From the Department of Molecular Biology and Biochemistry,
University of California, Irvine, California 92697
Poliovirus RNA replication requires the
activities of a viral RNA-dependent RNA polymerase,
3Dpol, in conjunction with several additional viral
and likely cellular proteins. The importance of both the 3A and 3B
coding regions has been documented previously by genetic tests, and
their biochemical activities have been the subject of several recent
investigations. In this study, we examined the previously reported
stimulation of 3D-catalyzed RNA synthesis by 3AB. We show that 3AB does
not stimulate RNA synthesis on templates that are stably base paired to
a primer, indicating that 3AB does not stabilize or otherwise activate
3Dpol for chain elongation. Similarly, it does not alter
the kinetic parameters or binding affinities of 3D for substrates. In
the absence of a primer, or in the presence of a primer that does not
form a stable hybrid with the template, 3AB increases the utilization
of 3'-hydroxyl termini as sites for chain elongation by 3D, and thereby
stimulates RNA synthesis. 3AB may interact with and stabilize these
sites and/or may recruit 3Dpol to the site, resulting in
stimulation of the initiation of elongation events. We propose that
this activity is required for stabilizing weak interactions that occur
during nucleotidyl-protein-primed initiation events in the viral RNA
replication complex.
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INTRODUCTION |
Poliovirus-infected cells contain an RNA-dependent RNA
polymerase activity that has been attributed to a virally encoded
protein, now known as 3Dpol (1-3). A great deal of work
over the past 25 years from many laboratories has been devoted to the
purification and characterization of this enzyme, and several
significant characteristics of 3Dpol and its biological
activities have been determined. The poliovirus RNA polymerase is a
52-kDa protein that has been purified in large quantities to yield a
protein with very few, if any, detectable contaminants (4-6). Detailed
x-ray structural studies of a major portion of the protein have been
completed (7) and establish the basic features common to all types of
nucleic acid polymerases (DNA-directed as well as RNA-directed); these
include "fingers," "palm," and "thumb" subdomains (8, 9).
The enzyme is dependent on both a template and a primer for activity
and is responsible for RNA chain elongation during poliovirus RNA
replication in infected cells. Although the natural primer for RNA
synthesis in poliovirus-infected cells remains to be definitively
identified, synthetic primers are commonly used in the laboratory for
assay and characterization of 3Dpol activity. It was
proposed some years ago that structural elements in the RNA template
may provide a mechanism for self-priming (10); however, a more current
model involves protein priming (11). The enzyme has no apparent
template specificity by itself, and yet it is known that in an infected
cell 3Dpol replicates only viral RNA.
The genome-linked protein, VPg (3B), plays an essential role in viral
RNA replication, as mutations in the 3B coding sequence may abrogate
RNA synthesis (12). It is found on the 5'-ends of all nascent RNA
strands, including those not yet completed (13-15), indicating that
attachment occurs at the time of or shortly after initiation of RNA
synthesis. These observations led to the suggestion that VPg may serve
as primer for initiation of RNA strand synthesis. Several polypeptides
containing VPg sequences, including 3AB, have been detected in infected
cells by immunoprecipitation with anti-VPg antibodies (16-18). These
polypeptides are enriched in membrane fractions containing the viral
RNA replication complexes isolated from infected cells. Using a
partially purified 3Dpol preparation, a "host factor,"
and a poliovirion RNA template, product RNA was digested with RNase,
and residual material was immunoprecipitated with anti-VPg serum;
polyacrylamide gel electrophoretic analysis of this material showed 49 and 14 kDa proteins, the latter presumably 3AB, associated with
residual RNA (19). This important result suggested that 3D and other
proteins resident in this 3D preparation were sufficient to catalyze
the transfer of 3AB to nascent RNA. Mutation of 3A generated a
temperature-sensitive virus characterized by decreased synthesis of
viral minus and/or plus strands at a nonpermissive temperature (20,
21). It remains unclear, however, whether VPg (3B), 3AB, or some other
precursor form serves as the donor of VPg to nascent RNA strands.
Previous reports have shown that 3AB stimulated 3D activity
50-100-fold in a poly(A)-oligo(dT) template-primer system (22, 23).
With a heteropolymeric template and primer there was also substantial
stimulation (approximately 10-fold) of product formed in the presence
of 3AB. For unexplained reasons, the products were of varied sizes,
both in the presence or absence of 3AB, including some quite large
products greater than dimer length. With a poliovirion RNA template
primed with oligo(dT), stimulation of 3D-catalyzed product formation by
3AB was 5-10-fold. In a different study, Plotch and Palant (24) used
poliovirus minus strands in a primer-independent reaction with a high
concentration of 3D; they reported that they observed about 75-fold
stimulation of 3D activity by 3AB with no increase in the rate of chain
elongation.
In addition to measurements of 3AB effects on
template-dependent RNA synthesis by 3Dpol,
other reported 3AB activities include: (1) 3AB binds to 3CD and 3D (24,
25). (2) 3AB assists 3CD in forming a complex with the cloverleaf
structure at the 5'-end of poliovirus plus strands and at the 3'-end of
plus strands (26). (3) 3AB stimulates cleavage of 3CD to form 3C plus
3D (25). (4) 3AB binds to RNA in a sequence nonspecific manner (27).
Interestingly a mutation, K107E, in 3AB decreased RNA binding and RNA
plus and minus strand synthesis in mutant virus-infected cells. (5) 3AB
associates with membranes as an integral membrane protein during
in vitro translation (28).
It is not known whether any or all of these activities observed
in vitro reflect significant contributions to the RNA
replication reaction in an infected cell. Indeed, a biochemical
mechanism for protein priming of RNA strand elongation has not been
defined. Several laboratories have reported the formation of
uridylylated forms of 3B (VPg), both in vivo and in
vitro, that could form weakly base paired hybrids with the
3'-terminal nucleotides of poliovirus plus and minus strand RNAs; and
it is assumed that these nucleotidyl-proteins serve as primers for RNA
synthesis. In this report we show that 3AB does not stimulate
3D-catalyzed reactions on templates that are stably base paired to a
primer. In the absence of primer (or in the presence of a primer that does not form a stable hybrid with the template), 3AB allows more efficient utilization of 3'-hydroxyl termini that transiently form
duplex regions. 3AB may interact with and stabilize these sites and/or
may recruit the 3D polymerase to the site, resulting in stimulation of
the initiation of elongation events. We suggest that this activity of
3AB allows initiation to occur on templates formed by weak interactions
during nucleotidyl-protein-primed initiation events.
