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Journal of Virology, March 2001, p. 2818-2824, Vol. 75, No. 6
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.6.2818-2824.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Sequences at the 3' Untranslated Region of Bamboo
Mosaic Potexvirus RNA Interact with the Viral RNA-Dependent RNA
Polymerase
Cheng-Yen
Huang,
Yih-Leh
Huang,
Menghsiao
Meng,
Yau-Heiu
Hsu, and
Ching-Hsiu
Tsai*
Graduate Institute of Agricultural
Biotechnology, National Chung Hsing University, Taichung 402, Taiwan
Received 3 October 2000/Accepted 18 December 2000
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ABSTRACT |
The 3' untranslated region (UTR) of bamboo mosaic potexvirus (BaMV)
genomic RNA was found to fold into a series of stem-loop structures
including a pseudoknot structure. These structures were demonstrated to
be important for viral RNA replication and were believed to be
recognized by the replicase (C.-P. Cheng and C.-H. Tsai, J. Mol. Biol.
288:555-565, 1999). Electrophoretic mobility shift and
competition assays have now been used to demonstrate that the
Escherichia coli-expressed RNA-dependent RNA polymerase domain (
893) derived from BaMV open reading frame 1 could
specifically bind to the 3' UTR of BaMV RNA. No competition was
observed when bovine liver tRNAs or poly(I)(C) double-stranded
homopolymers were used as competitors, and the cucumber mosaic virus 3'
UTR was a less efficient competitor. Competition analysis with
different regions of the BaMV 3' UTR showed that 893 binds to at
least two independent RNA binding sites, stem-loop D and the poly(A) tail. Footprinting analysis revealed that 893 could protect the sequences at loop D containing the potexviral conserved hexamer motif
and part of the stem of domain D from chemical cleavage.
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INTRODUCTION |
Bamboo mosaic potexvirus
(BaMV) has a flexuous rod-shaped morphology (17) and
comprises a single-stranded positive-sense RNA genome with a 5'
m7GpppG structure and 3' poly(A) tail. The
6,366-nucleotide (nt) genome [excluding the 3' poly(A) tail] consists
of five open reading frames and 94- and 142-nt untranslated regions
(UTR) at the 5' and 3' ends, respectively (19). Open
reading frame 1 (ORF1), encoding a 155-kDa polypeptide, can be
translated directly from the virion RNA in an in vitro rabbit
reticulocyte lysate (18). This polypeptide comprises three
domains, a methyltransferase-like domain (24), an RNA
helicase-like domain (12, 13), and an RNA-dependent RNA
polymerase domain (1, 15, 16), from the N to the C
terminus. The full-length 155-kDa polypeptide and the C-terminal 80-kDa
fragment (893) containing the RdRp domain were overexpressed in
Escherichia coli and demonstrated to have RNA-dependent RNA
polymerase activities (16).
The 3' UTR of single-stranded plus-sense viral genomic RNA, whether the
end structure is a tRNA-like structure (TLS), a poly(A) tail, or a
non-TLS heteropolymeric sequence, has been demonstrated to play
variable roles in minus-strand promotion, provision of telomere,
regulation of access to the minus-strand origin, and packaging
(10). There are several lines of evidence from studying the replication of turnip yellow mosaic virus (9, 25, 26), brome mosaic virus (4, 29), turnip crinkle virus (3,
27), and the picornaviruses (14, 20, 21, 22, 23)
showing that the specific sequence or the structure at the 3' UTR is
important for de novo minus-strand RNA synthesis. However, there are
only a few cases, including encephalomyocarditis virus (7,
8) and hepatitis C virus (6), demonstrating that
the viral RNA polymerase interacts directly with the 3' UTR of its own genome.
The 3' UTR of BaMV genomic RNA has been found by enzymatic and chemical
structural probing to fold into four independent stem-loops and a
tertiary pseudoknot structure (Fig. 1)
(5). With a series of mutations introduced into the 3' UTR of BaMV
genomic RNA, it has been shown that both nucleotide sequences and
structures are important for viral RNA accumulation in protoplasts
(5, 31). The potexviral conserved hexamer motif in the 3'
UTR of BaMV RNA and that in a defective RNA of clover yellow mosaic
potexvirus were shown to play an important role in viral RNA
replication (32). Although two tobacco proteins were
identified to interact with the U-rich sequence at the 3' UTR of potato
virus X (28), the interactions between the RdRp and the
sequence of the 3' UTR have not been experimentally mapped.
