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Journal of Virology, December 2000, p. 11201-11209, Vol. 74, No. 23
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Efficient and Specific Initiation of Subgenomic RNA Synthesis by
Cucumber Mosaic Virus Replicase In Vitro Requires an Upstream
RNA Stem-Loop
M.-H.
Chen,1
M. J.
Roossinck,2 and
C. C.
Kao1,*
Department of Biology, Indiana University,
Bloomington, Indiana 47405,1 and Plant
Biology Division, Samuel Robert Noble Foundation, Ardmore, Oklahoma
734022
Received 7 July 2000/Accepted 12 September 2000
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ABSTRACT |
We defined the minimal core promoter sequences responsible for
efficient and accurate initiation of cucumber mosaic virus (CMV)
subgenomic RNA4. The necessary sequence maps to positions 
28 to +15 relative to the initiation cytidylate used to initiate RNA
synthesis in vivo. Positions 28 to 5 contain a 9-bp stem and a
6-nucleotide purine-rich loop. Considerable changes in the stem and the
loop are tolerated for RNA synthesis, including replacement with a
different stem-loop. In a template competition assay, the stem-loop and
the initiation cytidylate are sufficient to interact with the CMV
replicase. Thus, the mechanism of core promoter recognition by the CMV
replicase appears to be less specific in comparison to the minimal
subgenomic core promoter of the closely related brome
mosaic virus.
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INTRODUCTION |
Viruses that encode multiple
proteins in an RNA have evolved different strategies to translate all
of the coding sequences. The Bromoviridae family of
plant viruses, with genomes of capped plus-stranded RNAs, transcribes subgenomic-length RNAs by
initiating synthesis from an internal sequence within the
minus-strand RNA (for a review, see reference 19).
The subgenomic promoter is defined as a sequence that
interacts with the viral replicase to direct the accurate
initiation of RNA synthesis.
In Brome mosaic virus (BMV), genus
Bromovirus, family Bromoviridae, several
sequence motifs appear to contribute to efficient subgenomic RNA synthesis in protoplasts. These include an
upstream A-U-rich sequence, a polyuridylate tract, a 20-nucleotide (nt) core promoter, and a downstream A-U-rich sequence (9, 18). The polyuridylate sequence is of variable length; it is required for
BMV infection in vivo (31) and binds to the replicase in vitro (1). The BMV core promoter directs the BMV replicase to recognize the initiation nucleotide. A mutation in the core promoter
can compensate for the lack of a polyuridylate sequence (31). Using a reductionist approach, our laboratory
determined that the core promoter alone is required and sufficient for
accurate and efficient in vitro initiation of transcripts less than 15 nt in length (1). A systematic mutational analysis of the
subgenomic core promoter revealed that four essential
nucleotides, at positions 17, 14, 13, and 11 relative to the
initiation cytidylate (+1C), are recognized by the RNA-dependent RNA
polymerase (RdRp) in a sequence-specific manner (25). The
base and some ribose moieties of these key nucleotides responsible for
RdRp recognition were identified using RNAs containing chemically
synthesized nucleotide analogs (26).
Jaspars (14) used nucleotide sequence alignment to
demonstrate that a structure upstream of the subgenomic RNA
initiation cytidylate is found in alfalfa mosaic virus (AMV) and
several other members of the Bromoviridae family. Haasnoot
et al. (13) demonstrated that a stem-loop structure is
correlated to AMV subgenomic RNA synthesis. We
biochemically examined the cucumber mosaic virus (CMV)
subgenomic promoter to expand the information on the
structure and sequence required for transcription by viral replicases.
CMV, genus Cucumovirus, family Bromoviridae, is
an economically important plant pathogen and can be classified into two
major serological subgroups, I and II. Nucleotide sequence alignment of
RNA3 indicated that subgroup I can be divided into two other subgroups,
IA and IB (24). The CMV RNA genome consists of RNA1 (~3.4
kb), RNA2 (~3 kb), and RNA3 (~2.2 kb). As in BMV and AMV, the
capsid is translated from subgenomic RNA4 (~1 kb)
(22). A second, low-abundance subgenomic
transcript, RNA4a, is produced from the minus strand of RNA2
(8). The cis-acting sequences for CMV RNA4
synthesis have been characterized (4). Using a construct
containing two subgenomic promoters for RNA4, Boccard and Baucombe (4) demonstrated that the sequence from 70 nt upstream to 30 nt downstream of the initiation cytidylate (+1C; complement of nt 1167 in the Kin strain of CMV) was required to direct
RNA synthesis in transfected protoplasts. A truncated promoter beginning 30 nt upstream of the initiation cytidylate also directed subgenomic RNA4 synthesis but at a reduced level
(4). Here we further define the sequence needed to
direct CMV subgenomic RNA synthesis using an enriched
CMV replicase that can accurately direct the synthesis of
genomic minus-strand, genomic plus-strand, and
subgenomic RNA in vitro (29). Deletion analysis
indicates that a stem-loop at nt 28 to 5 upstream of the +1C is
responsible for the efficient and accurate initiation of
subgenomic RNA synthesis. This stem-loop structure is
conserved in all subgroups of CMV.
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MATERIALS AND METHODS |
RNA synthesis and purification.
Transcription conditions
were as previously described (20). Briefly, the DNA strands
were purified via denaturing polyacrylamide gel electrophoresis (PAGE)
and then adjusted to a final concentration of 8 µM. One microliter of
each DNA was used in a 20-µl transcription reaction mixture
containing 40 mM Tris (pH 8.1), 1 mM spermidine, 0.01% Triton X-100,
80 mg of polyethylene glycol 8000, and 4 mM each nucleoside
triphosphate. The T7 RNA polymerase used was purified using the
protocol of Grodberg and Dunn (10). RNAs of the correct size
were purified by preparative denaturing gel electrophoresis and excised
from the gel after UV shadowing. The gel slices were crushed and ground
to small pieces, and the RNA was eluted from the polyacrylamide with
0.4 M ammonium acetate at 30°C overnight. Following precipitation
with ethanol, the RNA concentration was determined by spectrophotometry
and checked by staining with 0.25% toluidine blue on an analytical gel.