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EXPERIMENTAL PROCEDURES |
Materials--
Restriction enzymes were obtained from Boehringer
Mannheim, Life Technologies, Inc., or New England Biolabs, Inc. The
expression vectors, pGEM-4Z and pGEM-3Z, and RNasin were obtained from
Promega. Calf intestinal alkaline phosphatase, proteinase K, and T4 DNA ligase were from Boehringer Mannheim. Isopropyl
-D-thiogalactopyranoside was obtained from Sigma. DNA
primers were synthesized by Genemed Biotechnologies, Inc.
Escherichia coli JM110 was provided by Bert Semler,
University of California, Irvine. E. coli
BL21(DE3)pLysS-pT5T-3D was a gift from Steve Schultz, University of
Colorado, and E. coli BL21(DE3)-pT7 lac 3AB was a gift from
Aniko Paul and Eckard Wimmer, State University of New York, Stony
Brook. The in vitro transcription kits, T7 MEGAscript and
SP6 MEGAscript, were purchased from Ambion, Inc. [3H]UTP
(NET-380), [
-32P]CTP (BLU508H), and
[
-32P]UTP (BLU507H) were obtained from NEN Life
Science Products.
Construction of Expression Vectors--
Poliovirus genome
sequences 1-72 were inserted into the expression vector, pGEM-4Z,
which was modified to remove sequences between the SmaI and
HindIII sites in the multiple cloning site. The vector was
then digested with EcoRI and Asp718 and
dephosphorylated with calf intestinal alkaline phosphatase and the
large fragment was agarose gel purified and isolated with the Qiaex II
extraction kit (Qiaex, Inc.). The plasmid pT7N224- (29) was digested
with EcoRI and Asp718 and the
70-bp1 fragment was isolated
by agarose gel electrophoresis. This fragment was inserted into the
vector fragment with T4 DNA ligase and used to transform E. coli JM109 cells to obtain pGEM-4Z-PV1-72. The plasmid was
verified by restriction digests and gel analyses.
Poliovirus sequences 5522-5627 were inserted into the expression
vector, pGEM-3Z. The vector was digested with EcoRI and
HindIII and dephosphorylated with calf intestinal
phosphatase; the 2693-bp fragment was isolated by agarose gel
electrophoresis and extraction with Qiaex II beads, as above.
Poliovirus cDNA sequences were obtained by amplification by the
polymerase chain reaction (PCR) using the plasmid, pEXC-3D
(30) and the following primers:
PV5627(
)(5'-GAGCCCAAGCTTCGAGCGCTTTGGCATCC-3') and
PV5522(+)(5'-GCGAATTCGGAGTCCACGACAACGTGGC-3').
Taq polymerase reactions contained 10 mM
Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM
MgCl2, 0.3 µM of each primer, 15 ng of
pEXC-3D, 200 µM dNTPs (Boehringer Mannheim), and
Taq polymerase. Reactions were performed in 100 µl for 35 cycles of 30 s at 96 °C, 30 s at 55 °C, and 2 min at
72 °C. PCR products were extracted with phenol-CHCl3,
precipitated with isopropanol, and digested with EcoRI and
HindIII. The 109-bp fragment was resolved on a 1.5% agarose
gel, and the selected DNA was extracted with Qiaex II beads. Thereafter
the vector and poliovirus 109-bp cDNA fragment were ligated and
transformed into E. coli JM110. The correct plasmid, pGEM-3Z-PV5522-5627, was verified by restriction mapping.
In Vitro RNA Transcription and Preparation of Hybrid
Substrates--
Plasmid pGEM-4Z-PV1-72 was digested with
RsaI, and extracted DNA was transcribed with SP6 RNA
polymerase (Ambion, Inc.) to yield 36 nt plus strand RNA.
Alternatively, pGEM-4Z-PV1-72 was digested with EcoRI, and
extracted DNA was transcribed with T7 RNA polymerase (Ambion, Inc.) to
yield 88 nt minus strand RNA (complementary to poliovirus plus strand
at nt 1-72).
Plasmid pGEM-3Z-PV 5522-5627 was digested with HindIII and
SspI, and extracted DNA was transcribed with T7 RNA
polymerase to produce 118 nt poliovirus plus strand sequences.
Alternatively, the plasmid was digested with ClaI and
PvuII, and extracted DNA was transcribed with SP6 RNA
polymerase to produce 55 nt poliovirus minus strand sequences.
All transcription reactions (20 µl) contained 1 µl of 60 µM [3H]UTP to facilitate quantitation of
transcipts made. Hybrid structures were then formed based on a 1 to 1 molar ratio of plus and minus strands for either the poliovirus 1-72
hybrid or for the poliovirus 5522-5627 hybrid in 100 mM
NaCl to yield solutions of 40 pmol of hybrid per µl. Samples were
heated at 65 °C for 30 min and then slow cooled to 37 °C over 25 min; these solutions could be stored at
20 °C. Hybrid formation
was confirmed for similarly synthesized 32P transcripts and
analysis of putative hybrids in 6% polyacrylamide gels containing 7 M urea and TBE buffer (89 mM Tris base, 89 mM boric acid, and 2 mM EDTA, pH 8.3).
Preparation of Purified Transcripts or Transcripts Synthesized
from PCR-derived cDNA--
T7 or SP6 transcripts, synthesized from
DNA digested with the appropriate restriction enzymes, were
fractionated in 6% polyacrylamide, 7 M urea gels (TBE
buffer) that were 1.5-mm thick at 8 watts (constant) until the
bromphenol blue dye migrated about 80% to the bottom. The gel was then
stained with ethidium bromide (0.5 µg/ml) in TBE buffer at room
temperature for 30 min. Transcripts were visualized under ultraviolet
radiation and appropriate transcripts were excised. These gel bands
were dispersed through an 18 gauge needle, and RNA was extracted with 3 vol of 0.5 M ammonium acetate, 10 mM EDTA, pH
7.5, at 4 °C overnight. The mixture was centrifuged, the supernatant
collected, and RNA was precipitated with 2.5 vol of 100% ethanol; the
concentration of the transcripts was determined by absorbance at 260 nm.
