Poliovirus Protein 3AB Displays Nucleic Acid Chape
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病菌学杂志 2006年第4期
Department of Cell Biology and Molecular Genetics, University of Maryland—College Park, College Park, Maryland 20742
ABSTRACT
Poliovirus protein 3AB displayed nucleic acid chaperone activity in promoting the hybridization of complementary nucleic acids and destabilizing secondary structure. Hybridization reactions at 30°C between 20- and 40-nucleotide RNA oligonucleotides and 179- or 765-nucleotide RNAs that contained a complementary region were greatly enhanced in the presence of 3AB. The effect was nonspecific as reactions between DNA oligonucleotides and RNA or DNA templates were also enhanced. Reactions were optimal with 1 mM MgCl2 and 20 mM KCl. Analysis of the reactions with various 3AB and template concentrations indicated that enhancement required a critical amount of 3AB that increased as the concentration of nucleic acid increased. This was consistent with a requirement for 3AB to "coat" the nucleic acids for enhancement. The helix-destabilizing activity of 3AB was tested in an assay with two 42-nucleotide completely complementary DNAs. Each complement formed a strong stem-loop (G = –7.2 kcal/mol) that required unwinding for hybridization to occur. DNAs were modified at the 3' or 5' end with fluorescent probes such that hybridization resulted in quenching of the fluorescent signal. Under optimal conditions at 30°C, 3AB stimulated hybridization in a concentration-dependent manner, as did human immunodeficiency virus nucleocapsid protein, an established chaperone. The results are discussed with respect to the role of 3AB in viral replication and recombination.
INTRODUCTION
Picornaviruses are responsible for several diseases, including the common cold and chronic hepatitis in humans and foot-and-mouth disease in animals. These viruses contain positive-sense single-stranded RNA that is directly translated into a long polyprotein upon entry into the cytoplasm. Approximately 10 different proteins are produced by cleavage of the polyprotein at specific locations (16). Several of these proteins have been shown to participate in genome replication. These include 3Dpol; the viral RNA-dependent RNA polymerase; 3CD, 3AB, and 3B, the latter being a cleavage product of 3AB; and 3CD, the precursor of 3Dpol (1, 15, 28, 34, 46). Presumably these viral factors and some host proteins form a complex by interacting with a cloverleaf structure that forms near the 5' end of the viral RNA (1, 10, 12, 46). The exact sequence of events leading to release of 3B and 3Dpol from the precursor proteins is not clear, although 3C or the 3C portion of 3CD likely carries out the cleavage. Protein 3B (more commonly called VPg) is covalently linked to the 5' end of newly synthesized viral RNAs and is believed to serve as a primer for RNA synthesis by 3Dpol (5, 39, 40). It has been shown in vitro that 3Dpol catalyzes uridylylation of a specific tyrosine residue on 3B (27). Although poly(rA) can serve as the template for this process, 3Dpol-catalyzed uridylylation also occurs using a region near the center of the viral genome termed cre as the template and is stimulated by 3CD (29).
The exact role of 3AB is not clearly defined, although results indicate that it stimulates RNA synthesis by 3Dpol, perhaps by enhancing primer utilization (22, 28, 32, 34, 37). In addition, the 3A domain of 3AB may act to anchor the replication complex to membranes (42). Among the activities associated with 3AB are nonspecific RNA binding (22, 45, 46), direct binding to 3CD and 3Dpol (32), stimulation of cleavage of 3CD to 3C plus 3Dpol (25), and association with membranes as an integral membrane protein (42). In the presence of 3CD, 3AB demonstrates specific binding to poliovirus RNA, perhaps by a direct interaction with 3CD (12, 22). In contrast, 3AB from another picornavirus, hepatitis A virus, is able to specifically bind to structures at the 5' and 3' ends of the viral genome without the help of other viral proteins (7, 20).
Nucleic acid chaperones are classically defined as proteins that aid in the process of folding by preventing misfolding or by resolving misfolded species (14). They generally bind nonspecifically, disrupting weakly folded secondary structures and thereby promoting the formation of the most stably folded state of the nucleic acid molecule. They include, for example, human ribonucleoprotein A1 (hnRNP A1), single-strand binding protein of Escherichia coli, and T4 gene 32 protein (14). The most-studied viral chaperone is human immunodeficiency virus (HIV) nucleocapsid protein (NC) (for a review, see reference 23). This protein binds RNA or DNA nonspecifically and completely coats the nucleic acid strand. NC possesses helix-destabilization activity and can promote the formation of more stable hybrid structures at the expense of weaker ones. In addition, it can accelerate hybrid formation between complementary nucleic acid strands. HIV NC has many roles in viral replication, including among others, promoting binding of host tRNA to the primer-binding site, stimulating strong-stop positive- and negative-strand transfers, enhancing recombination, increasing the processivity (average number of nucleotides synthesized in a single binding event between the polymerase and primer-template) of reverse transcriptase, and coating and protecting the genome in the capsid (23). Like NC, 3AB is also a nonspecific RNA binding protein (see above), has been reported to interact with the primer and template, and might also have to coat the whole template to stimulate RNA synthesis (28). Another report indicated that 3AB stimulates RNA synthesis by 3Dpol only when primers that bind weakly to the template are used (34). This implied that 3AB may have a role in enhancing primer binding or the association of 3Dpol with weakly bound primers. Taken together, these results suggest that 3AB may possess nucleic acid chaperone activity.
In this report, we show that 3AB possesses properties consistent with classical chaperone proteins. Notably 3AB greatly stimulated the rate of binding between complementary nucleic acids. Stimulation was nonspecific with respect to sequence as well as nucleic acid type, as both DNA hybrid formation and RNA hybrid formation were enhanced. Protein 3AB also demonstrated helix-destabilizing activity. The exact role 3AB's chaperone activity plays in replication is unclear, but many roles are possible, including promoting proper folding of important structures in the nontranslated regions of the viral genome, enhancing recombination, and helping to "unwind" the genome for RNA synthesis. Interestingly, two other poliovirus proteins have been reported to possess RNA binding properties. Polymerase 3Dpol binds cooperatively to nucleic acids and can multimerize. It has been suggested that multimerization may be required for efficient nucleic acid binding and polymerization (6). Also, protein 2C from poliovirus and other picornaviruses possesses ATPase and GTPase activity and binds to RNA (3, 4, 19, 21, 24, 30, 35, 36, 44). Unlike 3AB, 3Dpol did not display chaperone activity in the assays presented here, while 2C was not tested.
MATERIALS AND METHODS
Materials. T3 and T7 RNA polymerase, DNase I (RNase free), and ribonucleotides were obtained from Roche. RNasin was obtained from Promega. Restriction enzymes and T4 polynucleotide kinase were from New England Biolabs. DNA oligonucleotides used as primers were purchased from Genosys, Inc. RNA oligonucleotides were purchased from Dharmacon Research, Inc., and Cyber Syn, Inc. Oligonucleotides for florescence resonance energy transfer (FRET) experiments were from Integrated DNA Technologies, Inc. Sephadex G-50 spin columns were from Harvard Apparatus. Radiolabeled compounds were from New England Nuclear or Amersham. All other chemicals were obtained from Sigma Chemical Co. or Fisher Scientific.
Methods. (i) Preparation of polymerase (3Dpol), protein 3AB, and HIV NC by expression in Escherichia coli. Protein 3AB and 3Dpol were purified using vectors kindly provided by Stephen Plotch (formerly of Lederle Pharmaceuticals). The polymerase of poliovirus type 1 (Mahoney strain) used for these studies was expressed in E. coli using plasmid pT7pol and purified as previously described (33). The recovered enzyme was homogenous, as determined by Coomassie blue staining (data not shown), and had a specific activity of approximately 1.5 x 106 U/mg [1 U is defined as 1 pmol of UMP incorporated into trichloroacetic acid-precipitable poly(U) in 30 min at 30°C using oligo(U)-primed poly(rA) as template]. Protein 3AB of poliovirus type 1 (Mahoney strain) was expressed in E. coli using plasmid pGEX-3AB. Expression and purification were performed as described previously (32). Purified 3AB was stored at –70°C in buffer containing 50 mM Tris-HCl (pH 8), 1 mM dithiothreitol (DTT), 0.05% Triton X-100, and 10% glycerol (3AB buffer). This protein was homogenous, as determined by silver staining (data not shown). HIV NC protein was produced using a vector kindly provided by Charles McHenry (University of Colorado). The protein was expressed in E. coli and purified as previously described (47).
(ii) 5'-end labeling of oligonucleotides. Reactions for primer labeling were done in a 50-μl volume containing 70 mM Tris-HCl, pH 7.6, 10 mM MgCl2, 5 mM DTT, 10 μl of [-32P]ATP (3,000 Ci/mmol, 10 μCi/μl), and 2 μl (20 U) of T4 polynucleotide kinase. The reaction mixture was incubated for 30 min at 37°C, and then the T4 polynucleotide kinase was heat inactivated for 10 min at 70°C according to the manufacturer's recommendation. The material was then run through a Sephadex G-50 spin column.