RdRp activity assay and product analysis.
CMV replicase was
enriched from CMV-infected tobacco by a method described previously
(29), modeled after previously published protocols (11,
36). A standard assay consisted of a 20-µl reaction mixture
containing 0.25 pmol of template (unless stated otherwise), 5 µl of
replicase preparation, 20 mM sodium glutamate (pH 8.2), 4 mM
MgCl2, 12.5 mM dithiothreitol, 0.5% (vol/vol) Triton X-100, 1 mM MnCl2, 200 µM ATP and UTP, 500 µM GTP, and
250 nM [
-P32]CTP (Amersham). Reaction mixtures were
incubated at 25°C for 60 min, and reactions were stopped by
phenol-chloroform extraction followed by ethanol precipitation in the
presence of 5 µg of glycogen and 0.4 M ammonium acetate. Products
were separated by electrophoresis on 10 to 20% denaturing (8 M urea)
polyacrylamide gels. Gels were wrapped in plastic and exposed to film
at 80°C. Product bands were quantified using a PhosphorImager
(Molecular Dynamics). All values presented are the mean of at least
three independent assays, all of which varied by less than 20%.
Percent synthesis is calculated based on the sum of the intensities
from 15- and 16-nt products relative to the wild-type proscript (see below).
Native gel analysis.
RNA conformation native gel analysis
was performed in 10% polyacrylamide gels (38% acrylamide and 2%
bisacrylamide) containing 0.5× Tris-borate-EDTA (30). After
preelectrophoresis for 20 min, a 100-pmol sample in RNA renaturing
buffer (50 mM Tris-HCl [pH 7.4], 100 mM KCl, 0.1 mM EDTA) was heated
to 90°C for 2 min before the samples were placed on ice for at least
10 min. The sample was then adjusted to contain 0.5× Tris-borate-EDTA,
5% glycerol, and bromophenol blue. Electrophoresis was performed at a
constant 150 V at 5°C until bromophenol blue migrated to within 3 to
5 cm from the bottom of the gel (17 by 15 by 0.08 cm). The gel was then
stained with toluidine blue to visualize the RNAs.
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RESULTS |
Sequence required for initiation of CMV subgenomic RNA
in vitro.
Boccard and Baulcombe (4) found that the
minimal necessary sequence for CMV subgenomic RNA4
synthesis maps to nt 30 to +30 relative to the initiation
cytidylate. While the results from protoplast analysis are
biologically relevant, it can be difficult to determine the
contributions from multiple effects, such as changes in stability of
the RNA. Biochemical analysis for RNA synthesis was done to complement
and extend the protoplast results.
To examine the promoter for subgenomic RNA synthesis, we
used CMV replicase enriched from tobacco plants infected with CMV strain Fny (29) and in vitro-transcribed proscripts. The
latter were so named because these RNAs contain both promoter and
template sequence. The first proscript is a 294-nt RNA containing the
complement to the entire intercistronic region of CMV RNA3 (Fig.
1A). The 3' end of RNA C4223/+71 is 1 nt downstream of the complement of the 3a translational termination
codon, and the 5' end is 5 nt upstream of the translation initiation
codon of the capsid protein-coding sequence. C4223/+71 directed the
synthesis of several RNAs, including a major product of ~71 nt, the
length expected from initiation at the site used predominantly in vivo (the complement of nt 1183 in RNA3) (Fig. 1B, lane 2). Faint
higher-molecular-weight bands may be due to either initiation at
alternative sites or RNA recombination by the CMV replicase after
correct initiation. Similar recombination events have been observed and
characterized with the BMV and cowpea chlorotic mottle virus replicases
(M.-J. Kim and C. C. Kao, submitted for publication).
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FIG. 1.
Sequences required to direct the initiation of CMV
subgenomic RNA synthesis. (A) Partial sequence containing
the complement to the intercistronic region of minus-strand CMV RNA3.
Numbers correspond to the nucleotide positions of CMV strain Fny RNA3.
The underlined sequences at the left and right correspond to the
complement of the termination and initiation codons of the CMV 3a and
capsid proteins, respectively. The sequence in bold contains the 29 nt
3' of the initiation cytidylate. The sequence between positions 1117 and 1128 corresponds to the complement of the B-box sequence, a motif
demonstrated to be important in BMV RNA replication (9, 23).
The arrow identifies the initiation cytidylate for the initiation of
CMV subgenomic RNA synthesis used in vivo (4).
(B) Effects of deletion of the sequence 3' of the initiation
cytidylate. Proscripts used in the reactions are indicated at the top.
" " indicates that no template was in the reaction. The expected
71-nt product is denoted to the right of the autoradiograph from a 7 M
urea-10% polyacrylamide gel. (C) Effects of deletion of the sequence
5' of the initiation cytidylate. Since the deletions decrease the
template lengths, the products of RNA synthesis are of different
lengths. The gel was of two densities; the bottom portion contained
20% acrylamide, and the top contained 10% acrylamide. The border of
the two gel densities lies slightly above the 71-nt band. (D)
Examination of the length 3' of the initiation cytidylate required for
accurate and efficient RNA synthesis in a 7 M urea-20% polyacrylamide
gel. The expected products should be 15- and 16-nt. The 9- and 10-nt
products were incorrectly initiated. (E) The +1C used in the initiation
of RNA synthesis, and the effects of the 3' sequence.