Defined transcripts were also obtained by transcription of
PCR-generated cDNA. Using pGEM-4Z-PV1-72 for a template and
the following primers,
GEM-4Z-2488(+)(5'-GCAGATTGTACTGAGAGTGCACCATATGC-3') and
PV1-72(
)(5'-CTCTAATACGACTCACTATAGGG-3'), cDNA was amplified and extracted, as described above. The cDNA was digested either with RsaI or with EcoRI, and the DNA was
fractionated in a 1.5% agarose gel. The 108-bp fragment was isolated
from the EcoRI-digested DNA, or the 285-bp fragment was
excised from the RsaI-digested DNA. The DNA was extracted
with the Qiaex II kit. The 108-bp DNA and the 285-bp DNA were
transcribed with T7 RNA polymerase and SP6 RNA polymerase,
respectively, with the Ambion MEGAscript kits, as above.
Preparation of Poliovirus 3D and 3AB--
Poliovirus 3D
polymerase was expressed in E. coli BL21(DE3)pLysS-pT5T-3D
cells (31) at room temperature in medium containing 16 g/liter
tryptone, 10 g/liter yeast extract, 4 g/liter NaCl, 30 mM
K2HPO4, and 5 mM glucose, pH 8.3, under ampicillin (100 µg/ml) and chloramphenicol (25 µg/ml)
selection and were induced with 0.5 mM isopropyl
-D-thiogalactopyranoside when the
A600 reached 0.5; induction was continued for
20 h. RNA polymerase was purified from these cells essentially as
described previously (6, 32), including preparation of a soluble
lysate, 0-40% saturation ammonium sulfate precipitation,
phosphocellulose chromatography, and two Mono Q column
purifications.
Poliovirus 3AB was expressed in E. coli BL21(DE3)-pT7lac3AB
and purified as described by Lama and Carrasco (33) and Lama et
al. (22). Both 3D and 3AB preparations were stored in Buffer A (50 mM Tris-HCl, pH 8.0, 50 mM KCl, 5 mM
-mercaptoethanol, 0.1% Nonidet P-40, and 10%
glycerol) at
70 °C.
Assay of 3D Elongation Activity--
Poliovirus 3D polymerase
assay mixes contained 50 mM HEPES, pH 8.0, 0.5 mM of each nonlabeled NTP, 10 µM of
[32P]CTP or [32P]UTP (0.2 µCi/µl of
assay mix), 4 mM dithiothreitol, 3 mM magnesium acetate, 0.8 µM 3H-hybrid or
3H-template, 1 unit/µl RNasin, plus or minus 0.72 µM 3AB, and variable amounts (3-600 nM, but
usually 10 nM) of 3D. Reactions were started by the
addition of 3D on ice, and aliquots (20 µl) were removed at zero time
and after 30 min at 30 °C for acid precipitation. Precipitates were
collected on Whatman GF/C filters and washed with 1 M HCl,
0.1 M sodium pyrophosphate and ethanol, dried, and counted
in scintillation fluid.
Oxidation of Template Strands--
3H-labeled T7 RNA
polymerase-generated (
) strands (PV1-72) (1000 pmol) in a 50-µl
reaction were incubated with 1 mM sodium periodate (Sigma)
and 2 mM HCl at room temperature for 60 min in the dark.
Then glycerol (0.68 M) was added for a further incubation at room temperature for 30 min in the dark to destroy excess periodate. Finally, the solution was diluted to 200 µl, containing 50 µg of
yeast tRNA and 0.5 M ammonium acetate, 10 mM
EDTA, and RNA was precipitated with an equal volume of isopropanol,
collected by centrifugation, washed with ethanol, and dried. The RNA
was dissolved in 10 µl of 10 mM Tris-HCl, pH 7.5, 1 mM EDTA. This oxidized template was used directly in 3D
reactions or annealed with [3H]SP6(+) strands to make
hybrid structures, as described above.
Analysis of Products of 3D Reactions--
3D reactions with
template alone or with hybrid substrates (PV1-72 or PV5522-5627) plus
or minus 3AB were incubated, as described above. Aliquots (20 µl)
were removed for analysis of native RNA products, supplemented with 50 µg of glycogen (Boehringer Mannheim) or yeast tRNA carrier, and
extracted with phenol-CHCl3. RNA was precipitated with
isopropanol, washed with ethanol, and dried in a Speed-Vac (Savant).
Pellets were dissolved in 90% formamide (Fluka), TBE buffer, 0.025%
bromphenol blue, and 0.025% xylene cyanol prior to gel analysis.
RNA samples were glyoxalated to analyze RNA strands (34). Aliquots (20 µl) of 3D reactions supplemented with 50 µg of yeast tRNA were
extracted, precipitated, and dried, as described above. Pellets were
dissolved in a total of 30 µl containing 10 mM sodium phosphate, pH 7.0, 50% dimethyl sulfoxide (Fluka), and 1 M
glyoxal (Fluka) freshly deionized with Bio-Rad 501-x8(D), and incubated at 50 °C for 60 min. Samples were diluted with an equal volume of
0.8 M LiCl, and RNA was precipitated with 2.5 vol of
ethanol. Precipitates were collected in a Biofuge 13 (Heraeus) at
4 °C, 13,000 rpm, 30 min, and dried; pellets were dissolved in TBE
buffer, 8 M urea, 0.025% bromphenol, 0.025% xylene cyanol
(TBE/urea sample buffer) prior to analysis in a polyacrylamide/urea
gel, as described below.
RNase A digestion of aliquots (20 µl) from 3D reactions was used to
determine lengths of duplex segments in RNA products. RNA samples were
adjusted to 0.3 M NaCl and digested directly with 20 µg/ml of RNase A (preheated in 0.1 M sodium acetate, pH 5.2, at 80 °C for 10 min) at 30 °C for 30 min. Then glycogen
carrier (50 µg) was added and samples were extracted with
phenol-CHCl3, precipitated with isopropanol, dried, and
finally dissolved in TBE/urea sample buffer, as described previously
for gel analysis.