(iii) Preparation of RNA templates. Runoff transcripts with T3 RNA polymerase were made using the manufacturer's protocol. Plasmid pBSM13+ was cleaved with PvuII or NdeI, and T3 polymerase was used to prepare runoff transcripts of approximately 179 or 765 nucleotides in length, respectively. After transcription for 2 h, 15 U of DNase I (RNase free) was added and incubation was continued for 20 min. The reaction mixtures were extracted with phenol-chloroform-isoamyl alcohol (25:24:1) and precipitated with ethanol. Material was run through two successive Sephedex G-50 spin columns, and the amount of RNA was determined using a spectrophotometer. The length and purity of the RNA were evaluated by gel electrophoresis to ensure that it was full length (data not shown). The RNA was then quantified by spectrophotometric analysis. The equation used to calculate the molecular weight was ([A x 382.2] + [G x 344.2] + [C x 304.2] + [U x 305]). The molecular weight was used to calculate the molar concentration of RNA using the standard conversion of 1 unit of optical density at 260 nm 40 μg/ml for single-stranded RNA.
(iv) Preparation of DNA template. Fifty micrograms of plasmid pBSM13+ was digested with 100 U each of BglI and BamHI. These enzymes cleave the 3,204-nucleotide plasmid at positions 709 and 2,337 for BglI and 902 for BamHI. The material was then electrophoresed on an 8% denaturing polyacrylamide gel in order to separate the single strands of the smallest fragment (resulting from the cuts at 709 and 902). The two strands in this case were 193 and 201 nucleotides in length. The 201-nucleotide strand was excised and eluted from the gel by crushing and soaking the gel fragment overnight in 550 μl of a mixture of 80% formamide, 40 mM Tris-HCl (pH 7), 400 mM NaCl, and 1 mM EDTA (pH 8). This material was filtered through a 0.45-μm syringe filter and precipitated by adding 1 ml of ethanol. Quantification was performed as described above for the RNA template, except that the molecular weight was calculated using the equation ([A x 312.2] + [G x 328.2] + [C x 288.2] + [T x 303.2]).
(v) Extension reactions with 3Dpol and 3AB. The 765-nucleotide RNA template (10 nM) described above was incubated with 5' 32P-end-labeled 40-nucleotide R40 primer (15 nM; see Fig. 1) and various amounts of 3Dpol (as described in the legend to Fig. 2) in 50 mM HEPES (pH 7), 5 mM DTT, 1 mM MgCl2, 119 μM rNTPs, and 12.5 mM KCl. This material was preincubated for 3 min at 30°C in a total volume of 21 μl. Four microliters of 3AB (final concentration of 650 nM in reaction) or 3AB buffer (50 mM Tris-HCl, pH 8, 1 mM DTT, 0.05% Triton X-100, 10% glycerol) was added to start the reaction, and incubations were continued for 1 h. Twenty-five microliters of 2x formamide loading buffer (90% formamide, 10 mM EDTA, pH 8.0, 0.1% xylene cyanol, 0.1% bromophenol blue) was added. In some reactions, the primer was prehybridized to the template before addition of 3AB or 3Dpol. This was done by heating the sample to 70°C and then cooling it slowly to room temperature. Samples were loaded onto a 6% polyacrylamide-7 M urea sequencing gel and subjected to electrophoresis as described below. Dried gels were used for autoradiography.
(vi) Hybridization reactions with 3AB. Various RNA or DNA transcripts (10 nM) (described above) and complementary 5' 32P-labeled RNA or DNA primers (10 nM; see Fig. 1) were incubated in a mixture of 50 mM HEPES (pH 7), 5 mM DTT, 1.4 mM MgCl2, and 28.5 mM KCl. This material was preincubated for 3 min at 30°C in a total volume of 7 μl. Three microliters of 3AB (concentration as indicated in Results) or 3AB buffer (see above) was added after the preincubation to start the reaction. In some assays, specific components of the reaction mixture were varied (as indicated). These included variation of the template and 3AB concentrations as well as MgCl2 and KCl. There were also three controls. In the first two assays, the preincubated reaction mixtures were added to 5 μl of stop mix (20% glycerol, 20 mM EDTA, pH 8, 0.2% sodium dodecyl sulfate [SDS], 0.4 μg/μl tRNA, 1% bromophenol blue) and then either 3AB or buffer was added. These controls were used to show that the stop mix was terminating the reactions. The third control contained the preincubated reaction mixture and 3AB buffer and was heated at 65°C for 5 min and then slow cooled. This control was used to show where the hybrid migrated on the gel. Reactions were stopped at various times with 5 μl of stop mix. Then the products were analyzed on a 6% native polyacrylamide gel as describe below. Dried gels were used for autoradiography. The products were also quantified using a Bio-Rad FX phosphoimager.
(vii) Helix-destabilizing activity detected by gel shift or FRET. Two complementary 42-nucleotide DNAs, one with a 5' fluorescein-6-carboxamidohexyl (FAM) (FAM-CATTATCGGATAGTGGAACCTAGCTTCGACTATCGGATAATC-3') group and the second with a 3' 4-[[(4-dimethylamino)phenyl]-azo] benzenesulfonicamino (DABCYL) group (5'-GATTATCCGATAGTCGAAGCTAGGTTCCACTATCCGATAATG-DABCYL), were used in the assays. Using mfold (48) and 20 mM KCl, 1 mM MgCl2, and 30°C, each strand was predicted to form a stem-loop structure with a G value of –7.2 kcal/mol (see Fig. 7A). Annealing assays were completed at 30°C using a Cary Eclipse fluorescent spectrophotometer (Varian). FAM and DABCYL DNAs (10 and 20 nM, respectively.) were separately incubated for 5 min at 30°C in the presence or absence of 3AB (concentration as indicated) in 35 μl of buffer containing 50 mM HEPES (pH 7), 20 mM KCl, 5 mM DTT, 1 mM MgCl2, 6 mM Tris-HCl (pH 8), 0.014% Triton X-100, and 2.9% glycerol. The reactions were started by mixing the FAM and DABCYL samples in a quartz cuvette (final concentrations of 5 and 10 nM for FAM and DABCYL DNAs, respectively). The excitation wavelength was 494 nm with a bandwidth of 5 nm. The emission bandwidth was 10 nm, and the spectrum was observed at 520 nm. The emission spectrum was taken every minute for 16 min. An intensity ratio (Ir) was determined by dividing the peak intensity at a given time (It) by the peak intensity at time zero (I0) (Ir = It/I0). This value was plotted versus time for the different concentrations of 3AB used. Assays were also conducted with poliovirus 3Dpol and HIV NC protein under the same conditions. Gel shift assays were performed as described above with the following changes: (i) the 5' end of the DABCYL complement from above was labeled with 32P, using PNK as described above; and (ii) the final reaction volume was 100 μl and aliquots of 15 μl were removed at 1, 2, 4, 8, and 16 min and added to 7.5 μl of the stop solution (0.25% bromophenol blue, 20% glycerol, 20 mM EDTA, pH 8, 0.2% SDS, 0.4 mg/ml of yeast tRNA). For the time zero point, 7.5 μl from each preincubation with the different complements was added directly to 7.5 μl of stop solution. All reactions were transferred to ice after addition of stop solution. Samples were run on a 12% native polyacrylamide gel as described below, and autoradiograms were prepared from dried gels.
(viii) Polyacrylamide gel electrophoresis. For polyacrylamide gel electrophoresis, 6 or 12% (wt/vol) native polyacrylamide (29:1 acrylamide-bisacrylamide) gels or 6% denaturing gels (19:1 acrylamide-bisacrylamide with 7 M urea) were prepared and electrophoresis was performed as described previously (38).
RESULTS
In the presence of 3AB, 3Dpol is able to extend primers that were not prehybridized to the template. Initial experiments were designed to test whether 3Dpol, in the presence or absence of 3AB, could extend a 40-nucleotide RNA primer (R40) using a 765-nucleotide template strand (see Fig. 1) if the primer was not prehybridized to the template. This would require primer hybridization and then 3Dpol extension. Significant hybridization did not occur in the absence of additional protein (see Fig. 3 below), so either 3Dpol or 3AB would presumably need to facilitate hybrid formation for extension to be observed. Shown in Fig. 2 is an experiment in which increasing amounts of 3Dpol (10 to 650 nM) were added to reactions in the presence or absence of 650 nM 3AB. In the absence of 3AB, no extension to full-length products was observed at any 3Dpol concentration, while with 3AB extension was observed with all concentrations. The level of fully extended 256-nucleotide products increased as the amount of 3Dpol increased up to about 81 nM and then remained fairly constant until dropping off slightly at the highest enzyme level (650 nM). At the higher 3Dpol concentrations, some primers extended by a few nucleotides were observed. This likely resulted from non-template-directed addition of nucleotides to the 3' end of the primer by 3Dpol terminal transferase activity, as has been observed by others (2). Since 3AB is known to stimulate 3Dpol synthesis, especially at low polymerase concentrations (see the introduction), it was possible that the observed extension resulted from this and not from 3AB causing the primer to hybridize to the template. However, control reactions with the primer prehybridized to the template by heating and slow cooling showed comparable extension in the absence (lane 1) or presence (lane 2) of 3AB and 163 nM 3Dpol. This suggested that 3AB's main effect in the reactions without prehybridized primer was to stimulate hybridization, and this hypothesis was tested below.