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Deletions of the 3' sequence of C4
223/+71 were tested to identify the
minimal promoter sequence required for correct initiation
of RNA
synthesis. The following proscripts contain identical template
sequences that end at nt +71. Truncation to make the 3' ends at
positions
147,
70,
55, and
29 all retained correct initiation
of the ca. 71-nt product. An increase in the abundance of the
correctly
initiated product was observed with this series of deletions
(Fig.
1B,
lanes 3 to 6). However, having the 3' end at
21 resulted
in
significant decrease in RNA synthesis and the appearance of
some
lower-molecular-weight bands (Fig.
1B, lane 7; Fig.
1C, lane
5). Thus,
the 3' end of the CMV subgenomic core promoter maps
to
between nt
29 and
21, in good agreement with the results
of Boccard
and Baulcombe (
4).
To map the 5' end of the functional proscript, the constructs contained
the same 3' end at nt
29 and different 5' ends at
positions +71, +28,
and +15 nt. All three proscripts directed
synthesis (Fig.
1C, lanes 2 to 4) at comparable levels after normalizing
for radiolabeled CMP
incorporation, indicating that the template
sequence from positions +15
to +71 does not contain a signal that
regulates RNA synthesis.
With proscript CA
29/+15, the expected
15-nt product and a 16-nt
product were resolved by PAGE. The latter
was likely generated by the
nontemplated addition of one nucleotide
after the replicase reaches the
5' end of the template (
25).
Nontemplated nucleotide
addition is a common property of several
RdRps and has been observed
with CMV-associated RNAs (
21,
25,
36,
38).
Three proscripts that have identical 3' ends at position
21 and
template lengths of 71, 28, and 15 nt were examined to determine
if
there is an effect on efficient and accurate initiation. Consistent
with the above observation made with C4
21/+71 (Fig.
1B, lane
7), a
proscript containing only 21 nt 3' of the initiation cytidylate
resulted in not only lower levels of the correct-size products
but also
an increase in the abundance of incorrectly initiated
products (Fig.
1C, lanes 5 to 7). However, truncations at the
5' end from nt +71 to
+15 did not affect the amount of the correctly
initiated products,
confirming that a 15-nt template sequence
is sufficient for efficient
RNA synthesis. Since this template
length allows high-resolution
analysis of the replicase products,
it was routinely used in subsequent
experiments.
The products from
29/+15 and
21/+15 were examined by
high-resolution PAGE. The 15- and 16-nt products from both proscripts
were well separated in a 20% denaturing gel, and the difference
in
their production from the two proscripts was reproducible (Fig.
1D,
lanes 1 and 4). Due to the use of CTP as the radiolabeled
nucleotide,
products that initiated from the +1C but were aborted
before position
+9 would not be radiolabeled. The reaction with
29/+15 produced only
the 15- and 16-nt RNAs. In contrast, proscript
21/+15 directed the
synthesis of 9- and 10-nt RNAs that could
have initiated from the
cytidylate at the +7 position (Fig.
1A;
Fig.
1D, lane 4). Mutation of
the +7C to a guanylate abolished
synthesis of the 9- and 10-nt products
from
21/+15, demonstrating
that misinitiation occurred when the core
promoter was only 21
nt (M.-H. Chen, unpublished
data).
To determine whether positions
29 to
22 contain a specific
sequence that contributes to replicase recognition, or simply
serves as
a required length, we added either eight adenylates
or uridylates
to the 3' end of
21/+15 to result in proscripts
of the same length as
29/+15. These RNAs not only were less efficient
in directing the
synthesis of the 15- and 16-nt products in comparison
to
29/+15 but
also produced the 9- and 10-nt misinitiated products
(Fig.
1D, lanes 2 and 3). Therefore, the sequence upstream from
29 to +15 is required
for efficient and accurate initiation of
CMV subgenomic RNA
synthesis.
The use of the +1C in the initiation of the 15- and 16-nt RNAs was
examined by changing the +1C to a G in the context of both
29/+15 and
21/+15 (Fig.
1E, lanes 3 and 5). These changes abolished
the
synthesis of the 15- and 16-nt products from proscripts
29/+15,
+1C/G and
21/+15,+1C/G (Fig.
1E, lanes 3 and 5), indicating
that
the 15-nt product is initiated from +1C and terminated at the
end
of the template. Also, this result supports our hypothesis
that the
16-nt band initiated correctly and likely has the addition
of a
nontemplated nucleotide at the 3' end of the RNA. Thus, the
initiation
of RNA synthesis from proscript
29/+15 takes place
at the cytidylate
used in vivo (
4). The 9- and 10-nt products
from
proscript
21/+15,+1C/G were unaffected in comparison to
21/+15,
confirming that they resulted from incorrect initiation
instead of
premature termination. With both
29/+15,+1C/G and
21/+15,+1C/G, prominent 12- and 13-nt products were observed.
Based
on their sizes, these RNAs likely initiated from the +4
uridylate. In
addition, proscript 29/+15, +1C/G also produced
the 9- and 10-nt RNAs.
These results suggest that the fully functional
core promoter will
dictate the site of initiation and that +1C
is the preferred initiation
site. However, initiation can take
place at an alternative cytidylate
and/or uridylate when the RNA
contains changes in the core promoter or
the authentic initiation
cytidylate.
Strain variation of the putative CMV core promoter sequence.
The biochemical analysis defined a sequence that could be analyzed for
the features (structure or sequence) that direct RNA synthesis. The
mfold RNA secondary structure prediction program (13) predicted that nt 28 to 5 upstream of the +1C in
CMV strain Fny should form a stem of 9 bp, with a central A-C mismatch and a 6-nt purine-rich loop (Fig. 2A).