S1 nuclease digestion was performed by dilution of a 20-µl aliquot of
a 3D reaction to 30 µl, containing 0.3 M NaCl, 30 mM sodium acetate, pH 4.5, 3 mM
ZnSO4, 10 µg/ml alkali-digested salmon sperm DNA, and 1 unit of S1 nuclease and incubating at 45 °C for 15 min. Samples were
prepared for gel analysis, as described for RNase A-treated
samples.
Proteinase K (Boehringer Mannheim) digestion of aliquots (20 µl) from
3D reactions was done by adjusting to 30-µl reactions containing
0.5% SDS, 10 mM Tris-HCl, pH 7.5, 10 mM NaCl,
and proteinase K (50 µg/ml) and incubating at 37 °C for 30 min.
Thereafter samples were prepared for gel analysis, as described for
RNase-treated samples.
Routine product analyses were in 6% polyacrylamide, 7 M
urea gels in TBE buffer (20-cm wide × 14-cm long × 0.8-mm
thick), and the bromphenol blue dye front was run about 80% down the
gel at 12 watts (constant power). Under these conditions (samples were
not boiled before application to the gel), stable RNA hybrids were not
denatured in 7 M urea; only intrastrand secondary
interactions were eliminated. After glyoxal treatment, RNA strands were
fully separated and prevented from subsequent interaction. The gel was dried directly on Whatman 3MM sheets at 80 °C in a vacuum (Bio-Rad gel dryer). [32P]RNA bands were detected by
autoradiography on Kodak BioMax MR film.
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RESULTS |
Characterization of a 3AB-stimulated 3D-catalyzed Reaction--
A
defined heteropolymeric substrate was constructed to facilitate
interpretation of the analysis of products formed under various
conditions and to allow systematic characterization of the reaction
properties. The substrate, PV1-72, shown schematically in Fig.
1A, contained a partial duplex
(25 bp) with two 5'-overhangs of 11 and 63 nt. The shorter strand in
the duplex included nt 1-21 of poliovirus plus strand RNA, and the
longer strand included 72 nt of poliovirus minus strand RNA. The
relative amounts of each strand required to form a hybrid were
titrated; hybrid formation was monitored by polyacrylamide/urea gel
electrophoresis. When used in 3D-catalyzed RNA polymerase reactions,
the hybrid substrate was usually present at 0.8 µM, at
which level the reaction rate was independent of substrate
concentration.

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Fig. 1.
Hybrid substrates used for poliovirus RNA
polymerase reactions and the expected products. Schematic
structures of RNA are depicted with bars for poliovirus
sequences, lines for vector sequences, and wavy
lines for 32P-labeled RNA strands filled in by the
polymerase reaction. Connecting lines between strands denote hybridized
segments. A, hybrid PV1-72 contains an 88-nt strand
containing poliovirus minus strand sequences and a 36-nt strand
containing plus strand sequences. Single-strand overhangs are 11 nt and
63 nt. Native products (1) and (2), denote
fill-in with 32P-labeled segments on the unpaired
overhangs. Detectable radioactive strands (1') and
(2') are derived from native products (1) and
(2), respectively. B, hybrid PV5522-5627
contains a 118-nt strand containing poliovirus plus strand sequences
and a 55-nt strand containing minus strand sequences. Single-strand
overhangs are 72 nt and 9 nt. As in A,
32P-labeled native products, (1) and
(2), and their labeled denatured strands, (1')
and (2') are shown, respectively.
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Addition of 3AB to the reaction resulted in an approximately 10-fold
stimulation of UMP incorporation (see below). In preliminary experiments, we confirmed that 3AB had no effect on chain elongation rates (24). The Km for NTPs or for hybrid substrate was unaffected by 3AB (Table I). In
addition, the concentration of 3AB required to achieve maximal
stimulation did not vary with the size of single-strand segments in the
template over a 50-fold range (63 nt compared with 3000 nt, data not
shown); thus 3AB did not appear to coat single-strand segments of the
template to form a more active substrate for 3Dpol.
Fig. 2, lane 2, shows an
autoradiogram of the products formed by poliovirus 3Dpol on
the PV 1-72 substrate. The major product represents copying of the long
5'-overhang to generate a mostly double-stranded RNA product (Fig.
1A, (1) fill-in product, see below). In addition, a less abundant product of slightly lower mobility is produced. A
fraction of the product (about 10%) remained in the sample well, representing either aggregated or very large RNA product. This was not
due to residual unincorporated nucleotide, since it formed during
incubation (Fig. 2, lanes 1 and 2). Addition of
3AB to the reaction resulted in an 11-fold stimulation of activity
(lane 4), although the vast majority of the stimulated
product was large or aggregated material. (Note that less
3Dpol was utilized in the 3AB-stimulated reaction.) In
addition, there was increased synthesis of the product with slightly
lower mobility than the fill-in product. No product was formed by 3AB,
in the absence of 3Dpol (lane 6).

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Fig. 2.
Products of poliovirus 3Dpol
reactions in the absence and presence of 3AB. PV1-72 hybrid
substrate, prepared from unpurified transcripts, was incubated with 15 nM 3D (lanes 1 and 2), 5 nM 3D and 720 nM 3AB (lanes 3 and
4), or 720 nM 3AB (lanes 5 and
6) for 0 min (lanes 1, 3, and
5) or 60 min (lanes 2, 4, and
6). Aliquots were removed at the indicated times, and RNA
was purified and analyzed in a 6% polyacrylamide, 7 M urea
gel. The gel was dried and autoradiographed.
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Utilization of the template shown in Fig. 1A required a
primer strand, as expected for 3D-catalyzed reactions, although a low
level of primer-independent activity generated two bands plus some
large or aggregated material (Fig. 3,
lane 1). 3AB greatly stimulated production of these products
plus additional ones (lane 3), suggesting an increased
utilization of transient self-primed template structures. As seen
above, in the presence of the hybrid substrate, 3D catalyzed the
fill-in of unpaired single-strand segments to generate duplex products
(Fig. 1A, (1) and (2)) in addition to
some large, unresolved material (Fig. 3, lane 2). 3AB
stimulated this reaction 5-fold, generating predominantly large
products and lesser amounts of two duplex species (Fig. 1A,
(1) and (2)), one of which was greatly enriched
in the 3AB-stimulated reaction (Fig. 3, lane 4). If
nonpoliovirus proteins (ovalbumin or cytochrome c) are
substituted for 3AB in the 3D reactions, the products are the same as
those formed with 3D alone, and there is no stimulation of activity or
change in distribution of products (data shown below).