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FIG. 1.
Secondary folding of the 3' UTR of BaMV RNA. Nucleotides
are numbered from the 3' end cytosine just upstream of the poly(A)
tail. The arrows indicate the locations and the orientations of the
primers used for PCR.
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Due to the availability of the E. coli-overexpressed BaMV
RdRp (16), we had an opportunity to investigate the
interactions between RdRp and the 3' UTR. Here, we present data derived
from electrophoretic mobility gel shift assay (EMSA) and footprinting analysis to reveal the interactions of the purified recombinant RdRp
893 with the 3' UTR of BaMV genomic RNA in vitro.
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MATERIALS AND METHODS |
Expression and purification of recombinant RdRp.
Purification of the recombinant protein 893 RdRp was done as
described previously (16), except that two columns were
used for further purification before loading into the Talon metal
affinity resin column. In brief, once the cells were pelleted and
resuspended in 10 ml of sonication buffer (50 mM Tris-HCl [pH 8.0],
10 mM MgCl2, 10 mM KCl, 0.1% Triton X-100, 10%
glycerol, 5 mM 2-mercaptoethanol), sonication and centrifigation were
used to clarify the extract. Then the cell extract containing
recombinant RdRp was run consecutively through columns
containing 20 ml of DEAE-Sepharose (Pharmacia Biotech) and 20 ml
of P11 cellulose phosphate (Whatman). Finally, the BaMV recombinant
RdRp in the extract was mixed with 10 ml of Talon metal affinity resin
(Clontech), washed, eluted, and dialyzed against storage buffer (50 mM
Tris-HCl [pH 8.0], 10 mM MgCl2, 150 mM NaCl,
0.1% Triton X-100, 10% glycerol).
Preparation of RNA transcripts.
To prepare the 3' UTR
transcripts derived from BaMV RNA for analyses, DNA fragments with
different subdomains of the 3' UTR containing the T7 promoter were
amplified from infectious cDNA clone pBaMV/40A (W.-W. Chiu, C.-W. Peng,
Y.-H. Hsu, and C.-H. Tsai, unpublished
data) by PCR with different sets of primers listed in Table 1.
Each set of primers, T7/Ba3'+138 and T40GG, T7/Ba3'+138 and Ba3'noA,
T7/Ba3'+84 and Ba3'noA, T7/Ba3'+138 and Ba3'RT-loop, T7/Ba3'+34 and
T40GG, and T7/Ba3'+34 and Ba3'noA, were used to synthesize the DNA
fragments for direct transcription to produce the RNAs r138/40A,
r138/noA, r84/noA, rABC, r34/40A, and r34/noA, respectively. Transcript
r34/20A was derived from recombinant plasmid pBa3'+34, which had been
constructed by PCR amplification with primers T7/Ba3'+34 and Ba3'(
)
d(CGGGATCCTTTTTTTTTTTTTTT) and cloned into the SmaI site of
pUC19. Therefore, r34/20 contained not only 20 adenylate residues but
also 5 nonviral nucleotides derived from a BamHI restriction
site when pBa3'+34 was linearized with BamHI for in vitro
transcription. The 3' tRNA-like structure (TLS) of cucumber mosaic
virus, CMV3'TLS, was transcribed with T7 RNA polymerase from
BstNI-linearized pT7CMV/tRNA (30). All transcripts were purified by gel electrophoresis, and the
concentrations were determined by spectrophotometry. To prepare the
32P-labeled riboprobe, 5 pmol of r138/40A or
r138/noA was 3' end labeled with [32P]pCp with
20 U of T4 RNA ligase as described by England and Uhlenbeck (11).
EMSA of the interactions between 893 RdRp and the labeled
nucleic acids.