The predicted RNA secondary structure of RNA C470/+15 also contained
the same stem-loop at positions 28 to 5 (6). The
stability of this secondary structure, as predicted by
mfold, is 7.6 kcal/mol. The sequence from 4 to the end
of the template was predicted to be unstructured.
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FIG. 2.
Comparison of predicted RNA secondary structures for the
sequence 3' of the initiation cytidylate in three CMV subgroups. The
initiation cytidylate used in vivo in strains Fny (subgroup I) and Kin
(subgroup II) and the corresponding cytidylates in other CMV strains
are in bold letters (4, 22). The CMV isolates used to
generate the prototype structure are indicated directly under the viral
subgroups, and strains that vary from the prototype are listed under
the heading "W/ 1 nt change." Nucleotides that diverged from the
prototype in each subgroup are indicated with an arrow. The white
triangle in the middle structure denotes the insertion of two
nucleotides in strain SD.
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CMV isolates can be divided into three phylogenetic subgroups: IA, IB,
and II (
24). To examine the possible relevance of
features
in the stem-loop, the intercistronic (minus-strand RNA3s
from 25 different CMV isolates were aligned. The high degree of
similarity in
the sequence encompassing the core promoter makes
the alignments
unambiguous. For example, the template sequence
of 3'-GCAA-5' is found
in all 25 strains at positions
1 to +3
(Fig.
2 and data not shown),
helping to align the sequences. The
sequences examined varied according
to the three phylogenetic
subgroups. Subgroup IA, which includes Fny,
has the same sequence
in all 12 strains examined. The seven subgroup IB
isolates examined
had several sequence changes relative to the IA
motif. First,
the predicted stem is shorter than those in the IA
subgroup, indicating
that some flexibility in the stem length is
acceptable for CMV
subgenomic RNA transcription. Also, the
central A-C mismatch in
the IA stem was not conserved in the IB
isolates, indicating that
it may not be an essential feature in
replicase interaction. Strain
SD in subgroup IB was particularly
divergent relative to the others.
In addition to numerous substitutions
in the stem sequence (including
a change of nt
11 from a G to a C
that could destabilize the
stem), strain SD also has two additional
purines in the already
purine-rich
loop.
Six subgroup II isolates were examined, including the Kin isolate
characterized by Boccard and Baulcombe (
4). The subgroup
II
stem-loop was similar to the one in subgroup IA, with a 8-bp
stem with
a central A-C mispair. Subgroup II isolate S has a change
in the
terminal GC base pair that shortens the stem by one base
pair. Strain
WL has a transition of the
16 A to a G. The minor
variations in the
stem-loop sequence of the three CMV subgroups
indicate some flexibility
in the requirements for this sequence
to act as a core promoter
element.
Effects of sequence changes on RNA conformations.
Previously
we found that nondenaturing gel electrophoresis was able to distinguish
different conformations of RNAs of the same length (30).
Five CMV proscripts were tested: C429/+15 (henceforth referred to as
C4WT), C421/+15, C4S-Hi, which has a mutation designed to destabilize
the upper portion of the stem, C4S-Low, which should be destabilized in
the lower stem, and C4AS, which contains a completely different 8-bp
stem but maintains the loop sequence of C4WT (Fig.
3A). The stem in C4-AS comes from the
core promoter for the BMV tRNA-like structure, SLC, whose structure was
solved using multidimensional nuclear magnetic resonance spectroscopy
and is known to exist in an A-form helix with a 3-nt loop
(16). In a denaturing gel, all of the RNAs tested migrate according to their expected lengths (Chen, unpublished). In a nondenaturing gel, proscript C4WT migrated as a discrete band, indicating that any RNA structure that is present is likely due to
intramolecular interactions and not due to the existence of a duplex of
two molecules of C4WT. The observed mobility of C4WT was similar to
that of SLC+8, a 45-nt RNA that forms a stable stem-loop and an 8-nt
single-stranded sequence (Fig. 3, lane 2) (6, 16). Proscript
C421/+15 migrated to a lower position in the gel, as expected for its
lower molecular mass, and was more smeared, suggesting that the RNA
exists as a mixture of molecular conformations (Fig. 3B, lane 4).
C4S-Low migrated at a position reproducibly lower than that of C4WT,
perhaps due to a longer single-stranded region (Fig. 3, lane 5). C4-Hi
migrated as a higher-molecular-weight band in the nondenaturing gel,
consistent with a larger loop that should make the RNA less compact or
with an alternative structure (Fig. 3, lane 6). C4AS has mobility
similar to that of C4WT, suggesting that both RNAs fold into a
stem-loop structure. The altered mobility from the changes in the
putative stem was also consistent with the existence of a stable
stem-loop C4WT.
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FIG. 3.
Analysis of RNA structure using native gel
electrophoresis. (A) Sequences of the RNAs tested. Each of the RNAs
tested is 44 nt in length, spanning from positions 29 to +15.
However, only the portions relevant to formation of the stem-loop are
shown, with the nucleotides that form the stem indicated by arrows.
Nucleotide changes that affect the stem are shown in bold letters. C4AS
contains a sequence from the stem of the BMV SLC, whose structure has
been solved by nuclear magnetic resonance spectroscopy (Kim and Kao,
submitted). (B) RNAs stained with toluidine blue after PAGE in a 10%
nondenaturing gel. B2()26G is a 27-nt RNA whose secondary structure
was reported by Sivakumaran et al. (30). SLC+8 is a 45-nt
RNA whose structure was reported by Kim and Kao (submitted).
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Effects of stem changes on RNA synthesis in vitro.
The role of
the stem-loop in C4WT in directing RNA synthesis was examined
systematically. The change of one base pair in the stem in proscript
S-1bp did not affect RNA synthesis (Fig.