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Fig. 3.
Native and denatured products from 3D
reactions in the absence and presence of 3AB. Hybrid or template
substrates were prepared from unpurified transcripts. After each 3D
reaction of 30 min, samples (20 µl) were removed,
phenol-CHCl3 extracted, and RNA was precipitated.
32P-Labeled products were untreated (lanes 1-4)
or glyoxal-denatured (lanes 9-12) and fractionated in a
polyacrylamide/urea gel and visualized by autoradiography.
32P-Labeled PV1-72 template (88 nt), primer (36 nt), and
hybrid were analyzed in lanes 5, 6, and
7, respectively, as markers, and lane 8 contains
denatured 32P-template.
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This initial characterization of products of the 3D reaction presents
the following issues, which will be addressed below. (1) Stimulation of
the 3D reaction by 3AB results in formation of large products. (2) In
reactions containing 3D plus 3AB, a second product migrating slower
than fill-in product is preferentially enhanced. (3) The addition of
3AB to a 3D-catalyzed reaction greatly amplifies a primer-independent
reaction.
Characterization of Large Products Formed in the Presence of
3AB--
Denaturation of the products from a reaction containing 3AB
(Fig. 3, lanes 11 and 12) with template alone or
hybrid substrates had little or no effect on the mobility of the
predominant large products. As previously noted under "Experimental
Procedures," glyoxal treatment was essential to fully denature
RNA-RNA duplexes and prevent subsequent strand interactions. The
persisting large products were, therefore, not the result of
aggregation of unit length strands. In reactions with template alone,
the same denatured strands were observed in the 3D reaction with and
without 3AB, although 3AB stimulated the reaction considerably
(lanes 9 and 11); thus 3AB appears to facilitate
utilization of terminal 3'-hydroxyls as primers for elongation, perhaps
stabilizing short snapback structures.
To examine the possibility that large product was formed by end
addition to the template strand, we prepared 32P-hybrid
substrate for a 3D-catalyzed reaction with unlabeled nucleotides in an
effort to observe conversion of starting template to large product.
Very little substrate is converted to product, however, even in the
presence of high concentrations of 3D, so no significant accumulation
of labeled product was detected (data not shown). As an alternate
approach, we oxidized the 3'-hydroxyl of the template strand with
periodate to prevent end addition and examined large product formation
(Fig. 4). In primer-independent reactions
with 3D plus 3AB (Fig. 4, lanes 3 and 6), large
products were not synthesized on the oxidized template (lane
3). However, with a hybrid substrate, large products were formed
with or without an oxidized template strand (lanes 2 and
5). In this case, synthesis must have occurred by addition
to primer strands. The end addition reaction is dependent on 3AB since
with 3D alone using the hybrid substrate, the expected fill-in products
(Fig. 1A, (1) and (2)) were
synthesized with or without oxidized template strands, and there was
little accumulation of large products (lanes 1 and
4). These data suggested that end addition to RNA present in
either the template or primer preparations generated the very large
product RNAs. Examination of 32P-labeled template, primer,
and hybrid extracted directly from the transcription mix revealed the
presence of a small population of contaminating long RNAs (Fig. 3,
lanes 5-8 and Fig. 4, lanes 7 and 8).
These transcripts are likely generated from a small amount of DNA that
remains uncut after intended linearization. Interestingly, this
subpopulation of RNA is efficiently recognized by 3AB and thereby
apparently made available for elongation by 3D. Their length and
possible snapback or other structural folds may provide multiple
opportunities for use as templates, especially with 3AB interaction and
allows for large amounts of labeled nucleotide incorporation into small
molar quantities of product.

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Fig. 4.
Effect of oxidation of the 3'-hydroxyl of the
template strand on product formation. Hybrid and template
substrates were prepared from unpurified PV1-72 transcripts. Oxidized
templates were used in the substrates in lanes 1-3, and
untreated template strands were used in substrates for lanes
4-6. 32P-Labeled products from 3D-catalyzed reactions
with or without 3AB were obtained after 30 min of incubation; RNA was
extracted, precipitated, and fractionated in a polyacrylamide/urea gel
and was detected by autoradiography. Lane 7 contains marker
32P-labeled unoxidized template (88 nt), and lane
8 contains marker 32P-labeled hybrid (unoxidized
template strand).
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Reactions Utilizing Hybrids Derived from Purified
Transcripts--
To test whether contaminating long transcripts were
the source of the large products, unit-length transcripts (88 nt and 36 nt) were purified from preparative acrylamide gels and used to form
hybrid substrates for 3D. The PV1-72 hybrid was used in reactions with
3D alone (Fig. 5, lanes 1-4)
or with 3D plus 3AB (lanes 5-8). With these purified RNAs,
there was no stimulation of 3D activity by 3AB, and large products were
not formed in the presence or absence of 3AB. These results confirmed
the prediction that the large products were formed by end addition to
contaminating transcripts. The reaction with 3D plus 3AB still yielded
two different species of product (Fig. 5, lanes 5-8; Fig.
1A, (1) and (2)).

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Fig. 5.
Products from PV1-72 hybrid substrate
prepared from gel-purified transcripts. Native products from
3D-catalyzed reactions were obtained from aliquots removed at 2, 5, 15, and 30 min as indicated. Reactions were incubated in the absence
(lanes 1-4) or presence (lanes 5-8) of 3AB.
Products were prepared and analyzed as in Fig. 4.