Various amounts of purified 893 RdRp were
incubated with 2 fmol of 32P-labeled riboprobe in
20 µl of binding buffer (25 mM Tris-HCl [pH 8.0], 150 mM NaCl,
0.05% Triton X-100, 5% glycerol) for 10 min at room temperature.
After incubation, 2 µl of gel loading buffer was added to each
sample, loaded onto a 5% native polyacrylamide gel, and run with 0.5×
Tris-boric acid-EDTA buffer at room temperature with ice pads. Results
were analyzed and quantified with a BAS-1500 bioimaging analyzer
(FUJIFILM). For competition assays, purified 893 RdRp was first
incubated with unlabeled competitor RNAs in binding buffer for 5 min at
room temperature, and then 2 fmol of 32P-labeled
riboprobe was added and the mixture was incubated for another 10 min
and electroporesed through a 5% native polyacrylamide gel. To improve
binding efficiency, the amount of RdRp was optimized for use with
different probes, with 14 and 30 ng of 893 RdRp used for
32P-labeled r138/40A and r138/noA, respectively.
All EMSAs were done at least three times.
Quantitation of bands on autoradiographs.
The quantitation
of images of labeled RNA bands was performed using a BAS-1500
bioimaging analyzer (FUJIFILM). The RNA competition efficiency,
expressed as percent RNA bound, was calculated from the optical density
of the bands corresponding to the bound and free RNA. The formula was
as follows:
where
c was no competitor added as a positive
control.
DEPC footprinting analysis.
The condition used for
modification on the N-7 position of adenine residues by
diethylpyrocarbonate (DEPC) in footprinting analysis was modified from
previous studies for structural probing (5, 31). The
20-µl reaction mixture containing the EMSA binding buffer described
above and 1 µl of the 5'-end-labeled r84/40A transcripts (10,000 cpm;
3 ng) was incubated at 30°C for 15 min, with the addition of 20 or
100 ng of RdRp and a serial dilution of pure DEPC (ca. 97%; Sigma)
from 0.5 to 4 µl to optimize the best condition. After the reaction,
the mixtures were ethanol precipitated directly with 3 µg of yeast
total RNAs (Boehringer Mannheim). The dried pellets were dissolved in
100 µl of water, phenol-chloroform extracted to remove the RdRp, and
then ethanol precipitated. The dried RNAs were then dissolved in 20 µl of 1 M aniline(redistilled)-acetic acid (pH 4.3) solution directly and incubated at 60°C for 20 min in the dark, and then the cleaved RNA fragments were frozen at 80°C and freeze-dried under vacuum. The cleaved RNA fragments were resuspended in urea-containing loading
buffer and resolved by electrophoretic separation on 8% denaturing (7 M urea) polyacrylamide gels.
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RESULTS |
Specific binding of recombinant 893 RdRp to the 3' UTR of BaMV
RNA.
To determine whether purified recombinant BaMV 893 RdRp
(16) could bind to the 3' UTR of BaMV RNA, the
32P-labeled 181-nt transcript r138/40A (Fig. 1,
nucleotides from positions 1 to 138, 40 adenylate residues at the 3'
end, and three nonviral guanylate residues derived from the T7 promoter
at the 5' end) was used as a probe for EMSA. In the presence of 20 ng of 893 RdRp in the purification buffer (25 mM Tris-HCl [pH 8.0], 5 mM MgCl2, 75 mM
NaCl, 0.05% Triton X-100, 5% glycerol) (16), a major and
a minor slower-migrating band were observed (Fig. 2A, lane 1) to
migrate above the free probe (lane 7). When the concentration of 893
RdRp in the reaction was increased, multiple shifted bands were
observed (Fig. 2A) that might have resulted from protein-protein
interaction, as the protein tends to aggregate during purification. No
shifted band could be observed when up to 200 ng of bovine serum
albumin was added in the reaction along with the r138/40A probe under
the same conditions to test the specificity of these interactions
(lanes 4 to 6).
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FIG. 2.
EMSA of the RNA-protein interaction for specificity and
dependence on salt concentration. Shown are autoradiographs of binding
reactions electrophoresed in 5% native polyacrylamide gels. Two
femtomoles (126.5 pg) of r138/40A transcript was used in all reactions.