3AB accelerates the hybridization of cRNA or DNA nucleic acids. To determine if 3AB enhances the rate of hybridization between complements, several 5'-end-labeled RNA and DNA oligonucleotides (20 to 40 nucleotides in length) were incubated with cRNA and DNA templates (see Fig. 1). Incubations were carried out for 30 min in the presence or absence of 3AB (650 nM), and the material was run on a native polyacrylamide gel to separate bound and unbound oligonucleotides. Control reactions in which the oligonucleotide was hybridized to the template strand by heating and slow cooling were run to mark the hybrid positions on the gel (Fig. 3, lanes labeled B). The results showed that little or no hybridization occurred with any of the templates during a 30-min incubation in the absence of 3AB (lanes labeled C), while hybridized products were evident with all substrates in the presence of 3AB (lanes labeled D). For the DNA template and DNA primer (far-right lanes), a band migrating above the predicted hybrid location is also evident. The nature of this band is not clear, but it could be "supershifted" material that is bound to 3AB. The buffer used on the gel is designed to inhibit protein-nucleic acid interactions, but the inhibition may not be complete. Note that some material also stuck in the wells when the 765-nucleotide RNA template was used. The amount remaining in the wells seemed to be proportional to the amount of hybrid that formed rather than the presence or absence of 3AB as preformation of the hybrid (lanes labeled B) also resulted in some sticking. Taken together, the results show that 3AB can promote hybridization of complements irrespective of the type of nucleic acid or the sequence of the hybrid region.
An experiment was also performed with 3Dpol (650 nM) in the presence or absence of 3AB (650 nM) using the 765-nucleotide RNA and R40 as described above. The results showed that 3Dpol did not stimulate annealing and had no effect on stimulation by 3AB (data not shown).
Stimulation of hybrid formation results from 3AB and not a "contaminant." Since the 3AB preparation used here was purified after overexpression in E. coli, it is possible that a bacterial protein that contaminated the preparation was responsible for the observations. The procedure used yields homogenous preparations of 3AB as judged by Coomassie blue staining (32) and silver staining (data not shown). However, the possibility of a low-level contaminant cannot be ruled out. To test this, a sample was prepared using bacteria that were transformed with pGEX vector that did not contain the 3AB coding region (see Materials and Methods). Fractions from this purification that corresponded to the 3AB-containing fractions used in the assays were tested for hybridization activity using the 179-nucleotide RNA template and R20-1 (Fig. 4). These fractions (lanes 4, 5, and 6, at 1x, 2x, and 4x, respectively) showed no stimulation even when added at four times the equivalent amount (lane 6) relative to the 3AB-containing fraction (lane 2). In addition, mixing a 1x equivalent amount of the 3AB-negative fraction with 3AB had no effect on 3AB's stimulatory activity (lane 3). These results show that 3AB and not a contaminant is responsible for hybrid stimulation.
Optimal conditions determination for 3AB stimulation. The 179-nucleotide template and 20-nucleotide primer (R20-1) were used to determine optimal salt and divalent cation conditions for 3AB's annealing activity. A graph for these experiments is shown in Fig. 5. The 3AB concentration was 1,200 nM, and reactions were performed for 8 min. Optimal concentrations were 1 mM and 20 mM for MgCl2 and KCl, respectively. Note that in the absence of 3AB, annealing was slightly stimulated at higher MgCl2 and KCl concentrations as expected. With 3AB, there was a pronounced decrease in stimulation at higher (above 1 mM) MgCl2 concentrations, while increasing KCl between 20 and 80 mM had a more gradual effect.
A time course assay was also performed with the 179-nucleotide template and R20-1 using optimal conditions over 32 min with 1,200 nM 3AB. The results showed that 3AB stimulation was nearly linear for the first 10 min, followed by a gradual decline in rate (data not shown). Hybrid formation had not saturated after 32 min. Assays to determine optimal conditions and those used to test the rate of hybrid formation with various nucleic acid and 3AB concentrations (see below) were performed for 8 min. At this time point, products were easily measurable and the reaction was essentially in the linear phase.
3AB stimulation is concentration dependent and probably requires coating of the nucleic acid strands. Nucleic acid chaperones work by coating the nucleic acid strand rather than through a catalytic mechanism (14, 23). Therefore, large amounts of protein relative to the nucleic acid strands are typically needed to observe optimal chaperone activity. To test this, assays with different concentrations of 3AB (75, 300, and 1,200 nM) were performed with various levels of the 179-nucleotide RNA (2.5 to 80 nM) and a fixed concentration of primer R20-1 (10 nM). If coating is required for stimulation of annealing, then lower concentrations of 3AB should only stimulate with lower RNA concentrations, while larger amounts of 3AB should be able to stimulate at both low and high RNA concentrations. Representative autoradiograms from experiments with the three different 3AB concentrations are shown in Fig. 6A (75, 300, and 1,200 nM 3AB). At 1,200 nM, 3AB stimulated annealing with all concentrations of RNA (compare minus and plus 3AB lanes in the 1,200 nM experiment). For this amount of 3AB, at the lowest RNA concentration (2.5 nM) the level of stimulation was much lower than at 5 nM. This probably resulted from material stuck in the wells and "streaking" through the gel at this high 3AB concentration. This was not as prevalent when 75 or 300 nM 3AB was used. At 1,200 nM, the level of stimulation decreased as the concentration of RNA increased. This likely resulted from a lower proportion of the RNAs being adequately coated. At 300 nM 3AB, significant stimulation was only observed at the lower RNA concentrations (2.5, 5, 10, and 20 nM) while with 75 nM 3AB some stimulation was observed at the low RNA concentrations (2.5 and 5 nM), but not very much. This result is consistent with 3AB having to coat the template to exert its effect.
A second experiment in which the concentration of 3AB was varied and R20-1 and 179-nucleotide RNA were held constant at 10 nM and 5 nM, respectively, is shown in Fig. 6B. Significant stimulation was not observed at 3AB concentrations below 75 nM, and stimulation peaked when 300 to 600 nM 3AB was added. At these concentrations, there was approximately 1 3AB molecule per 13.3 (300 nM) and 6.6 (600 nM) total nucleotides in the reactions. A decrease in stimulation was observed at the 1,200 nM 3AB concentration, but streaking (see above) was also prevalent in this sample. Overall the experiment supports a mechanism that requires coating of the RNA for stimulation of annealing. This is especially true given the large amount of 3AB needed to observe stimulation.
3AB demonstrates helix-destabilizing activity and can "unwind" structured nucleic acid strands. Nucleic acid chaperones possess helix-destabilizing activity (14, 23). The strength of this activity can vary depending on the protein, with, for example, HIV NC possessing relatively weak destabilizing activity (23). To test the ability of 3AB to unwind nucleic acid secondary structures, an assay used for HIV NC protein (13) was adapted for 3AB. A diagram of the substrates is shown in Fig. 7A. In this assay, two 42-nucleotide complementary nucleic acid strands that form a defined stem-loop structure are used. One strand contained a DABCYL group (DAB in the figure) at the 3' end, while the other had a FAM group at the 5' end (see Materials and Methods). In this case, the predicted G value for each of the folded single strands was approximately –7.2 kcal/mol. Two types of assay systems were used: a gel shift assay or fluorescence quench-based assay (see below). For the gel shift assay, the 5' end of the DABCYL strand was labeled with 32P and the complementary strands (5 nM FAM and 10 nM DABCYL, respectively) were mixed in the presence or absence of 3AB (250 or 1,000 nM). In order to hybridize, the nucleic acids must first unwind to expose the complementary bases. After hybridizing, the larger more rigid hybrid migrates more slowly in the gel than the single-stranded DNA. An autoradiogram from an assay is shown in Fig. 7B. In the absence of 3AB, no hybridization was observed. Reactions with 3AB showed an increase in hybrid formation over time that was greater with the higher 3AB concentration (graphed in Fig. 7C). Control reactions in which 3AB was added along with the stop solution (time zero) showed that the solution prevented hybridization in the presence and absence of 3AB. These results indicate that 3AB can unfold and promote the hybridization of folded nucleic acid substrates.
