenomenon. RNA binding assays using short peptides that together cover the sequences in these two regions of protein 2C could locate more precisely the residues involved in RNA binding.

For defining the exact function of protein 2C in the virus replication cycle, we need to consider the two known biochemical roles of this protein, i.e. the NTPase (10, 12) and RNA binding activities. Viral proteins that interact with RNA may participate in replication, recombination, or transcription of viral RNA genomes; the formation of virions; and the transport of genomes via virus-encoded movement proteins (43, 44) . Several suggestions implicating poliovirus protein 2C in some of these processes have been advanced. Such functions require NTPase and RNA binding activities, and these may participate in RNA helicase action, traffic of viral RNA through the vesicular system, or virion morphogenesis (10, 12, 14) . In fact, genetic evidence suggests that 2C may be a multifunctional protein with an involvement in several processes during virus growth (5) . However, these genetic data could also be explained if 2C has a single biochemical function whose alteration causes pleiotropic effects in the viral life cycle. Certainly, 2C is involved in poliovirus RNA replication, and this protein might perhaps participate in the structural organization of the replication complex by attaching the viral RNA to membranes of the vesicular system, where viral RNA replication takes place (18) .

Movement of plant viruses from cell to cell is mediated by the so-called movement proteins (43, 44) . These proteins bind RNA when assayed either as such or as fusion proteins with MBP (45) . The function of these proteins in plant virus replication is still obscure, although it has been speculated that they can either transport the viral RNA genome through plasmodesmata or suppress the host responses that limit virus replication (44) . Some of these proteins share structural and functional properties with picornavirus 2C. Thus, for some plant viruses, there are relatively small (28-38 kDa) nonstructural transport proteins (44) . Of these, one of the best studied is the movement protein of tobacco mosaic virus, known as p30. Binding of p30 to viral nucleic acid involves two RNA-binding domains and causes unfolding of the RNA and formation of long complexes (28) . Moreover, potex-, hordei-, and furoviruses constitute a plant virus group whose members possess movement proteins that contain an NTP-binding motif similar to that of 2C (44) . Homology of the NTP-binding motif has been also observed among the CI proteins of potyvirus, the 58-kDa B-RNA coded protein of cowpea mosaic virus, and picornavirus protein 2C (46) . Even though the transport function has been assigned to proteins other than CI and the 58 kDa B-RNA coded protein in potyvirus and comovirus, the possibility that the CI and 58-kDa proteins are involved in genome transport has not been ruled out. Clearly, animal viruses do not need ``plant-like'' transport proteins to transmit the infection from cell to cell. However, it seems plausible to suggest that viral proteins exist that would mediate the traffic of genomes intracellularly. 2C has several properties that are presumably necessary for such a function including RNA affinity, NTPase activity, and its compartmentalization and association with the vesicular membranes, where the replication of genomes takes place.


FOOTNOTES

*
This work was supported in part by Plan Nacional Project BIO 92-0715 and Direcion General de Investigacion Científica y Tecnica Project PB90-0177 and by an institutional grant to the Centro de Biologia Molecular of the Fundación Ramón Areces. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a Formacion de Personal Investigador and a Residencia de Estudiantes fellowship.

To whom correspondence should be addressed. Tel.: 34-1-397-8450; Fax: 34-1-397-4799.

The abbreviations used are: MBP, maltose-binding protein; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; RNP, ribonucleoprotein.


ACKNOWLEDGEMENTS

The expert technical assistance of M. A. Sanz is acknowledged.


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