(A) The indicated amounts of purified 893 RdRp and bovine serum
albumin (BSA) were used; the control consisted of buffer only. (B)
Effect of salt concentration. Twenty nanograms of 893 RdRp was used,
and the indicated concentrations of MgCl2 and NaCl were
added. A nonsalt addition (lane 1) and a buffer-only control (lane 8)
were included.
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To prevent the formation of multiple shifted bands that could result
from unwanted protein-protein interactions, each component
in the
reaction buffer was optimized. The pH of Tris buffer ranged
from 5.2 to
9.5, the temperature ranged from 0 to 37°C, and the
concentration of
Triton X-100 ranged from 0 to 0.1%; no obvious
effects were observed
(data not shown). The concentrations of
MgCl
2 or
NaCl were, however, critical for binding, with 0 or 10
mM
MgCl
2 interfering most with binding (Fig.
2B).
Band retardation
was maximal at 150 mM NaCl (Fig.
2B, lane 6) and
weaker in the
presence of a lower salt concentration (lane 5). The
finally optimized
binding reaction was set as 20 ng of RdRp with 25 mM
Tris (pH
8.0), 150 mM NaCl, 0.05% Triton X-100, and 5% glycerol,
producing
one major shifted band that could be easily observed and
quantified.
To investigate the binding specificity of
893 RdRp with r138/40A
RNA, unlabeled RNAs were used as competitors during the
binding
reaction. Unlabeled r138/40A was shown to be a strong
competitor as
expected, whereas the double-stranded poly(I)(C),
bovine liver tRNAs,
and CMV 3' UTR (
30) could not compete with
the labeled
probe efficiently (Fig.
3 and Table
2). The binding
of the radiolabeled
r138/40A probe by
893 RdRp was inhibited
almost completely by a
10-fold molar excess of unlabeled r138/40A
(Fig.
3A, lane 6). The
competition of bovine liver tRNAs and poly(I)(C)
double-stranded RNA
was very ineffective even when a 100-fold
mass excess of RNAs was added
in the reaction (Fig.
3B). The 254
nt of the CMV 3' UTR was a weak
competitor, with 50- and 100-fold
mass excesses competing out only 10 and 30%, respectively, of
the binding (Fig.
3B and Table
2). By
contrast, poly(A) RNA (ca.
1.5 kb; Sigma) competed significantly, with
75% of the shifted
band competed out in the presence of a 10-fold mass
excess (Table
2). Poly(G) could compete out 30 and 50% of the shifted
band
with 10- and 100-fold mass excesses, respectively. Poly(C) showed
even less competitive activity, with 20% at the 100-fold excess
(Table
2). Poly(U) was also tested as a competitor that the runs
of uridylate
residues could pair with the probe containing 40
adenylate residues,
and it aggregated at the well (not shown).
To further test the
efficiency of the poly(A) sequence as a competitor
with the 3' UTR of
BaMV RNA, 0.5-, 1-, 2.5-, 5-, and 10-fold mass
excesses of poly(A) were
used, and they showed 70.7% ± 1.9%, 49.9%
± 3.4%, 37.1% ± 1.7%, 30.9% ± 1.2%, and 25.5% ± 1.8% (means ±
standard
deviations) of competition values, respectively. These
results
indicated that poly(A) could compete out 30% of the probe
which was
bound to
893 RdRp with as low as a 0.5-fold mass excess.
Overall,
these results suggested that
893 RdRp binding to the
3' UTR of BaMV
RNA was specific and perhaps involved the poly(A)
tail.
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FIG. 3.
Competition assays of 893 RdRp and 3' UTR interaction
with different RNAs. Purified 893 (14 ng) was incubated with buffer
only (lane 1 in panel A and lanes 1, 5, 9, and 13 in panel B) or with
indicated amounts of unlabeled RNAs for 5 min at room temperature,
followed by the addition of 2 fmol (126.5 pg) of
32P-labeled r138/40A riboprobe and further incubation for
10 min. The competition activities were analyzed by EMSA. (A)
Competition assays with the indicated molar fold excesses of unlabeled
r138/40A (lanes 2 to 6). Lane 7, r138/40A riboprobe only. (B)
Competition assays with mass fold excesses of unlabeled poly(I)(C)
double-stranded homopolymer (lanes 2 to 4), poly(A) homopolymer (lanes
6 to 8), bovine liver tRNA (lanes 10 to 12), and CMV 3' UTR. Lane 17, r138/40A riboprobe only.