A second more sensitive and quantitative assay based on fluorescence quenching was also used. As described above, one complement contained a fluorescing FAM probe at the 5' end while the other had a DABCYL quencher at the 3' end. Upon annealing, the DABCYL group is brought into close proximity to the FAM group and can quench fluorescence from FAM. This results in a decreasing level of fluorescence upon hybridization of the complements. We have previously used this assay to measure the helix-destabilizing activity of several HIV NC mutants (13). Plots for reactions with various concentrations of 3AB are shown in Fig. 7D, while 7E and F show plots for 3Dpol and HIV NC, respectively. Control reactions without 3AB or without the FAM-labeled complement showed essentially no fluorescence decrease under the conditions used (optimal conditions from above at 30°C with 5 nM FAM- and 10 nM DABCYL-derivatized nucleic acid strands). Annealing was stimulated by 3AB in a concentration-dependent manner, with the greatest stimulation observed at the largest amount of 3AB (1,000 nM). The results were consistent with the gel assay. Protein 3Dpol, which showed no annealing stimulation in the assays with RNA shown in Fig. 2 and 3, also showed no stimulation in this assay. However, since 3Dpol is an RNA binding protein and the substrate used here was DNA, this negative result would be expected even if 3Dpol had chaperone activity. HIV NC, an established chaperone protein, showed greater stimulation than 3AB, and only relatively small amounts of protein (about 125 nM) were required for nearly complete hybridization in less than 1 min (note different time scales and protein amounts for assays with HIV-NC and 3AB or 3Dpol). Increasing the HIV NC concentration beyond 125 nM had no significant effect on the reaction rate (data not shown). Overall the results show that 3AB has helix-destabilizing activity that can unwind nucleic acids and promote hybridization, though this activity is modest.
DISCUSSION
In this report, we show that poliovirus 3AB protein has properties consistent with a nucleic acid chaperone. The "classic" role for an RNA chaperone is to aid in folding of RNA to promote the most thermodynamically stable and generally functional structure. This is accomplished through stabilizing this structure as well as preventing or resolving misfolded RNAs that can lead to kinetically trapped nonoptimal species (14). Protein 3AB could serve such a role in poliovirus replication. In addition, there are many other places in the picornavirus life cycle where chaperone activity could play a role. These include (i) coating and protecting the nucleic acid, (ii) enhancing 3Dpol polymerization by helping to destabilize secondary structures in the genome, (iii) aiding in the initial binding of 3Dpol and other host and viral proteins through unwinding the RNA and making it more accessible, (iv) helping to separate positive and negative strands during synthesis, (v) and enhancing recombination or the completion of RNA synthesis by promoting the binding of incompletely synthesized positive- or negative-sense RNAs to complementary templates. Protein 3AB's roles in anchoring the replication complex to membranes, serving as a primer for RNA synthesis, and enhancing 3Dpol synthesis, are already well established (see the introduction); chaperone activity could be a factor in these and other functions not previously attributed to 3AB.
Protein 3AB accelerated the binding of oligonucleotides to complementary regions of RNA or DNA templates (Fig. 2 and 3). Previous reports indicated that 3AB is an RNA binding protein (see the introduction). Its ability to promote binding to both DNA and RNA indicates that it can also bind DNA. In this regard, it is similar to HIV NC, showing chaperone activity on both RNA and DNA (23). Note that enhancement of annealing by 3AB has been previously reported (34). In these experiments, the authors attributed the stimulation of 3Dpol synthesis by 3AB to promotion or stabilization of annealing for primers that normally anneal relatively poorly. Although the present work does not directly address the mechanism by which 3AB stimulates 3Dpol, a role in promoting primer binding is consistent with chaperone activity. The present work also indicates that 3AB can promote the hybridization of primers that form very stable hybrids with the template (R40, for example).
No chaperone-like activity was found for 3Dpol (see Fig. 2 and the Results section), despite previous results showing that this protein can bind to and coat nucleic acids (6). It is possible that the conditions used here did not allow 3Dpol coating or chaperone activity. No attempt was made to optimize conditions for 3Dpol, so chaperone activity for this protein cannot be ruled out. It should also be noted that complete coating in previous experiments required very high concentrations of 3Dpol, above the amounts used in these experiments (6). Therefore, it is possible that 3Dpol may have chaperone activity at concentrations well beyond where 3AB shows an effect. Protein 3Dpol also showed no helix-destabilizing activity in the assay shown in Fig. 7E; however, as was noted, the substrate was DNA and 3Dpol has only been shown to be an RNA binding protein. Therefore, no conclusions can be drawn from this assay.
The helix-destabilizing activity of 3AB was much less than that measured for HIV NC under the same conditions (Fig. 7D and F). Although this suggests that NC has greater helix-destabilizing activity than 3AB, only measurements performed under cellular conditions could confirm this. The assays were performed under optimal conditions for 3AB hybridization, and these may differ from conditions in the cell (see below). We know, for example, that NC's helix-destabilizing activity is extremely salt sensitive in the assay used here. Only a fraction of the activity remains at 80 mM KCl, as opposed to the 20 mM level used in these experiments (unpublished data). Currently 3AB is being tested under several conditions to determine how salt, pH, and divalent cation concentrations affect its helix-destabilizing activity. Also, the substrate used in the helix-destabilization assays was DNA, and it is possible that 3AB could show greater activity on RNA, the presumed substrate in vivo. Since HIV NC's activity is considered relatively weak (23), it is safe to say that 3AB's activity is also weak. A modest activity may be better suited to viral replication for a few reasons. First, viruses typically have several secondary structure motifs that are important to replication and a strong helix-destabilizing protein may disrupt these. Second, presumably a chaperone protein must be displaced by the polymerase during nucleic acid replication. A protein that binds too tightly, as a strong helix-destabilizing protein may do, could make this difficult.
In general, chaperone proteins work by coating nucleic acid strands. Results suggested that 3AB likely works this way as well. This is the most plausible explanation for the results observed in Fig. 6. Very large amounts of 3AB relative to the nucleic acid concentrations were required to observe significant stimulation. In the reactions with 5 nM 179-nucleotide RNA and 10 nM 20-base RNA oligonucleotide, peak stimulation was observed at about 300 to 600 nM 3AB (Fig. 6B). These concentrations correspond to approximately 6.5 and 13 nucleotides per 3AB molecule, respectively. It is known that each molecule of HIV NC coats approximately 7 nucleotides of nucleic acid (23). Although no experiments were done to directly determine the binding site size of 3AB, the rough estimates from the data above are in the range of what would be expected for complete coating of the nucleic acid in the reactions given that 3AB is approximately twice the molecular weight of NC. In addition, the rapid falloff of stimulation as the concentration of nucleic acid was increased in the experiment shown in Fig. 6A also supports a coating mechanism. When 300 nM 3AB was used, significant stimulation was only observed at lower nucleic acid concentrations (20 nM or below; Fig. 6A). With 75 nM 3AB, just a small amount of stimulation was observed at the lowest nucleic acid concentrations (2.5 and 5 nM; Fig. 6A). The lack of stimulation at the higher nucleic acid concentrations with 300 nM 3AB and the inability of 75 nM 3AB to stimulate strongly at any concentration clearly argue against a catalytic mechanism. In addition, a catalytic protein with helix-destabilizing activity would probably require an energy source such as nucleotide hydrolysis to function. No nucleotides or other potential energy sources were included in the reactions (except for those shown in Fig. 2). Note that this also argues strongly against the activity being a contaminant from E. coli. Because the 3AB preparations were essentially homogenous, as judged from gel analysis, any contaminants must have been present in very small amounts. Only an energy-requiring catalytic protein present at such low levels could have possibly demonstrated the chaperone-like activity found here.
The optimal conditions for 3AB annealing stimulation were 1 mM MgCl2 and 20 mM KCl (Fig. 5). Increasing the MgCl2 concentration resulted in a rapid falloff of stimulation, while higher KCl concentrations had a more gradual effect. Still, significant stimulation in comparison to that in reactions without 3AB was observed at all tested MgCl2 concentrations and all KCl concentrations below 160 nM. Although the concentration of Mg2+ in cells varies depending on the cell type and may change during infection, measurements of approximately 1 mM free and 8 mM complexed Mg2+ are essentially average (43). Therefore, the free Mg2+ concentration in cells is near optimal for 3AB activity. The concentration of K+ and other monovalent salts would be considerably higher than the 20 mM optimal. However, significant stimulation was observed even at much higher KCl concentrations. It is also possible that the concentrations of mono- and divalent cations in the vesicular structures where virus replication occurs are different from concentrations in other parts of the cell.
The need for an activity that helps anneal "primers" to nucleic acid is not immediately clear in the picornavirus replication process; however, it could help in recombination. In poliovirus, recombination is frequent and is proposed to occur by either "copy choice" or direct joining of RNAs (8, 9, 11, 17, 18, 26, 31, 41). In the copy choice mechanism, partially completed nucleic acid strands switch templates by binding to a complementary region on another RNA strand. The annealing and helix-destabilizing components of the chaperone activity could play an important role in this process or any of the others noted above. Further experiments, perhaps using mutants that lack chaperone activity while retaining other functions, will be required to understand the potential role(s) of this new activity.
ACKNOWLEDGMENTS
We thank Stephen Plotch (formerly of Lederle Pharmaceuticals) for expression clones for 3AB and 3Dpol and for help in purification of these proteins. We thank Charles McHenry (University of Colorado) for the HIV NC overexpression plasmid.