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A second method to prepare transcripts without contaminating long RNA
strands was investigated. PCR-generated cDNA, which contained the
coding sequences for selected RNAs (108 bp and 285 bp for minus and
plus strand transcripts, respectively), was prepared for transcription
reactions to yield RNA for the formation of the same PV1-72 hybrid
structure (Fig. 6, lanes
5-7). Under similar reaction conditions where the unpurified
transcripts of linearized plasmids yielded large products in reactions
with 3D plus 3AB (Fig. 6, lanes 3 and 4),
transcripts obtained from PCR-derived cDNA gave hybrid substrates
that did not yield large products with 3D plus 3AB and demonstrated no
3AB stimulation of 3D activity (lanes 8-11). Thus, large
products are due to contaminating RNAs that form potential templates in
primer-independent reactions; stimulation by 3AB is due to enhanced
recognition and utilization of these potential substrates.

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Fig. 6.
Products from 3D reactions using hybrid
substrates containing nonpurified transcripts (lanes 1-4)
or transcripts from PCR-generated cDNA (lanes
8-11). Products were obtained from reactions in the absence
(lanes 1, 2, 8, and 9) and
presence (lanes 3, 4, 10, and
11) of 3AB after 5 and 30 min. Aliquots (20 µl) were
removed, and RNA was isolated by phenol-CHCl3 extraction
and precipitation. 32P-Labeled template (lane 5,
88 nt), primer (lane 6, 36 nt), and hybrid (lane
7) served as RNA markers. RNA was fractionated in a
polyacrylamide/urea gel and analyzed by autoradiography.
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3AB Stimulation of the 3D Reaction as a Function of 3D
Concentration--
Other investigators have reported maximal 3AB
stimulation of 3D activity at low 3D concentrations using a
heteropolymeric hybrid or template substrate (22-24). We examined 3AB
stimulation as a function of 3D concentration between 3 nM
and 600 nM 3D. 3AB added to hybrid derived from unpurified
transcripts of linearized plasmids gave increasing stimulation with
decreasing 3D concentration with a maximum of 5-10-fold stimulation at
3 nM 3D in repeated experiments (Fig.
7). Above 30 nM 3D there was
no 3AB stimulation. With hybrid substrate derived from purified
transcripts there was no 3AB stimulation over the entire 3D
concentration range, supporting the conclusion that 3AB stimulation
occurred on contaminating primer-independent templates. Furthermore,
3AB does not serve to stabilize active 3D at low protein
concentrations.

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Fig. 7.
Stimulation of 3D polymerase elongation
activity as a function of 3D concentration. 3D activity was
measured in the presence and absence of 3AB at each 3D concentration
with PV1-72 hybrid substrate. No stimulation by 3AB is depicted as
0 on the ordinate. Crude transcripts refer to
hybrid substrates formed from nonpurified transcripts, and purified
transcripts (Purif. transcripts) refer to hybrids formed
from gel-purified transcripts.
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Characterization of the Slower Mobility Species Induced by
3AB--
A second characteristic of the 3AB-stimulated reaction is the
marked enrichment of a product migrating as a discrete band slightly
slower than the fill-in product (Fig. 1A, (1);
e.g. Fig. 3, lane 4; Fig. 5, lanes
5-8; Fig. 6, lanes 4 and 11). To
characterize this slower mobility species, the products of a 3D plus
3AB reaction with PV1-72 hybrid substrate, formed from transcripts
synthesized from PCR-generated cDNA, were subjected to various
treatments (Fig. 8). The previously
observed fill-in products were formed in a reaction with 3D alone (Fig.
8, lane 1; Fig. 1A, (1)); the predominant product co-migrates, coincidentally, with the template strand (lane 5). The same fill-in products, plus a slower
migrating species, were formed in the reaction with 3D plus 3AB (Fig.
8, lane 2). (The formation of minor amounts of larger RNA
species, generated by end addition to unhybridized template or primer
strands that form transient snapback structures apparently stabilized by 3AB, can also be seen in this gel.) When these products were denatured with glyoxal, unit-length strands were formed for each reaction (Fig. 8, lanes 3 and 4; Fig.
1A, (1') and (2')). Thus the slower
migrating species formed with 3D plus 3AB had only unit-length
32P-labeled strands. S1 nuclease digestion of products was
incomplete (lanes 8 and 9) but generated for both
reactions RNA with a faster mobility. Single-strand segments were
completely digested with RNase A in high salt and showed, almost
exclusively, a single duplex segment for RNA derived from the 3D
reaction (lane 10), consistent with fill-in of the longer
5'-overhang of the hybrid substrate (Fig. 1A,
(1)). In contrast, the products from the 3D plus 3AB
reaction contained two major species of duplex segments, consistent
with the fill-in at either 5'-overhang in the hybrid but only one
overhang per molecule to yield duplexes of 88 and 36 bp (lane
11; Fig. 1A, (1) and (2),
respectively). This result further supports the interpretation that 3AB
increases utilization of 3'-hydroxyls, which appear unavailable for
utilization as primer for 3D alone. Finally, proteinase K digestion of
products did not alter the mobility of products (compare Fig. 8,
lanes 1 and 2 with lanes 12 and
13). Therefore 3AB association with RNA products does not
survive phenol-CHCl3 extraction and does not account for
the slower mobility species associated with the 3D plus 3AB reaction.

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Fig. 8.
Characterization of products from
3D-catalyzed reactions in the presence and absence of 3AB. PV1-72
hybrid substrate (0.8 µM) was formed from transcripts
synthesized from PCR-generated cDNA. Two 3D reactions were
prepared, one in the absence of 3AB and one in the presence of 3AB.
After 30 min of incubation, aliquots (20 µl) were removed, and RNA
products were untreated (lanes 1 and 2),
glyoxal-denatured (lanes 3 and 4), S1
nuclease-digested (lanes 8 and 9), RNase
A-digested (lanes 10 and 11), or proteinase
K-treated (lanes 12 and 13). Samples were
fractionated in a polyacrylamide/urea gel and analyzed by
autoradiography. Marker [32P]RNAs were template
(lane 5, 88 nt), primer (lane 6, 36 nt), and
hybrid (lane 7).
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These results show that the slower mobility species enriched among the
native products of the 3D plus 3AB reaction contains an extension of
the minus strand of the PV1-72 hybrid with the starting plus strand
unaltered (Fig. 1A, (2)). After denaturation only
minus strand from this species contains 32P and hence
denaturation yields full-length strands (Fig. 1A, (2')).