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TABLE 2.
Competition analyses of the interaction between 893
and the 3' UTR of BaMV RNA with different competitors
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The poly(A) tail in the 3' UTR of BaMV RNA is involved in 893
RdRp binding.
Of all the heterologous competitors tested, the
poly(A) RNA was shown to be the most efficient competitor (Table 2),
suggesting that BaMV RdRp could bind the poly(A) sequence which is also
part of the BaMV genomic RNA. Initiation of minus-strand synthesis within the poly(A) might be expected to involve RdRp binding. In order
to test the involvement of the poly(A) sequence of BaMV RNA in RdRp
binding, constructs with different numbers of adenylate residues or
different parts of the 3' UTR were created for use as competitor
ligands (Fig. 1).
Unlabeled r138/40A with 40 adenylate residues at the 3' end competed
the binding of probe r138/40A up to 50% with a 0.5-fold
molar excess
relative to the probe. A 5-fold molar excess of r138/noA,
containing no
adenylate residues at the 3' end, was required to
obtain the same
competition efficiency (Fig.
4). These
results
suggested that the presence of 40 adenylate residues could
produce
a 10-fold difference in competition efficiency; however, we
could
not rule out the possibility that this effect may also be
contributed
by the structural difference of the upstream sequence when
40
adenylate residues were removed from r138/40A. To prevent the
possible interference of upstream sequence, transcript r34/40A,
containing only the pseudoknot region (Fig.
1), was designed and
used
as a competitor. Results showed that the contribution of
the upstream
sequence of the 3' UTR (domains ABC and D) to competition
was around
2.5-fold (compare the amount of competitor needed for
50% competition)
between r138/40A and r34/40A (Fig.
4). Based
on the competition
activity of r34/40A, two constructs, r34/20A
and r34/noA, were designed
to have 20 and no adenylate residues,
and these showed five- to sixfold
less competitive activity than
that of r34/40A and no competitive
activity, respectively (Fig.
4). Overall, these results suggested that
the poly(A) sequence
of the BaMV 3' end does play an important role in
893 RdRp binding.
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FIG. 4.
Binding of 893 RdRp to the 3' UTR, investigated in
competitive binding assays using different domains of the 3' UTR RNAs
and different lengths of poly(A) tail. Purified 893 RdRp (14 ng) was
incubated with buffer only (100% control) or with the indicated molar
excesses of unlabeled RNAs for 5 min at room temperature, followed by
the addition of 2 fmol (126.5 pg) of 32P-labeled r138/40A
riboprobe for further incubation for 10 min. The competition activities
were analyzed by EMSA. (A) Representative experiments that have
contributed to the quantitative data in panel B. All EMSAs were done at
least three times, and the percentages of RNA bound were determined as
described in Materials and Methods.
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Stem-loop D is another region of the binding sites for 893
RdRp.
Results from competition analysis of r138/40A and r34/40A
revealed that the 3' UTR of BaMV RNA was involved in binding with 893 RdRp (Fig. 4). In order to identify any specific region in the
3' UTR capable of binding RdRp independent of the poly(A) tail,
r138/noA was [32P]pCp labeled and used as a
probe for gel shift analysis. Transcripts r138/noA, rABC, r84/noA, and
r34/noA (Fig. 1, positions 1 to 138, 39 to 138, 1 to 84, and 1 to 34, respectively) were synthesized and used as competitors. Results showed
that r34/noA and rABC had no or little competitive activity on the
competition analysis and that r84/noA with the complete stem-loop D was
an effective competitor (Fig. 5).
Although r84/noA was a good competitor, it was still fivefold less
efficient than r138/noA comprising the ABC and D domains. This
suggested that there might be an interaction between the ABC and D
domains that would enhance the competition activity. These results
would also imply that stem-loop D could be one of the binding regions
for 893 RdRp.
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FIG. 5.