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ABSTRACT
Poliovirus protein 3AB displayed nucleic acid chaperone activity in promoting the hybridization of complementary nucleic acids and destabilizing secondary structure. Hybridization reactions at 30°C between 20- and 40-nucleotide RNA oligonucleotides and 179- or 765-nucleotide RNAs that contained a complementary region were greatly enhanced in the presence of 3AB. The effect was nonspecific as reactions between DNA oligonucleotides and RNA or DNA templates were also enhanced. Reactions were optimal with 1 mM MgCl2 and 20 mM KCl. Analysis of the reactions with various 3AB and template concentrations indicated that enhancement required a critical amount of 3AB that increased as the concentration of nucleic acid increased. This was consistent with a requirement for 3AB to "coat" the nucleic acids for enhancement. The helix-destabilizing activity of 3AB was tested in an assay with two 42-nucleotide completely complementary DNAs. Each complement formed a strong stem-loop (G = –7.2 kcal/mol) that required unwinding for hybridization to occur. DNAs were modified at the 3' or 5' end with fluorescent probes such that hybridization resulted in quenching of the fluorescent signal. Under optimal conditions at 30°C, 3AB stimulated hybridization in a concentration-dependent manner, as did human immunodeficiency virus nucleocapsid protein, an established chaperone. The results are discussed with respect to the role of 3AB in viral replication and recombination.
INTRODUCTION
Picornaviruses are responsible for several diseases, including the common cold and chronic hepatitis in humans and foot-and-mouth disease in animals. These viruses contain positive-sense single-stranded RNA that is directly translated into a long polyprotein upon entry into the cytoplasm. Approximately 10 different proteins are produced by cleavage of the polyprotein at specific locations (16). Several of these proteins have been shown to participate in genome replication. These include 3Dpol; the viral RNA-dependent RNA polymerase; 3CD, 3AB, and 3B, the latter being a cleavage product of 3AB; and 3CD, the precursor of 3Dpol (1, 15, 28, 34, 46). Presumably these viral factors and some host proteins form a complex by interacting with a cloverleaf structure that forms near the 5' end of the viral RNA (1, 10, 12, 46). The exact sequence of events leading to release of 3B and 3Dpol from the precursor proteins is not clear, although 3C or the 3C portion of 3CD likely carries out the cleavage. Protein 3B (more commonly called VPg) is covalently linked to the 5' end of newly synthesized viral RNAs and is believed to serve as a primer for RNA synthesis by 3Dpol (5, 39, 40). It has been shown in vitro that 3Dpol catalyzes uridylylation of a specific tyrosine residue on 3B (27). Although poly(rA) can serve as the template for this process, 3Dpol-catalyzed uridylylation also occurs using a region near the center of the viral genome termed cre as the template and is stimulated by 3CD (29).
The exact role of 3AB is not clearly defined, although results indicate that it stimulates RNA synthesis by 3Dpol, perhaps by enhancing primer utilization (22, 28, 32, 34, 37). In addition, the 3A domain of 3AB may act to anchor the replication complex to membranes (42). Among the activities associated with 3AB are nonspecific RNA binding (22, 45, 46), direct binding to 3CD and 3Dpol (32), stimulation of cleavage of 3CD to 3C plus 3Dpol (25), and association with membranes as an integral membrane protein (42). In the presence of 3CD, 3AB demonstrates specific binding to poliovirus RNA, perhaps by a direct interaction with 3CD (12, 22). In contrast, 3AB from another picornavirus, hepatitis A virus, is able to specifically bind to structures at the 5' and 3' ends of the viral genome without the help of other viral proteins (7, 20).
Nucleic acid chaperones are classically defined as proteins that aid in the process of folding by preventing misfolding or by resolving misfolded species (14). They generally bind nonspecifically, disrupting weakly folded secondary structures and thereby promoting the formation of the most stably folded state of the nucleic acid molecule. They include, for example, human ribonucleoprotein A1 (hnRNP A1), single-strand binding protein of Escherichia coli, and T4 gene 32 protein (14). The most-studied viral chaperone is human immunodeficiency virus (HIV) nucleocapsid protein (NC) (for a review, see reference 23). This protein binds RNA or DNA nonspecifically and completely coats the nucleic acid strand. NC possesses helix-destabilization activity and can promote the formation of more stable hybrid structures at the expense of weaker ones. In addition, it can accelerate hybrid formation between complementary nucleic acid strands. HIV NC has many roles in viral replication, including among others, promoting binding of host tRNA to the primer-binding site, stimulating strong-stop positive- and negative-strand transfers, enhancing recombination, increasing the processivity (average number of nucleotides synthesized in a single binding event between the polymerase and primer-template) of reverse transcriptase, and coating and protecting the genome in the capsid (23). Like NC, 3AB is also a nonspecific RNA binding protein (see above), has been reported to interact with the primer and template, and might also have to coat the whole template to stimulate RNA synthesis (28). Another report indicated that 3AB stimulates RNA synthesis by 3Dpol only when primers that bind weakly to the template are used (34). This implied that 3AB may have a role in enhancing primer binding or the association of 3Dpol with weakly bound primers. Taken together, these results suggest that 3AB may possess nucleic acid chaperone activity.
In this report, we show that 3AB possesses properties consistent with classical chaperone proteins. Notably 3AB greatly stimulated the rate of binding between complementary nucleic acids. Stimulation was nonspecific with respect to sequence as well as nucleic acid type, as both DNA hybrid formation and RNA hybrid formation were enhanced. Protein 3AB also demonstrated helix-destabilizing activity. The exact role 3AB's chaperone activity plays in replication is unclear, but many roles are possible, including promoting proper folding of important structures in the nontranslated regions of the viral genome, enhancing recombination, and helping to "unwind" the genome for RNA synthesis. Interestingly, two other poliovirus proteins have been reported to possess RNA binding properties. Polymerase 3Dpol binds cooperatively to nucleic acids and can multimerize. It has been suggested that multimerization may be required for efficient nucleic acid binding and polymerization (6). Also, protein 2C from poliovirus and other picornaviruses possesses ATPase and GTPase activity and binds to RNA (3, 4, 19, 21, 24, 30, 35, 36, 44). Unlike 3AB, 3Dpol did not display chaperone activity in the assays presented here, while 2C was not tested.
MATERIALS AND METHODS
Materials. T3 and T7 RNA polymerase, DNase I (RNase free), and ribonucleotides were obtained from Roche. RNasin was obtained from Promega. Restriction enzymes and T4 polynucleotide kinase were from New England Biolabs. DNA oligonucleotides used as primers were purchased from Genosys, Inc. RNA oligonucleotides were purchased from Dharmacon Research, Inc., and Cyber Syn, Inc. Oligonucleotides for florescence resonance energy transfer (FRET) experiments were from Integrated DNA Technologies, Inc. Sephadex G-50 spin columns were from Harvard Apparatus. Radiolabeled compounds were from New England Nuclear or Amersham. All other chemicals were obtained from Sigma Chemical Co. or Fisher Scientific.
Methods. (i) Preparation of polymerase (3Dpol), protein 3AB, and HIV NC by expression in Escherichia coli. Protein 3AB and 3Dpol were purified using vectors kindly provided by Stephen Plotch (formerly of Lederle Pharmaceuticals). The polymerase of poliovirus type 1 (Mahoney strain) used for these studies was expressed in E. coli using plasmid pT7pol and purified as previously described (33). The recovered enzyme was homogenous, as determined by Coomassie blue staining (data not shown), and had a specific activity of approximately 1.5 x 106 U/mg [1 U is defined as 1 pmol of UMP incorporated into trichloroacetic acid-precipitable poly(U) in 30 min at 30°C using oligo(U)-primed poly(rA) as template]. Protein 3AB of poliovirus type 1 (Mahoney strain) was expressed in E. coli using plasmid pGEX-3AB. Expression and purification were performed as described previously (32). Purified 3AB was stored at –70°C in buffer containing 50 mM Tris-HCl (pH 8), 1 mM dithiothreitol (DTT), 0.05% Triton X-100, and 10% glycerol (3AB buffer). This protein was homogenous, as determined by silver staining (data not shown). HIV NC protein was produced using a vector kindly provided by Charles McHenry (University of Colorado). The protein was expressed in E. coli and purified as previously described (47).
(ii) 5'-end labeling of oligonucleotides. Reactions for primer labeling were done in a 50-μl volume containing 70 mM Tris-HCl, pH 7.6, 10 mM MgCl2, 5 mM DTT, 10 μl of [-32P]ATP (3,000 Ci/mmol, 10 μCi/μl), and 2 μl (20 U) of T4 polynucleotide kinase. The reaction mixture was incubated for 30 min at 37°C, and then the T4 polynucleotide kinase was heat inactivated for 10 min at 70°C according to the manufacturer's recommendation. The material was then run through a Sephadex G-50 spin column.