4B, lane 3). No effects on synthesis were
observed when both sides of the stem were switched to their
complementary sequences (lane 4). A change that should form a stem of a
different sequence, C4AS, reproducibly directed about 1.5-fold of
the synthesis from C4WT (Fig. 4A; Fig. 4B, lane 5). Furthermore, the
absence of the 9- and 10-nt products indicates that these changes did
not result in misinitiation. These results indicate that the sequence
of the stem is not important for efficient and accurate initiation or
the level of RNA synthesis, which is in agreement with the analysis of
the sequences of CMV strains that showed some variations in the stem
(Fig. 2). Also, the A-C mispair present in subgroups IA and II, but
absent in subgroup IB isolates, appears unimportant in vitro.
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FIG. 4.
Effects of changes in the stem in the CMV
subgenomic core promoter on the level and accuracy of RNA
synthesis. (A) Summary of most of the RNA constructs tested for the
ability to direct RNA synthesis. Nucleotides changed from the prototype
C4WT are indicated in bold letters. The predicted RNA secondary
structures that resulted from the changes are also shown. (B)
Autoradiograph of the results from RNA synthesis assays from several
proscripts. The proscripts tested are indicated above the lanes, and
sizes of the products are indicated in nucleotides at the left. "%
of syn" indicates the percentage of the CMV replicase products made
from the specified template relative to C4WT tested in the same set of
reactions; "" indicates that no template was added to the
reaction. A 7 M urea-20% polyacrylamide gel was used for analysis of
the 15- and 16-nt products shown in this and subsequent figures.
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Since C4
21/+15 directed a significant amount of misinitiation (Fig.
1D), we wanted to determine what changes in C4WT would
result in
misinitiation. Mutants that retained 28, 25, or 23 nt
upstream of the
initiation cytidylate were examined. Proscripts
C4
25/+15 and
C4
23/+15 resulted in reduced synthesis (Fig.
4B,
lanes 7 and 8).
However, the 9- and 10-nt misinitiated products
were less abundant in
reactions with C4
23/+15, while they were
easily observed C4WT (Fig.
1E, lane 4; Fig.
4B, lane 9). This
result suggests that a truncated
stem of ~3 bp near the loop might
be sufficient to prevent
misinitiation.
To confirm the importance of the stem, changes that should destabilize
the stem were also tested. Proscript C4S-Hi has nt
22 to
20 changed
to a sequence no longer complementary to
11
to
13. Proscript
C4S-Low contains changes of
5 to
9 and is
predicted by the
mfold program to form an alternative structure.
Both changes
affected RNA structure, as determined by changes
in the mobility of the
RNA in a nondenaturing gel in comparison
to C4WT (Fig.
3), and both
resulted in the reduced ability to
direct RNA synthesis (Fig.
4B, lanes
13 and 14). With C4S-Low,
small amounts of the 9- and 10-nt products
were observed (Fig.
4B, lane 14). Finally, proscripts that lack 2 (C4S-
2) or 4 (C4S-
4)
bp of the stem were reduced in RNA synthesis
(Fig.
4B, lanes 17
and 18). C4S-
4 had not only a more severe
reduction of the expected
15- and 16-nt products in comparison to
C4S-
2 but also an increase
in the amount of misinitiated 9- and
10-nt products (Fig.
4B,
lane 18). Taken together, these results
confirm our previous hypothesis
based on the comparison of CMV RNA
sequences that a stem of ca.
7 bp in length is more than sufficient for
efficient RNA synthesis.
However, changes in the sequence of the stem
can be tolerated
as long as a stem is
formed.
Effect of loop nucleotide changes.
The loop nucleotides in the
different CMV isolates were rich in purines, but the SD isolate had two
additional purines inserted in the loop (Fig. 2). To examine the
features in the loop required for RNA synthesis, a relatively
conservative change of each of the six nucleotides (3' ACGAAG 5')
to its Watson-Crick (W-C) transition was made separately and
examined for RNA synthesis by the CMV replicase (Fig.
5A). Most of the nucleotides could be
individually changed without a significant effect on the efficiency and
specificity of initiation, except for C4L15A/G, whose change
decreased synthesis to 57% relative to C4WT (Fig. 5A, lane 7). Next,
each of the six nucleotides was individually changed to their W-C
transversions. Changes at positions 19, 18, and 17 did not affect
RNA synthesis, but slight reductions were observed with changes of the
individual purines at positions 16 and 14 (lanes 3 to 8). Reduced
synthesis was also observed with W-C transitional mutant C4L14G/A
(78%) (lane 8) but not with C4L16A/G (112%) (lane 6), although the reduction was not as significant as that from transversions. More significant reduction was observed with a change at position 15 (57%) (Fig. 5B, lane 7). The accuracy of initiation was not affected by these mutations. These results indicate that a specific adenylate at
position 15 and probably accompanied with purine bases are favored
even though the specific nucleotide requirements at other positions in
the loop for RNA synthesis in vitro are relatively lax.
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FIG. 5.
Effects of changes in the hexanucleotide loop within the
CMV core promoter. (A) Effects of single-base W-C transversions of the
six loop nucleotides. The sequence of the wild-type loop from positions
19 to 14 is in bold. The constructs used are named so as to
indicate the identity of the original base before the slash and the
nucleotide of the substitution after the slash. Positions of the 15- and 16-nt replicase products are shown at the right. "% of syn"
indicates the percentage of the CMV replicase products made from the
specified template relative to C4WT tested in the same set of
reactions; "" indicates that no template was added to the RNA
synthesis reaction. (B) Effects of single-base W-C transversions of the
six loop nucleotides. (C) Effects of multiple nucleotide substitutions
and deletions in the loop. C4-Ts has all of the loop nucleotides
changed to 3' GUAGGA 5'; C4-Tv has all of the loop
nucleotides changed to 3' UGUUUC 5'. Changes in the other
constructs are indicated according to the final sequence of the loop.