Primer-independent Reaction in the Presence of 3D Plus 3AB--
As
shown above, hybrid substrate prepared from transcripts of
PCR-generated cDNA produced no large products and no stimulation of
3D activity by 3AB, although there was increased utilization of the
minus strand 3'-hydroxyl to produce the partially duplex product with
reduced mobility (Fig. 9, lanes
1 and 2). Previous data (Fig. 3, lanes 1 and
3) also showed that addition of 3AB to a 3D-catalyzed
reaction greatly stimulated utilization of template as substrate in the
absence of primer. Fig. 9 (lanes 9 and 10) illustrates this stimulation of primer-independent 3D activity, using
PCR-generated cDNA transcripts to prevent generation of large RNA
products. With a substrate of template alone, the same products were
made in a 3D reaction (Fig. 9, lane 9) as in a 3D reaction
supplemented with 3AB (lane 10), ovalbumin (lane
11), or cytochrome c (lane 12), but only 3AB
specifically stimulated synthesis of each of the products, confirming
that 3AB promotes utilization of 3'-hydroxyls that may form transient
intramolecular base pairs. 3AB may stabilize these structures by
binding RNA, as well as binding 3D to create a potential site for
elongation.

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Fig. 9.
Effect of various proteins on 3D-catalyzed
reactions with hybrid or template alone. Transcripts for
substrates were synthesized from PCR-amplified cDNA. After a 3D
reaction of 30 min with the indicated supplements, samples (20 µl)
were removed, phenol-CHCl3 extracted, and RNA was
precipitated. The resulting 32P-labeled products were
analyzed in a polyacrylamide/urea gel by autoradiography. Samples in
lanes 1-4 were from reactions containing hybrid substrate.
Samples in lanes 9-12 were from reactions containing
template substrate alone. 32P-Labeled marker RNAs are
template (lane 5, 88 nt), primer (lane 6, 36 nt),
hybrid (lane 7), and two times unit length template
(lane 8, 176 nt).
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3AB Effects on the 3D Reaction Using a Different Hybrid
Substrate--
Another hybrid substrate, PV5522-5627 (Fig.
1B), was prepared to ensure that the 3AB effects on the 3D
reaction were not unique to the PV1-72 substrate. This substrate also
contained 5'-overhangs that potentially could be filled in with 3D in
elongation reactions. The poliovirus RNA sequences were derived from a
portion of the 3C coding region. Using gel-purified transcripts for
preparation of hybrid and template substrates, there was no
accumulation of large products and no 3AB stimulation of 3D elongation
activity (Fig. 10, lanes
1-4 and 9-12). Reaction with hybrid substrate gave fill-in product (lanes 2 and 4; Fig.
1B, (1) and (2)), which upon denaturation gave predominantly unit-length strands with or without 3AB, as before (lanes 10 and 12; Fig.
1B, (1') and (2')). The strands were 9 nt longer than the starting template strand because the primer strand
has a 9-nt extension beyond the 3'-end of the template. In an identical
experiment using crude transcripts for preparation of hybrid and
template substrates, large products comprised the predominant product
and 3AB stimulated the 3D reaction 12-fold with a hybrid substrate
(data not shown). Thus, the same pattern of results was observed for
both substrates.

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Fig. 10.
Utilization of PV5522-5627 hybrid and
template substrates in 3D-catalyzed reactions. Substrates were
obtained from gel-purified transcripts. After each 3D reaction of 30 min, samples (20 µl) were removed, phenol-CHCl3
extracted, and RNA was precipitated. 32P-Labeled products
were fractionated in a polyacrylamide/urea gel and analyzed by
autoradiography. Lanes 1-4 contain native products, and
lanes 9-12 contain glyoxal-denatured products. Marker
[32P]RNAs were template (lane 5, 118 nt),
primer (lane 6, 55 nt), hybrid (lane 7), and
glyoxal-denatured template (lane 8), all derived from
unpurified transcripts.
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However, some differences were observed with the two substrates. For
example, PV5522-5627 template alone produced very little product with
or without 3AB (Fig. 10, lanes 1 and 3 or
lanes 9 and 11), suggesting that no transient
snapbacks are formed that could be stabilized by 3AB. In addition,
little or no second product with slightly slower mobility was formed or
enriched by 3AB supplementation of the 3D reaction with the
PV5522-5627 hybrid substrate (Fig. 10, lanes 2 and
4). In some experiments, some slower mobility product was
detected (Fig. 11, lane
2).Thus, 3AB appeared to only poorly promote utilization of the
3'-hydroxyl of the plus strand in this hybrid over and above what 3D
could do alone.

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Fig. 11.
Characterization of products from
3D-catalyzed reactions with PV5522-5627 hybrid substrate. Hybrid
substrate was formed from gel-purified transcripts. Reactions in the
absence or presence of 3AB were incubated for 30 min. Samples (20 µl)
were removed, and RNA products were untreated (lanes 1 and
2), glyoxal-denatured (lanes 3 and 4),
or digested with RNase A in high salt (lanes 9 and
10). Samples were fractionated in a polyacrylamide/urea gel
and analyzed by autoradiography. Marker [32P]RNAs were
from unpurified transcripts, glyoxal-denatured template (lane
5), untreated template (lane 6, 118 nt), primer
(lane 7, 55 nt), and hybrid (lane 8).
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Additional characterization of the products of the PV5522-5627 hybrid
reactions without and with 3AB are illustrated in Fig. 11. Native
products were predominantly fill-in products, i.e. there was
conversion of hybrid substrate (marker shown in lane 8) to fill-in product (lanes 1 and 2); both end fill-in
products are detectable in the 3D plus 3AB reaction in this experiment
(lane 2; Fig. 1B, (1) and
(2)). Denaturation with glyoxal produced unit-length strands
(about 9 nt longer than denatured template strands; compare marker
template in lane 5 with lanes 3 and 4;
Fig. 1B, (1') and (2')). RNase A
digestion of products for both the 3D and 3D plus 3AB reactions showed
a predominant duplex segment co-migrating with fill-in product
(lanes 9 and 10; Fig. 1B,
(1)); it cannot be established whether there is fill-in of
both overhangs or only the longer overhang from these results. A small
amount of the fill-in of the shorter 5'-overhang to produce a 55-bp
duplex segment is also observed (lane 10; Fig.