Mapping of the binding domains of 893 RdRp on the
BaMV 3' UTR by competition assays. Purified 893 RdRp (40 ng) was
incubated with buffer only (100% for the control) or with the
indicated molar fold excesses of unlabeled RNAs for 5 min at room
temperature, followed by the addition of 2 fmol (95.88 pg) of
32P-labeled r138/noA riboprobe for further incubation for
10 min. The competition activities were analyzed by EMSA. (A)
Representative experiments that have contributed to the quantative data
in panel B. All EMSAs were done at least three times, and the
percentages of RNA bound were determined as described in Materials and
Methods.
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The potexviral conserved hexamer motif interacts with RdRp.
To
address which region of domain D could be the possible binding site of
BaMV RdRp, we performed a footprinting analysis. The condition used for
footprinting was optimized for protein binding and chemical probing
with DEPC to modify N7 of adenylate residues. Transcript r84/40A was 5'
end labeled and then treated with DEPC in the absence or presence of 20 or 100 ng of 893 RdRp, followed by aniline cleavage (Fig.
6). Nucleotides A60 in the bulge, A33 to
A28 (AAUAAA, the putative polyadenylation signal) in the internal loop,
and A12 and A10 in the loop of the 3' pseudoknot showed equal densities
of banding with or without the 893 RdRp addition, indicating that
they were not protected by the RdRp. By contrast, the first adenylate
residue, A52, of the hexamer motif in apical loop D showed either less
detectable banding signal in the presence of RdRp (Fig. 6, lane 2) than
in the absence of RdRp (Fig. 6, lane 1) or no detectable banding signal
in the presence of RdRp (Fig. 6, lane 3). The last two adenylate
residues of the hexamer motif, A48 and A47, showed lower cleavage
efficiency in either the absence or presence of RdRp. In addition, some
nonspecific cleavages (the nonadenylate residues, C50 in loop D and C40
and G37 in stem D) were observed in the absence of RdRp but protected in the presence of 893 RdRp. Overall, these results suggested that
the polymerase domain of ORF1 of BaMV (893 RdRp) could specifically bind to loop D containing the potexviral conserved hexamer motif (AC[C/U]UAA) and part of stem D of the 3' UTR of BaMV RNA.
Unfortunately, the poly(A) binding region could not be resolved from
this probe. Maybe there is protection on each probe molecule,
but the location within the poly(A) tail varies, so that the overall
modification cannot be noticeably diminished.
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FIG. 6.
DEPC footprint of the 893 RdRp binding site on the
r84/40 RNA. Show is an autoradiograph of the aniline cleavage products
on a polyacrylamide gel containing 8% urea. Lanes: 1, aniline cleavage
of DEPC-modified r84/40A RNA without 893 RdRp; 2 and 3, aniline
cleavage of DEPC-modified r84/40A RNA in the presence of 20 and 100 ng
of 893 RdRp, respectively; 4, aniline cleavage of r84/40A RNA
without DEPC treatment. The positions of selected nucleotides in the
r84/40A sequence are indicated on the left for reference.
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DISCUSSION |
The 3' UTRs of positive-sense viral genomic RNAs are believed to
contain sequences or structures participating in template recognition
with viral RNA-dependent RNA polymerase to initiate minus-strand RNA
synthesis. With results derived from EMSA and competition experiments,
we demonstrate that purified recombinant RdRp could bind specifically
to the 3' UTR of BaMV RNA without any host factor from plants or other
virus-encoded polypeptides. Hepatitis C virus NS5B and
encephalomyocarditis virus 3Dpol are two other cases in which
overexpressed RdRp was demonstrated to bind the conserved stem-loop
structures of the viral 3' end RNA (6) and both the 3' UTR
and poly(A) tail (7), respectively, without any other
factor being involved. This differs from phage Q
, in which the
phage-encoded RNA polymerase subunit must form a complex with at least
three host proteins to attain specificity for binding to Q RNA and
related RNAs (2).