The putative closing base pairs are in lowercase letters. Products of
the RNA synthesis reaction are indicated at the left. The asterisk
denotes a misinitiation product of ca. 25 nt. (D) Effects of changes in
both the stem and the loop nucleotides. C4AS contains an alternative
stem sequence shown in Fig. 4. The nucleotides in the loop are
identified in the names of the proscripts. Where changed, the
identities of the closing nucleotides are indicated in lowercase
letters.
|
|
More drastic changes to the loop nucleotides were examined. W-C
transitions of all six nucleotides in the loop were made in
a
proscript named C4L-Ts. C4L-Ts directed RNA synthesis at 37%
relative
to C4WT and resulted in small amounts of misinitiated
products
(Fig.
5C, lane 2). Changing the loop sequence to their
W-C
transversions (3'UGCUUC5') in proscript C4L-Tv also resulted
in approximately 37% of the level of synthesis in comparison to
C4WT.
Minor amounts of 9- and 10-nt misinitiated products were
observed with
both C4L-Ts and C4L-Tv. However, a prominent longer
misinitiated
product was observed with C4L-Tv (lane 4). We decided
to next alter the
number of nucleotides in the loop. Two different
proscripts, with U-A
closing base pairs and four loop nucleotides
of ACAA and GAAG (same as
those in C4WT), were found to have only
a minor detrimental effect on
the efficiency and accuracy of RNA
synthesis (lanes 5 and 6, respectively). However, since a U-A
closing base pair is relatively
unstable and could result in a
6-nt loop (with one fewer base pair in
the stem), we changed the
loop to a tetraloop of the GNRA class
(
34) with a CG closing
base pair. The latter proscript
directed 45% synthesis of the
15- and 16-nt products in comparison to
C4WT, with a noticeable
increase in the abundance of the 9- and 10-nt
misinitiated products.
Next, a 3-nt loop of the sequences of GGG, AAA,
and CCC were tested
in the context of a UA closing base pair. All three
constructs
had an increased amount of the 9- and 10-nt misinitiated
products
relative to C4WT (lanes 8 to 10). However, the amount of the
15-
and 16-nt products differed more significantly. A loop of AAA
was
able to direct 45% synthesis of C4WT (lane 9), while loops
of GGG or
CCC resulted in RNA synthesis of only 16 or 6%, respectively,
relative
to C4WT (lanes 8 and 10). All of these changes indicate
that one or
more adenylates are preferred for efficient and accurate
synthesis by the CMV replicase. The only RNA that directed
synthesis
without an adenylate in the loop was C4-Tv (lane 4).
Whether this
is due to some compensatory effects of the neighboring
nucleotides
is not
clear.
We next changed both the stem and the loop (Fig.
5D). Consistent with
previous results, C4AS, containing an altered stem sequence
and the
normal 6-nt loop, directed 1.5-fold more synthesis than
C4WT without
incorrectly initiated products (Fig.
4B, lane 5;
Fig.
5D, lane 3).
However, an altered stem with a triloop of UAU
and a UA closing base
pair reduced RNA synthesis to 27% relative
to synthesis from C4AS and
also produced the 9- and 10-nt misinitiated
RNAs (Fig.
5D, lane 4). A
triloop with two adenylates (AUA) and
a CG closing base pair was able
to accurately direct synthesis
of only the 15- and 16-nt products, but
at 55% relative to C4AS
(Fig.
5D, lane 5). These results are in
agreement with our previous
observation that there is a preference for
one or more adenylates
at the 5' portion of the
loop.
Proscript sequence needed to interact with replicase.
The
observation that some changes in the stem-loop will affect the
use of an initiation site indicates that both the stem-loop and the
initiation cytidylate may interact with the replicase. To determine if
the sequence interacted directly with the CMV replicase, we used a
template competition assay. In this assay, increasing concentrations of
a competitor RNA that does not produce visible products were added to
reaction mixtures containing a constant amount of C4WT. RNA containing
nt 29 to +2, with the 2C changed to U to remove a potential
alternative initiation cytidylate, decreased the synthesis from C4WT
significantly. C429/+2 was able to reduce synthesis from C4WT to 50%
when present at 52 nM. This concentration, the IC50, could
be used to compare the relative inhibitory activities of different
competitors. The IC50 for 29/+2, with the +1C changed to
an adenylate, was 107 nM (Fig. 6),
demonstrating that the initiation cytidylate does contribute to the
interaction with the replicase. An RNA containing the sequence from
28 to 5, with only the stem-loop necessary for efficient and
accurate initiation, was a poor competitor, with an IC50 of
>1 µM (Fig. 6). Therefore, both the stem within the CMV core
subgenomic promoter and the initiation cytidylate are
needed to stably interact with the CMV replicase.
View larger version (36K):
[in this window]
[in a new window]
|
FIG. 6.
Minimal proscript sequence required to interact with the
CMV replicase, as identified by a template competition assay. The
reaction measures the synthesis from C4WT as affected by the three
competitor RNAs listed at the top. The IC50s were
calculated from three independent experiments, and 1 standard deviation
from the mean is listed after the mean. For SL-28/+5, reduction of
synthesis to 50% was never obtained in three independent
experiments.
|
|
Spatial requirements between the stem-loop and the initiation
cytidylate.