1B, duplex segment of (2)). The latter shows that
to a limited extent 3AB enhances 3D-catalyzed addition to this
5'-overhang. In summary, 3AB affects product formation from two
different substrates in similar ways. However, the second substrate
(Fig. 1B) exhibited more primer dependence even in the
presence of 3AB, probably due to less secondary structure at the 3'
terminus.
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DISCUSSION |
Understanding of the essential components and reactions leading to
the complete replication of poliovirus RNA is incomplete. In the
absence of a defined system capable of achieving the complete replication reaction in vitro, several laboratories have
conducted studies of activities of individual proteins and combinations of proteins known to be involved in RNA replication by genetic tests.
In this way it is hoped that partial reactions can be defined that,
when appropriately constituted together, will reveal the replication
picture in toto.
Evidence for the role of 3AB in poliovirus RNA replication stems from
observation of the RNA-negative phenotype conferred by mutations in
either the 3A or 3B coding regions, from biochemical observations of
3AB associated with intracellular replication complexes, and from the
presence of 3B on incomplete nascent RNA strands. These data prompted
several recent efforts to examine the effects of 3AB on various
putative steps in the RNA replication reaction. For example, it was
shown that 3AB interacts with RNA (26, 27) and with 3CD and 3D (24, 25,
36) and that it can form a complex with 3CD and the termini of
poliovirus plus strand RNA (26, 35). In addition, several investigators
have reported that 3AB stimulates 3D polymerase activity (22-24, 27). The biological relevance of these observations to the viral RNA replication mechanism has yet to be determined.
In this report, we have examined the stimulation of 3D-catalyzed RNA
synthesis by 3AB in some detail. We conclude that 3AB does not
stimulate 3D activity on a template that is stably base paired to a
primer. Thus, 3AB has no inherent effect on 3D stability or catalysis.
If no primer is available or if the primer is not stably base paired to
the template, 3AB stimulates 3D activity significantly. We postulate
that 3AB interacts with 3'-hydroxyl ends that are transiently base
paired, resulting in stabilization of a hybrid region containing a site
for elongation. Recruitment of 3D to the site by protein-protein
interaction would result in increased elongation activity. As
predicted, stimulation is greatest when 3D concentration is low, since
3AB-3D interactions would have a greater impact on localizing 3D to the
initiation site than when 3D is in excess. The hypothesis that 3AB-3D
interaction is responsible for the increased polymerase activity is
consistent with our recent observations that 3AB can stimulate
polymerase activity catalyzed by the closely related Coxsackievirus B3
RNA polymerase but has no effect on polymerase activity catalyzed by
avian myeloblastosis virus reverse transcriptase on the same template
(data not shown).
Templates with high degrees of internal structure frequently fail to
form stable hybrids with primer sequences even after denaturation and
renaturation in primer excess. We have examined "hybrids" intended
for use as substrates in the 3D reactions in polyacrylamide/urea gels.
Although the PV1-72 substrate, used extensively in our studies,
produced very stable hybrids, we were unable to generate a stable
hybrid using the entire 5'-terminal cloverleaf structure (37) spanning
nt 1-108. Similarly, stable hybrids did not form from the 3'-end of
the poliovirus genome with transcripts from nt 7332 through the poly(A)
segment into vector sequences and a variety of primers spanning the
poly(A) tail with heteronucleotides on either side.
It is likely that heteropolymeric substrates used by others (23, 24),
which were reported to support significant stimulation of 3D activity
by 3AB, were unstable hybrids, behaving in a fashion similar to the
primer-independent reactions described here. In addition,
poly(A)-oligo(dT) substrate produced a 50-100-fold stimulation of the
reaction by 3AB (22, 23). This large stimulation may result from
transient hybrid formation on a template with multiple potential start
sites.
The large products formed in 3D reactions supplemented with 3AB have
been observed previously (23). We have shown that these products are
synthesized from contaminating transcripts produced by SP6 and T7 RNA
polymerases from a population of DNAs. To form functional hybrid
substrates for 3D reactions without production of large products,
transcripts must be gel-purified or cDNAs must be constructed of
small size and these must be gel-purified prior to transcription with
SP6 or T7 RNA polymerase. We have repeatedly found that DNA digested
with restriction endonucleases, seemingly to completion by analytical
gel analysis, does not produce a homogeneous population of transcripts.
The large products are favored in 3D plus 3AB reactions since they are
primer-independent and because radioactive incorporation into product
is length-dependent. 3D alone does not amplify production
of large products because transient hybrid structures that include the
3'-hydroxyl terminus are apparently not accessible to 3D at the
concentration used in the absence of 3AB.
The primary product synthesized by 3D alone on the PV1-72 substrate was
a fill-in complementary to the longer 5'-overhang (Fig. 1A,
(1)). Addition of 3AB enriched for a slower mobility band,
which represented fill-in complementary to the shorter 5'-overhang (Fig. 8, lanes 10 and 11; Fig. 1A,
(2)). There is some indication (Figs. 3 and 5) that both
termini are not utilized simultaneously, perhaps merely due to the low
statistical probability of one molecule being bound twice in a reaction
where overall template utilization is extremely low.
The biochemical roles for 3AB in the process of viral RNA replication
in infected cells are yet undefined. Certainly its potential to serve
as the VPg donor for RNA strand initiation is critical. Its association
with intracellular membranes (16-18) may anchor the replicating
structures in the replication complex. Its ability to bind RNA, whether
or not the interactions are sequence-specific, may facilitate
positioning of 3AB on the replicating RNAs. The activity described in
this report may result in stabilization of the weak hybrid formed
between the template and a uridylylated form of 3AB or 3B. Finally,
affinity for 3CD and stimulation of cleavage to generate 3D or direct
binding and recruitment of 3D itself could help localize the polymerase
and thereby increase chain initiation rates. There appear to be no
contributions of 3AB once chain elongation is initiated. Further
studies will be required to dissect the importance of individual 3AB
activities in the overall RNA replication reaction.
We thank Yolanda Bell for valuable assistance
in preparation of figures. We also thank Bert Semler, Karla
Kirkegaard, Yolanda Bell, and Aniko Paul for helpful comments
on the manuscript.