(iii) Preparation of RNA templates. Runoff transcripts with T3 RNA polymerase were made using the manufacturer's protocol. Plasmid pBSM13+ was cleaved with PvuII or NdeI, and T3 polymerase was used to prepare runoff transcripts of approximately 179 or 765 nucleotides in length, respectively. After transcription for 2 h, 15 U of DNase I (RNase free) was added and incubation was continued for 20 min. The reaction mixtures were extracted with phenol-chloroform-isoamyl alcohol (25:24:1) and precipitated with ethanol. Material was run through two successive Sephedex G-50 spin columns, and the amount of RNA was determined using a spectrophotometer. The length and purity of the RNA were evaluated by gel electrophoresis to ensure that it was full length (data not shown). The RNA was then quantified by spectrophotometric analysis. The equation used to calculate the molecular weight was ([A x 382.2] + [G x 344.2] + [C x 304.2] + [U x 305]). The molecular weight was used to calculate the molar concentration of RNA using the standard conversion of 1 unit of optical density at 260 nm 40 μg/ml for single-stranded RNA.
(iv) Preparation of DNA template. Fifty micrograms of plasmid pBSM13+ was digested with 100 U each of BglI and BamHI. These enzymes cleave the 3,204-nucleotide plasmid at positions 709 and 2,337 for BglI and 902 for BamHI. The material was then electrophoresed on an 8% denaturing polyacrylamide gel in order to separate the single strands of the smallest fragment (resulting from the cuts at 709 and 902). The two strands in this case were 193 and 201 nucleotides in length. The 201-nucleotide strand was excised and eluted from the gel by crushing and soaking the gel fragment overnight in 550 μl of a mixture of 80% formamide, 40 mM Tris-HCl (pH 7), 400 mM NaCl, and 1 mM EDTA (pH 8). This material was filtered through a 0.45-μm syringe filter and precipitated by adding 1 ml of ethanol. Quantification was performed as described above for the RNA template, except that the molecular weight was calculated using the equation ([A x 312.2] + [G x 328.2] + [C x 288.2] + [T x 303.2]).
(v) Extension reactions with 3Dpol and 3AB. The 765-nucleotide RNA template (10 nM) described above was incubated with 5' 32P-end-labeled 40-nucleotide R40 primer (15 nM; see Fig. 1) and various amounts of 3Dpol (as described in the legend to Fig. 2) in 50 mM HEPES (pH 7), 5 mM DTT, 1 mM MgCl2, 119 μM rNTPs, and 12.5 mM KCl. This material was preincubated for 3 min at 30°C in a total volume of 21 μl. Four microliters of 3AB (final concentration of 650 nM in reaction) or 3AB buffer (50 mM Tris-HCl, pH 8, 1 mM DTT, 0.05% Triton X-100, 10% glycerol) was added to start the reaction, and incubations were continued for 1 h. Twenty-five microliters of 2x formamide loading buffer (90% formamide, 10 mM EDTA, pH 8.0, 0.1% xylene cyanol, 0.1% bromophenol blue) was added. In some reactions, the primer was prehybridized to the template before addition of 3AB or 3Dpol. This was done by heating the sample to 70°C and then cooling it slowly to room temperature. Samples were loaded onto a 6% polyacrylamide-7 M urea sequencing gel and subjected to electrophoresis as described below. Dried gels were used for autoradiography.
(vi) Hybridization reactions with 3AB. Various RNA or DNA transcripts (10 nM) (described above) and complementary 5' 32P-labeled RNA or DNA primers (10 nM; see Fig. 1) were incubated in a mixture of 50 mM HEPES (pH 7), 5 mM DTT, 1.4 mM MgCl2, and 28.5 mM KCl. This material was preincubated for 3 min at 30°C in a total volume of 7 μl. Three microliters of 3AB (concentration as indicated in Results) or 3AB buffer (see above) was added after the preincubation to start the reaction. In some assays, specific components of the reaction mixture were varied (as indicated). These included variation of the template and 3AB concentrations as well as MgCl2 and KCl. There were also three controls. In the first two assays, the preincubated reaction mixtures were added to 5 μl of stop mix (20% glycerol, 20 mM EDTA, pH 8, 0.2% sodium dodecyl sulfate [SDS], 0.4 μg/μl tRNA, 1% bromophenol blue) and then either 3AB or buffer was added. These controls were used to show that the stop mix was terminating the reactions. The third control contained the preincubated reaction mixture and 3AB buffer and was heated at 65°C for 5 min and then slow cooled. This control was used to show where the hybrid migrated on the gel. Reactions were stopped at various times with 5 μl of stop mix. Then the products were analyzed on a 6% native polyacrylamide gel as describe below. Dried gels were used for autoradiography. The products were also quantified using a Bio-Rad FX phosphoimager.
(vii) Helix-destabilizing activity detected by gel shift or FRET. Two complementary 42-nucleotide DNAs, one with a 5' fluorescein-6-carboxamidohexyl (FAM) (FAM-CATTATCGGATAGTGGAACCTAGCTTCGACTATCGGATAATC-3') group and the second with a 3' 4-[[(4-dimethylamino)phenyl]-azo] benzenesulfonicamino (DABCYL) group (5'-GATTATCCGATAGTCGAAGCTAGGTTCCACTATCCGATAATG-DABCYL), were used in the assays. Using mfold (48) and 20 mM KCl, 1 mM MgCl2, and 30°C, each strand was predicted to form a stem-loop structure with a G value of –7.2 kcal/mol (see Fig. 7A). Annealing assays were completed at 30°C using a Cary Eclipse fluorescent spectrophotometer (Varian). FAM and DABCYL DNAs (10 and 20 nM, respectively.) were separately incubated for 5 min at 30°C in the presence or absence of 3AB (concentration as indicated) in 35 μl of buffer containing 50 mM HEPES (pH 7), 20 mM KCl, 5 mM DTT, 1 mM MgCl2, 6 mM Tris-HCl (pH 8), 0.014% Triton X-100, and 2.9% glycerol. The reactions were started by mixing the FAM and DABCYL samples in a quartz cuvette (final concentrations of 5 and 10 nM for FAM and DABCYL DNAs, respectively). The excitation wavelength was 494 nm with a bandwidth of 5 nm. The emission bandwidth was 10 nm, and the spectrum was observed at 520 nm. The emission spectrum was taken every minute for 16 min. An intensity ratio (Ir) was determined by dividing the peak intensity at a given time (It) by the peak intensity at time zero (I0) (Ir = It/I0). This value was plotted versus time for the different concentrations of 3AB used. Assays were also conducted with poliovirus 3Dpol and HIV NC protein under the same conditions. Gel shift assays were performed as described above with the following changes: (i) the 5' end of the DABCYL complement from above was labeled with 32P, using PNK as described above; and (ii) the final reaction volume was 100 μl and aliquots of 15 μl were removed at 1, 2, 4, 8, and 16 min and added to 7.5 μl of the stop solution (0.25% bromophenol blue, 20% glycerol, 20 mM EDTA, pH 8, 0.2% SDS, 0.4 mg/ml of yeast tRNA). For the time zero point, 7.5 μl from each preincubation with the different complements was added directly to 7.5 μl of stop solution. All reactions were transferred to ice after addition of stop solution. Samples were run on a 12% native polyacrylamide gel as described below, and autoradiograms were prepared from dried gels.
(viii) Polyacrylamide gel electrophoresis. For polyacrylamide gel electrophoresis, 6 or 12% (wt/vol) native polyacrylamide (29:1 acrylamide-bisacrylamide) gels or 6% denaturing gels (19:1 acrylamide-bisacrylamide with 7 M urea) were prepared and electrophoresis was performed as described previously (38).
RESULTS
In the presence of 3AB, 3Dpol is able to extend primers that were not prehybridized to the template. Initial experiments were designed to test whether 3Dpol, in the presence or absence of 3AB, could extend a 40-nucleotide RNA primer (R40) using a 765-nucleotide template strand (see Fig. 1) if the primer was not prehybridized to the template. This would require primer hybridization and then 3Dpol extension. Significant hybridization did not occur in the absence of additional protein (see Fig. 3 below), so either 3Dpol or 3AB would presumably need to facilitate hybrid formation for extension to be observed. Shown in Fig. 2 is an experiment in which increasing amounts of 3Dpol (10 to 650 nM) were added to reactions in the presence or absence of 650 nM 3AB. In the absence of 3AB, no extension to full-length products was observed at any 3Dpol concentration, while with 3AB extension was observed with all concentrations. The level of fully extended 256-nucleotide products increased as the amount of 3Dpol increased up to about 81 nM and then remained fairly constant until dropping off slightly at the highest enzyme level (650 nM). At the higher 3Dpol concentrations, some primers extended by a few nucleotides were observed. This likely resulted from non-template-directed addition of nucleotides to the 3' end of the primer by 3Dpol terminal transferase activity, as has been observed by others (2). Since 3AB is known to stimulate 3Dpol synthesis, especially at low polymerase concentrations (see the introduction), it was possible that the observed extension resulted from this and not from 3AB causing the primer to hybridize to the template. However, control reactions with the primer prehybridized to the template by heating and slow cooling showed comparable extension in the absence (lane 1) or presence (lane 2) of 3AB and 163 nM 3Dpol. This suggested that 3AB's main effect in the reactions without prehybridized primer was to stimulate hybridization, and this hypothesis was tested below.