Since both the stem-loop and the initiation cytidylate
are required to interact with the CMV replicase, we examined the
effect of altering the number of nucleotides between the two elements. The single-stranded sequence between 5 and 1 was targeted for changes because it lies between the stem and the +1C. Two
uridylates or four adenylates inserted between positions 5 and 4 in
proscripts C4I2U and C4I4A, respectively, had little effect on the
efficiency of RNA synthesis, except for a minor amount of a longer
product from C4I2U, which likely initiated from 6C, based on the
length of the product (Fig. 7A; Fig. 7B,
lanes 3 and 4). Deletion of the two adenylates at 4 and 3 in
proscript C4AA resulted in decreased synthesis, and some
misinitiated products were produced (Fig. 7B; lane 5). Removal of the
three nucleotides from 4 to 2 in proscript C4AAC
ignificantly reduced accurate initiation, and increased amounts
of RNAs of 9, 10, 11, and 12 nt were observed, probably due
to misinitiation at +6C and +3C (Fig. 7B, lane 6). These results
indicate that there is some flexibility in the spatial requirements
between the stem-loop and the initiation site, and insertions are
better tolerated than deletions.
View larger version (59K):
[in this window]
[in a new window]
|
FIG. 7.
Effects of changing the spacing between the stem-loop
and the initiation cytidylate. (A) Schematic of the region affected by
insertions and deletions. The initiation cytidylate is in bold and a
larger font. Asterisks identify potential alternative initiation sites.
Underlined bold letters indicate insertions; nucleotides deleted are
indicated by dashes. (B) RNA synthesis from the mutant proscripts
identified in panel A. Sizes of the initiation products are indicated
in nucleotides at the right. The symbol # identifies a longer product
in the reaction with C4I2U.
|
|
Some spatial flexibility in the position of the initiation
template nucleotide is tolerated by the CMV replicase in vitro,
which
raises the question of why the
2C is not used as an initiation
site,
especially in the presence of 2-nt insertions between
5
and
4. Two
previous observations may be relevant to the role
of the
2C. First,
Adkins et al. (
2) previously showed that
an adenylate is
strongly preferred at the +2 position in the initiation
of
genomic and subgenomic plus-strand synthesis of
alpha-like
viruses. Second, Sivakumaran et al. (
30)
established that guanylates
and cytidylates are generally not found in
the first four positions
after the initiation of genomic
plus-strand RNA synthesis in alphaviruses.
These observations suggest
that the
2C is not a suitable initiation
site because the
1
position is a G. Therefore, C4
1G/A was made,
changing the
1G to an
A. C4
1G/A initiated RNA synthesis from
the
2C, as evidenced by the
products that are 17 and 18 nt in
length, at 35% in comparison to the
correctly initiated products
of 15 and 16 nt (Fig.
7B, lane 9). The
1G is conserved in all
of the CMV strains examined (Fig.
2) and
is also found in the
templates for subgenomic RNAs of BMV
and CMV. It may be required
to lend specificity to the
recognition of the +1C.
| |
DISCUSSION |
Due to its importance as a plant pathogen, CMV has been well
characterized in terms of host range, symptom determinants, and other
biological properties. However, the biochemical characterization of the
CMV RNA replication signals has lagged behind that of other viruses. We
seek to take a reductionist's approach to define the sequences
responsible for directing CMV RNA synthesis. The results can be
compared and contrasted to those for other members of the Bromoviridae. For the CMV subgenomic RNA
synthesis, we have defined a stem-loop sequence from 28 to 5
relative to the initiation cytidylate that contributes to efficient and
accurate initiation of subgenomic RNA synthesis.
Our results are in general agreement with and extend the analysis of
CMV replication in protoplasts by Boccard and Baulcombe (4).
In protoplasts, nt 30 to +67 containing the CMV
subgenomic promoter could direct synthesis of the native
subgenomic RNA4 and of a second copy of the
subgenomic promoter placed within the capsid-coding
sequence, although synthesis is lower than an RNA with 70 nt upstream
of the initiation cytidylate (4). We found that in vitro,
the presence of sequence 3' of position 28 did not improve RNA
synthesis. The differences observed by Boccard and Baulcombe
(4) may have been due to some factor(s) that enhances RNA
synthesis which is absent in our replicase preparation or to the fact
that the sequence from 70 to 30 has some effects in vivo other than
on RNA synthesis. Nonetheless, the finding that only 28 nt upstream of
the initiation site is sufficient to direct basal levels of RNA
synthesis indicates that the core subgenomic promoter is
contained within this sequence. We have demonstrated that the stem-loop
and the initiation cytidylate are minimal replicase recognition
elements. Both contribute to the specific selection of the initiation
template nucleotide.
While the stem-loop structure upstream of the CMV
subgenomic RNA4 initiation site contributes to the
efficiency and accuracy of initiation, it is somewhat surprising that
many of the changes made in the sequence allowed RNA synthesis. While
the presence of a stem-loop is required, complete replacement of the
stem and loop nucleotides with at least some alternative sequences
retained efficient and accurate RNA synthesis (Fig. 5D). The sequences found in various CMV strains support the claim that some variations in
the stem and the loop are tolerated.
Yoshinari et al. (37) and Deiman et al. (7) have
previously proposed that a nonspecific structure upstream of an
initiation site may be sufficient for the specific recognition of the
initiation site. Despite the fact that some mutations in the stem-loop
were still recognizable and able to direct RNA synthesis by CMV
replicase, we note that not all nonspecific secondary structures can
direct efficient and accurate initiation (e.g., structures in which the loops were changed to CCC or GGG [Fig. 5C]). In fact, several changes
in the stem and the loop caused initiation to occur at both +1 and
other positions. Therefore, while the sequences in the stem and loop
can be changed and retain significant levels of RNA synthesis, the
wild-type sequence has already apparently been optimized for efficient
and accurate initiation of RNA synthesis.
CMV, BMV, and AMV subgenomic promoters.
RNA
structures that appear to direct RNA synthesis by viral replicases have
been demonstrated in several species (for review, see references
5 and 19). In several of these
cases, the sequence that directs correct initiation of RNA synthesis
includes a stem-loop upstream of the initiation site (e.g., reference
32). Also, for subgenomic RNA synthesis
of barley yellow dwarf virus and AMV, stem-loops are apparently
required (12, 17). The identification of a stem-loop
directing efficient and accurate CMV subgenomic RNA
synthesis contributes to this paradigm.