Although encephalomyocarditis virus 3Dpol was shown to have specific
binding activity, it was unable to form a complex with the poly(A)
alone or with the 3' UTR RNA without a poly(A) tail (7). A
pseudoknot structure derived from the 3' UTR and poly(A) was proposed
to be the signal for 3Dpol binding. Our findings indicate that BaMV
893 RdRp could specifically interact with the poly(A) sequence and
stem-loop D of the 3' UTR independently (Fig. 3b and Table 2). These
results suggested that RdRp of BaMV ORF1 could contact at least two RNA
binding sites, one comprising the hexamer conserved sequence and the
other comprising the poly(A) tail. It has been shown that a sufficient
length of poly(A) tail is required to maintain the integrity of the 3'
pseudoknot structure, which is important for efficient replication of
BaMV RNA in vivo (31). It would be interesting to
determine whether a similar poly(A) length is required for maintaining
the pseudoknot structure and for RdRp binding. It is possible that the
poly(A) sequence could be used as a template for the minus-sense RNA
initiation. A mutant with a guanylate residue insertion at the fourth
position of the poly(A) tail was inoculated into protoplasts and
plants. The progeny recovered from cells was found to retain the
guanylate residue at the same position (Chiu et al., unpublished). This result indicated that the initiation site of minus-sense RNA synthesis could use the poly(A) tail as a template and is possibly located downstream of the fourth nucleotide of the poly(A) tail. Therefore, the
poly(A) tail of BaMV RNA might play several functional roles, including
the formation of the pseudoknot structure for replication (31), acting as template for minus-sense RNA initiation
(Chiu et al., unpublished), and simply in RNA stability, as that of mRNA in eukaryotic cells.
The other binding site on the RNA was located at stem-loop D, covering
the entire loop and part of the stem. Since the conserved potexviral
hexamer motif is housed in this loop, it is possible that this hexamer
motif could play the role of a specificity determinant for RdRp
recognition. Through this interaction, the RdRp could dock at the right
position to bind the poly(A) region to initiate minus-sense RNA
synthesis. The major drawback of our results was that the overexpressed
RdRp used in experiments was only the C-terminal domain of BaMV ORF1.
It is unknown whether the full-length form of this polypeptide,
containing the N-terminal and middle portions of the methyltransferase
and helicase domains, respectively, would show the same RNA binding.
However, since 893 RdRp was demonstrated to be competent for
polymerization activity (16) and the interaction with the
3' UTR was specific, the interference of the other two domains would be
expected to be low. Besides, the hexamer motif protected by RdRp in the
footprinting analysis has been observed to play an important role in
the replication of BaMV RNA in protoplasts. The specificity of each
nucleotide has shown that the first nucleotide is purine specific, the
second is pyrimidine specific, the third can be tolerated with
mutations, and the rest of the nucleotides of the hexamer are
restricted to UAA (12). The results of the in vivo
protoplast assay involving full-length RdRp and the in vitro
footprinting and gel shifting analyses with the truncated form of RdRp
are correlated.
Functional analysis of the tertiary structure of the 3' UTR of BaMV RNA
revealed that maintaining the integrity of the terminal pseudoknot is
important, and the deletion of the ABC domain seriously impaired virus
accumulation in protoplasts (6, 32). It is possible that
these regions could also interact with either a host- or virus-encoded
factor(s). It will be interesting to identify this factor(s) and the
relationship with RdRp, especially for the virus-encoded
methyltransferase and helicase domains.
Overall, our results suggest that BaMV RdRp could specifically
recognize the 3' UTR, which provides the loop D conserved hexamer sequence (ACCUAA) and the poly(A) tail. This step could be important for the virus to initiate minus-sense RNA synthesis.
| |
ACKNOWLEDGMENTS |
We thank Theo Dreher at Oregon State University for discussion
and editorial help.
This work was supported by grants from the National Science Council
(projects NSC 88-2311-B-005-040 and 88-2311-B-005-002-B11).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Graduate
Institute of Agricultural Biotechnology, National Chung Hsing
University, Taichung 402, Taiwan. Phone: 886-4-284-0451. Fax:
886-4-286-0260. E-mail: chtsai1{at}dragon.nchu.edu.tw.
| |
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Journal of Virology, March 2001, p. 2818-2824, Vol. 75, No. 6
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.6.2818-2824.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
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