3AB accelerates the hybridization of cRNA or DNA nucleic acids. To determine if 3AB enhances the rate of hybridization between complements, several 5'-end-labeled RNA and DNA oligonucleotides (20 to 40 nucleotides in length) were incubated with cRNA and DNA templates (see Fig. 1). Incubations were carried out for 30 min in the presence or absence of 3AB (650 nM), and the material was run on a native polyacrylamide gel to separate bound and unbound oligonucleotides. Control reactions in which the oligonucleotide was hybridized to the template strand by heating and slow cooling were run to mark the hybrid positions on the gel (Fig. 3, lanes labeled B). The results showed that little or no hybridization occurred with any of the templates during a 30-min incubation in the absence of 3AB (lanes labeled C), while hybridized products were evident with all substrates in the presence of 3AB (lanes labeled D). For the DNA template and DNA primer (far-right lanes), a band migrating above the predicted hybrid location is also evident. The nature of this band is not clear, but it could be "supershifted" material that is bound to 3AB. The buffer used on the gel is designed to inhibit protein-nucleic acid interactions, but the inhibition may not be complete. Note that some material also stuck in the wells when the 765-nucleotide RNA template was used. The amount remaining in the wells seemed to be proportional to the amount of hybrid that formed rather than the presence or absence of 3AB as preformation of the hybrid (lanes labeled B) also resulted in some sticking. Taken together, the results show that 3AB can promote hybridization of complements irrespective of the type of nucleic acid or the sequence of the hybrid region.
An experiment was also performed with 3Dpol (650 nM) in the presence or absence of 3AB (650 nM) using the 765-nucleotide RNA and R40 as described above. The results showed that 3Dpol did not stimulate annealing and had no effect on stimulation by 3AB (data not shown).
Stimulation of hybrid formation results from 3AB and not a "contaminant." Since the 3AB preparation used here was purified after overexpression in E. coli, it is possible that a bacterial protein that contaminated the preparation was responsible for the observations. The procedure used yields homogenous preparations of 3AB as judged by Coomassie blue staining (32) and silver staining (data not shown). However, the possibility of a low-level contaminant cannot be ruled out. To test this, a sample was prepared using bacteria that were transformed with pGEX vector that did not contain the 3AB coding region (see Materials and Methods). Fractions from this purification that corresponded to the 3AB-containing fractions used in the assays were tested for hybridization activity using the 179-nucleotide RNA template and R20-1 (Fig. 4). These fractions (lanes 4, 5, and 6, at 1x, 2x, and 4x, respectively) showed no stimulation even when added at four times the equivalent amount (lane 6) relative to the 3AB-containing fraction (lane 2). In addition, mixing a 1x equivalent amount of the 3AB-negative fraction with 3AB had no effect on 3AB's stimulatory activity (lane 3). These results show that 3AB and not a contaminant is responsible for hybrid stimulation.
Optimal conditions determination for 3AB stimulation. The 179-nucleotide template and 20-nucleotide primer (R20-1) were used to determine optimal salt and divalent cation conditions for 3AB's annealing activity. A graph for these experiments is shown in Fig. 5. The 3AB concentration was 1,200 nM, and reactions were performed for 8 min. Optimal concentrations were 1 mM and 20 mM for MgCl2 and KCl, respectively. Note that in the absence of 3AB, annealing was slightly stimulated at higher MgCl2 and KCl concentrations as expected. With 3AB, there was a pronounced decrease in stimulation at higher (above 1 mM) MgCl2 concentrations, while increasing KCl between 20 and 80 mM had a more gradual effect.
A time course assay was also performed with the 179-nucleotide template and R20-1 using optimal conditions over 32 min with 1,200 nM 3AB. The results showed that 3AB stimulation was nearly linear for the first 10 min, followed by a gradual decline in rate (data not shown). Hybrid formation had not saturated after 32 min. Assays to determine optimal conditions and those used to test the rate of hybrid formation with various nucleic acid and 3AB concentrations (see below) were performed for 8 min. At this time point, products were easily measurable and the reaction was essentially in the linear phase.
3AB stimulation is concentration dependent and probably requires coating of the nucleic acid strands. Nucleic acid chaperones work by coating the nucleic acid strand rather than through a catalytic mechanism (14, 23). Therefore, large amounts of protein relative to the nucleic acid strands are typically needed to observe optimal chaperone activity. To test this, assays with different concentrations of 3AB (75, 300, and 1,200 nM) were performed with various levels of the 179-nucleotide RNA (2.5 to 80 nM) and a fixed concentration of primer R20-1 (10 nM). If coating is required for stimulation of annealing, then lower concentrations of 3AB should only stimulate with lower RNA concentrations, while larger amounts of 3AB should be able to stimulate at both low and high RNA concentrations. Representative autoradiograms from experiments with the three different 3AB concentrations are shown in Fig. 6A (75, 300, and 1,200 nM 3AB). At 1,200 nM, 3AB stimulated annealing with all concentrations of RNA (compare minus and plus 3AB lanes in the 1,200 nM experiment). For this amount of 3AB, at the lowest RNA concentration (2.5 nM) the level of stimulation was much lower than at 5 nM. This probably resulted from material stuck in the wells and "streaking" through the gel at this high 3AB concentration. This was not as prevalent when 75 or 300 nM 3AB was used. At 1,200 nM, the level of stimulation decreased as the concentration of RNA increased. This likely resulted from a lower proportion of the RNAs being adequately coated. At 300 nM 3AB, significant stimulation was only observed at the lower RNA concentrations (2.5, 5, 10, and 20 nM) while with 75 nM 3AB some stimulation was observed at the low RNA concentrations (2.5 and 5 nM), but not very much. This result is consistent with 3AB having to coat the template to exert its effect.
A second experiment in which the concentration of 3AB was varied and R20-1 and 179-nucleotide RNA were held constant at 10 nM and 5 nM, respectively, is shown in Fig. 6B. Significant stimulation was not observed at 3AB concentrations below 75 nM, and stimulation peaked when 300 to 600 nM 3AB was added. At these concentrations, there was approximately 1 3AB molecule per 13.3 (300 nM) and 6.6 (600 nM) total nucleotides in the reactions. A decrease in stimulation was observed at the 1,200 nM 3AB concentration, but streaking (see above) was also prevalent in this sample. Overall the experiment supports a mechanism that requires coating of the RNA for stimulation of annealing. This is especially true given the large amount of 3AB needed to observe stimulation.
3AB demonstrates helix-destabilizing activity and can "unwind" structured nucleic acid strands. Nucleic acid chaperones possess helix-destabilizing activity (14, 23). The strength of this activity can vary depending on the protein, with, for example, HIV NC possessing relatively weak destabilizing activity (23). To test the ability of 3AB to unwind nucleic acid secondary structures, an assay used for HIV NC protein (13) was adapted for 3AB. A diagram of the substrates is shown in Fig. 7A. In this assay, two 42-nucleotide complementary nucleic acid strands that form a defined stem-loop structure are used. One strand contained a DABCYL group (DAB in the figure) at the 3' end, while the other had a FAM group at the 5' end (see Materials and Methods). In this case, the predicted G value for each of the folded single strands was approximately –7.2 kcal/mol. Two types of assay systems were used: a gel shift assay or fluorescence quench-based assay (see below). For the gel shift assay, the 5' end of the DABCYL strand was labeled with 32P and the complementary strands (5 nM FAM and 10 nM DABCYL, respectively) were mixed in the presence or absence of 3AB (250 or 1,000 nM). In order to hybridize, the nucleic acids must first unwind to expose the complementary bases. After hybridizing, the larger more rigid hybrid migrates more slowly in the gel than the single-stranded DNA. An autoradiogram from an assay is shown in Fig. 7B. In the absence of 3AB, no hybridization was observed. Reactions with 3AB showed an increase in hybrid formation over time that was greater with the higher 3AB concentration (graphed in Fig. 7C). Control reactions in which 3AB was added along with the stop solution (time zero) showed that the solution prevented hybridization in the presence and absence of 3AB. These results indicate that 3AB can unfold and promote the hybridization of folded nucleic acid substrates.