The requirement for AMV subgenomic RNA synthesis in vitro
differs from the situation with the CMV subgenomic core
promoter,
where the stem sequence can be totally changed with no
adverse
effects on the efficiency and accuracy of RNA synthesis.
Exchanging
the two sides of the AMV stem, especially in the lower part
of
the stem, caused significant reduction in RNA synthesis. Also,
the
structure of the AMV stem-loop was insufficient for the initiation
of
AMV subgenomic RNA synthesis, since a single-stranded
region
from positions
37 to
31 was required (
11). It is
possible
that the specific nucleotides in the AMV stem and
single-stranded
regions, or a structure other than the one proposed,
contribute
to replicase
recognition.
With the BMV subgenomic core promoter, several specific
nucleotides at positions
17,
14,
13, and
11 that exist in an
apparently
unstructured region of the core promoter direct replicase
interaction
and accuracy of initiation (
25). Nucleotide
moieties at these
four positions that can affect replicase recognition
have been
identified (
26). Within positions
17 to
11,
stringent spatial
requirements exist with respect to the BMV
subgenomic core promoter.
One or two nucleotides inserted
or deleted between positions
17
and
14 and between positions
14
and
13 resulted in severe decreases
in RNA synthesis (
33).
The situation is quite different with
the CMV core
subgenomic promoter, where removal of several loop
nucleotides and even replacement of the endogenous stem-loop with
a
different stem-loop still retained more than 50% of the synthesis
of
the wild-type proscript (Fig.
5). We conclude that the CMV
replicase
has more relaxed recognition requirements compared to
the BMV
replicase. Thus, the previous hypothesis of a common recognition
mode
for the subgenomic promoters proposed by our lab
(
25) was
incorrect.
Haasnoot et al. (
11) suggested that the recognition
nucleotides in the BMV subgenomic core promoter also exist
in a stem-loop
secondary structure. The stem was proposed to be formed
by the
base pairing of nt
22 to
17 and
14 to
9, partly because
transversions
of both the
17 and
13 mutations resulted in 17% of
wild-type
core promoter activity in comparison to the single changes at
either position (
11). This claim is in contrast to our
analysis
of the BMV subgenomic core promoter (
1,
25-27,
33), and such
low RNA synthesis by the double mutation
indicates a specific
requirement for the bases at those positions, not
the formation
of a base pair. Furthermore, mutations affecting both
sides of
the putative stem could retain significant levels of RNA
synthesis
in comparison to a wild-type RNA (
1,
28). Finally,
UV spectrometry
performed continuously from 0 to 100°C revealed that
a functional
BMV subgenomic proscript,
20/
13, does not
possess a stable structure
in solution, even at 10°C (our RNA
synthesis assays can be performed
at up to 40°C (C. C. Kao,
unpublished data). A stable structure
in the BMV subgenomic
promoter does not appear to exist a priori
in solution to direct
recognition by the BMV replicase. This is
not to say that RNA structure
does not play a role in BMV subgenomic
RNA initiation. We
have reported evidence consistent with the
model that intramolecular
RNA interactions may form after replicase
binding in an induced-fit
mechanism (
3,
33). A prominent
difference between the CMV
and BMV subgenomic core promoters is
the presence of a
polyuridylate tract 3' of the BMV core promoter.
The polyuridylate
tract may keep the core sequence accessible
for replicase interaction
and obviate the need to evolve a specific
structure that then dictates
subgenomic RNA synthesis (
1).
In CMV, the
presence of eight uridylates was unable to compensate
for the
truncation of the stem-loop (Fig.
1D). Thus, even closely
related
viruses could adapt different strategies for the initiation
of RNA
synthesis.
Despite the differences in the initial recognition of the CMV and BMV
subgenomic core promoters by the appropriate viral
polymerases,
several features are common to both promoters. First, they
both
have relatively short sequences that interact with the replicase
and direct accurate initiation of RNA synthesis in vitro. Second,
there
is a flexible spacer sequence between the key recognition
elements and the initiation cytidylate. Third, the initiation
cytidylates are required by both replicases to increase the
stability
of the interaction between the proscripts and the replicases
(Fig.
7 and reference
26). Fourth, the cytidylate
used in vivo is
apparently the optimal one for initiation due to the
spacing between
the recognition element and the +1C. For example, the
1G renders
the
2C an ineffective initiation nucleotide, and the
A-U-rich
sequence immediately after initiation seems to play a role in
efficient RNA synthesis. Given these similarities, it is quite
likely
that an induced-fit model will best describe the interaction
between
the CMV replicase and its core promoter. Finally, motifs
other than the
core promoter may be capable of directing viral
subgenomic RNA synthesis. Long-range interactions
between RNA
elements have been reported to regulate
subgenomic RNA synthesis
in several viruses (
15,
19,
28).
| |
ACKNOWLEDGMENTS |
Helpful discussions and encouragement from K. Sivakumaran and
editing by L. Kao are much appreciated.
The Kao laboratory is supported by the NSF (MCB9507344) and USDA
(9702126) and by a fellowship from the Samuel Noble Foundation. M.J.R.
is supported by funding from the Samuel Robert Noble Foundation. C.C.K.
acknowledges a Linda and Jack Gill fellowship.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Indiana
University, Department of Biology, 1001 E. Third St., Bloomington, IN
47405. Phone: (812) 855-7959. Fax: (812) 855-6705. E-mail:
ckao{at}bio.indiana.edu.
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Journal of Virology, December 2000, p. 11201-11209, Vol. 74, No. 23
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