A second more sensitive and quantitative assay based on fluorescence quenching was also used. As described above, one complement contained a fluorescing FAM probe at the 5' end while the other had a DABCYL quencher at the 3' end. Upon annealing, the DABCYL group is brought into close proximity to the FAM group and can quench fluorescence from FAM. This results in a decreasing level of fluorescence upon hybridization of the complements. We have previously used this assay to measure the helix-destabilizing activity of several HIV NC mutants (13). Plots for reactions with various concentrations of 3AB are shown in Fig. 7D, while 7E and F show plots for 3Dpol and HIV NC, respectively. Control reactions without 3AB or without the FAM-labeled complement showed essentially no fluorescence decrease under the conditions used (optimal conditions from above at 30°C with 5 nM FAM- and 10 nM DABCYL-derivatized nucleic acid strands). Annealing was stimulated by 3AB in a concentration-dependent manner, with the greatest stimulation observed at the largest amount of 3AB (1,000 nM). The results were consistent with the gel assay. Protein 3Dpol, which showed no annealing stimulation in the assays with RNA shown in Fig. 2 and 3, also showed no stimulation in this assay. However, since 3Dpol is an RNA binding protein and the substrate used here was DNA, this negative result would be expected even if 3Dpol had chaperone activity. HIV NC, an established chaperone protein, showed greater stimulation than 3AB, and only relatively small amounts of protein (about 125 nM) were required for nearly complete hybridization in less than 1 min (note different time scales and protein amounts for assays with HIV-NC and 3AB or 3Dpol). Increasing the HIV NC concentration beyond 125 nM had no significant effect on the reaction rate (data not shown). Overall the results show that 3AB has helix-destabilizing activity that can unwind nucleic acids and promote hybridization, though this activity is modest.
DISCUSSION
In this report, we show that poliovirus 3AB protein has properties consistent with a nucleic acid chaperone. The "classic" role for an RNA chaperone is to aid in folding of RNA to promote the most thermodynamically stable and generally functional structure. This is accomplished through stabilizing this structure as well as preventing or resolving misfolded RNAs that can lead to kinetically trapped nonoptimal species (14). Protein 3AB could serve such a role in poliovirus replication. In addition, there are many other places in the picornavirus life cycle where chaperone activity could play a role. These include (i) coating and protecting the nucleic acid, (ii) enhancing 3Dpol polymerization by helping to destabilize secondary structures in the genome, (iii) aiding in the initial binding of 3Dpol and other host and viral proteins through unwinding the RNA and making it more accessible, (iv) helping to separate positive and negative strands during synthesis, (v) and enhancing recombination or the completion of RNA synthesis by promoting the binding of incompletely synthesized positive- or negative-sense RNAs to complementary templates. Protein 3AB's roles in anchoring the replication complex to membranes, serving as a primer for RNA synthesis, and enhancing 3Dpol synthesis, are already well established (see the introduction); chaperone activity could be a factor in these and other functions not previously attributed to 3AB.
Protein 3AB accelerated the binding of oligonucleotides to complementary regions of RNA or DNA templates (Fig. 2 and 3). Previous reports indicated that 3AB is an RNA binding protein (see the introduction). Its ability to promote binding to both DNA and RNA indicates that it can also bind DNA. In this regard, it is similar to HIV NC, showing chaperone activity on both RNA and DNA (23). Note that enhancement of annealing by 3AB has been previously reported (34). In these experiments, the authors attributed the stimulation of 3Dpol synthesis by 3AB to promotion or stabilization of annealing for primers that normally anneal relatively poorly. Although the present work does not directly address the mechanism by which 3AB stimulates 3Dpol, a role in promoting primer binding is consistent with chaperone activity. The present work also indicates that 3AB can promote the hybridization of primers that form very stable hybrids with the template (R40, for example).
No chaperone-like activity was found for 3Dpol (see Fig. 2 and the Results section), despite previous results showing that this protein can bind to and coat nucleic acids (6). It is possible that the conditions used here did not allow 3Dpol coating or chaperone activity. No attempt was made to optimize conditions for 3Dpol, so chaperone activity for this protein cannot be ruled out. It should also be noted that complete coating in previous experiments required very high concentrations of 3Dpol, above the amounts used in these experiments (6). Therefore, it is possible that 3Dpol may have chaperone activity at concentrations well beyond where 3AB shows an effect. Protein 3Dpol also showed no helix-destabilizing activity in the assay shown in Fig. 7E; however, as was noted, the substrate was DNA and 3Dpol has only been shown to be an RNA binding protein. Therefore, no conclusions can be drawn from this assay.
The helix-destabilizing activity of 3AB was much less than that measured for HIV NC under the same conditions (Fig. 7D and F). Although this suggests that NC has greater helix-destabilizing activity than 3AB, only measurements performed under cellular conditions could confirm this. The assays were performed under optimal conditions for 3AB hybridization, and these may differ from conditions in the cell (see below). We know, for example, that NC's helix-destabilizing activity is extremely salt sensitive in the assay used here. Only a fraction of the activity remains at 80 mM KCl, as opposed to the 20 mM level used in these experiments (unpublished data). Currently 3AB is being tested under several conditions to determine how salt, pH, and divalent cation concentrations affect its helix-destabilizing activity. Also, the substrate used in the helix-destabilization assays was DNA, and it is possible that 3AB could show greater activity on RNA, the presumed substrate in vivo. Since HIV NC's activity is considered relatively weak (23), it is safe to say that 3AB's activity is also weak. A modest activity may be better suited to viral replication for a few reasons. First, viruses typically have several secondary structure motifs that are important to replication and a strong helix-destabilizing protein may disrupt these. Second, presumably a chaperone protein must be displaced by the polymerase during nucleic acid replication. A protein that binds too tightly, as a strong helix-destabilizing protein may do, could make this difficult.
In general, chaperone proteins work by coating nucleic acid strands. Results suggested that 3AB likely works this way as well. This is the most plausible explanation for the results observed in Fig. 6. Very large amounts of 3AB relative to the nucleic acid concentrations were required to observe significant stimulation. In the reactions with 5 nM 179-nucleotide RNA and 10 nM 20-base RNA oligonucleotide, peak stimulation was observed at about 300 to 600 nM 3AB (Fig. 6B). These concentrations correspond to approximately 6.5 and 13 nucleotides per 3AB molecule, respectively. It is known that each molecule of HIV NC coats approximately 7 nucleotides of nucleic acid (23). Although no experiments were done to directly determine the binding site size of 3AB, the rough estimates from the data above are in the range of what would be expected for complete coating of the nucleic acid in the reactions given that 3AB is approximately twice the molecular weight of NC. In addition, the rapid falloff of stimulation as the concentration of nucleic acid was increased in the experiment shown in Fig. 6A also supports a coating mechanism. When 300 nM 3AB was used, significant stimulation was only observed at lower nucleic acid concentrations (20 nM or below; Fig. 6A). With 75 nM 3AB, just a small amount of stimulation was observed at the lowest nucleic acid concentrations (2.5 and 5 nM; Fig. 6A). The lack of stimulation at the higher nucleic acid concentrations with 300 nM 3AB and the inability of 75 nM 3AB to stimulate strongly at any concentration clearly argue against a catalytic mechanism. In addition, a catalytic protein with helix-destabilizing activity would probably require an energy source such as nucleotide hydrolysis to function. No nucleotides or other potential energy sources were included in the reactions (except for those shown in Fig. 2). Note that this also argues strongly against the activity being a contaminant from E. coli. Because the 3AB preparations were essentially homogenous, as judged from gel analysis, any contaminants must have been present in very small amounts. Only an energy-requiring catalytic protein present at such low levels could have possibly demonstrated the chaperone-like activity found here.
The optimal conditions for 3AB annealing stimulation were 1 mM MgCl2 and 20 mM KCl (Fig. 5). Increasing the MgCl2 concentration resulted in a rapid falloff of stimulation, while higher KCl concentrations had a more gradual effect. Still, significant stimulation in comparison to that in reactions without 3AB was observed at all tested MgCl2 concentrations and all KCl concentrations below 160 nM. Although the concentration of Mg2+ in cells varies depending on the cell type and may change during infection, measurements of approximately 1 mM free and 8 mM complexed Mg2+ are essentially average (43). Therefore, the free Mg2+ concentration in cells is near optimal for 3AB activity. The concentration of K+ and other monovalent salts would be considerably higher than the 20 mM optimal. However, significant stimulation was observed even at much higher KCl concentrations. It is also possible that the concentrations of mono- and divalent cations in the vesicular structures where virus replication occurs are different from concentrations in other parts of the cell.
The need for an activity that helps anneal "primers" to nucleic acid is not immediately clear in the picornavirus replication process; however, it could help in recombination. In poliovirus, recombination is frequent and is proposed to occur by either "copy choice" or direct joining of RNAs (8, 9, 11, 17, 18, 26, 31, 41). In the copy choice mechanism, partially completed nucleic acid strands switch templates by binding to a complementary region on another RNA strand. The annealing and helix-destabilizing components of the chaperone activity could play an important role in this process or any of the others noted above. Further experiments, perhaps using mutants that lack chaperone activity while retaining other functions, will be required to understand the potential role(s) of this new activity.
ACKNOWLEDGMENTS
We thank Stephen Plotch (formerly of Lederle Pharmaceuticals) for expression clones for 3AB and 3Dpol and for help in purification of these proteins. We thank Charles McHenry (University of Colorado) for the HIV NC overexpression plasmid.
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