Functional Analysis of the Cloverleaf-like Structure in the 3' Untranslated Region of Bamboo Mosaic
Potexvirus RNA Revealed Dual Roles in Viral RNA Replication and Long Distance Movement
I-Hsuan Chen, MengHsiao Meng, Yau-Heiu Hsu, and Ching-Hsiu Tsai*
Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, 402, TAIWAN
running title: Functional Analysis of the Cloverleaf-like Structure in the 3' UTR of BaMV RNA
*Corresponding author
Phone number: (886)-4-22840451
Fax number: (886)-4-22860260
e-mail: chtsai1@dragon.nchu.edu.tw
Abstract
The 3' untranslated region (UTR) of bamboo mosaic potexvirus (BaMV) RNA was identified to fold into a
tertiary structure comprising a cloverleaf-like structure designated ABC domain followed by a major stem-loop
D, which in turn is followed by a pseudoknot E and a poly (A) tail.
The coat protein accumulation level of the
mutant, BaMV40A/ΔABC, lacking ABC domain was just 15% that of wild type when inoculated into
protoplasts of Nicotiana benthamiana.
BaMV RNA replication.
This suggested that ABC domain might play an important role in
In order to define the precise role of each of the three stem-loops of ABC domain in
RNA replication, three mutants BaMV40A/ΔA, -/ΔB, and -/ΔC each lacking stem-loop A, B, and C,
respectively, was created.
Our results showed that accumulation of viral products of mutants
BaMV40A/ΔB and -/ΔC were not as efficient as wild type.
On the contrary, level of accumulation of viral
products of BaMV/ΔA was similar to that of wild type in protoplasts and inoculated leaves.
Interestingly, the
accumulation of viral products was not as efficient as that of wild type in systemic leaves implying that
stem-loop A is dispensable for replication, but signifies a role in systemic accumulation.
Using UV
cross-linking and competition experiments, it was demonstrated that the E. coli-expressed helicase domain of
BaMV ORF1 can preferentially interact with the ABC domain.
Keywords: bamboo mosaic potexvirus; RNA replication; helicase-like domain; long distance movement
Introduction
Bamboo mosaic potexvirus (BaMV) is a flexuous rod-shaped virus (Lin et al., 1977) possessing a
single-stranded positive-sense RNA genome with a 5' m7GpppG structure and a 3' poly (A) tail.
The
6,366-nucleotide (nt)-long genome [excluding the 3' poly (A) tail] consists of five open reading frames and a
94- and a 142-nt untranslated region (UTR) at the 5'- and 3'-ends, respectively (Lin et al., 1994).
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 (Lin et al., 1992).
It has been shown that the 3' UTR of BaMV RNA could fold into a tertiary structure with a
cloverleaf-like structure, a major stem-loop, and a pseudoknot designated as ABC, D, and E domains,
respectively (Cheng and Tsai, 1999).
Functional analysis of the tertiary structure revealed that maintaining
the integrity of the pseudoknot and the stem structure of D domain are important for viral RNA replication in
protoplasts (Tsai et al., 1999; Cheng and Tsai, 1999).
A mutant lacking ABC domain was defective in coat
protein accumulation in protoplasts (Cheng and Tsai, 1999).
Results derived from footprinting,
electrophoretic mobility shift, and competition analyses showed that the truncated RdRp domain encoded by
BaMV ORF1 could specifically interact at D and E domains of the 3' UTR (Huang et al., 2001).
In this study,
we focus on the role of ABC domain in the replication of BaMV RNA in protoplasts and plants.
Results
Stem-loops B and C play an important role in viral RNA replication.
A mutant lacking the cloverleaf-like structure (ABC domain) in the 3'UTR of BaMV RNA (Fig. 1) was
shown to be defective in the accumulation of viral products in protoplasts (Cheng and Tsai, 1999).
determine which of the three stem-loops of ABC domain is the major determinant in
To
BaMV RNA replication,
each stem-loop A, B, and C was deleted in each mutant and designated as BaMV40A/A, -/B, and -/C,
respectively.
Transcripts derived from pBaMV40A and its derivatives were inoculated into N. benthamiana
protoplasts to examine the accumulation of coat protein, genomic RNA, and subgenomic RNAs (Fig. 2).
Total proteins and RNAs were extracted from the inoculated protoplasts at 16, 24, 32, and 48 h
postinoculation (hpi) and analyzed on Western and Northern blots, respectively.
These results indicated
that the accumulation level of viral RNA at 8 hpi was undetectable but increased significantly from 16 to 48
hpi (Fig. 2).
It was noted that the level of RNA accumulation was still increasing at the 48h time point.
If we
compared the relative accumulation efficiency of the genomic RNA at each time point (Fig. 2C, the numbers
in parenthesis), we have found that the relative ratio of mutants to wild type is quite similar at each time point.
Therefore, we took the 48 h time point with the strongest signal for a comparison (Fig. 2C).
BaMV40A/A
was just like wild type, and BaMV40A/ABC, -/B and -/C were only about 20 to 30% that of wild type.
To rule
out the possibility of a structural change due to the stem-loop deletion, we have predicted the secondary
structure of the 3' UTR with each stem-loop deletion of ABC domain using STAR (Abrahams et al., 1990) and
MFOLD (Zuker, 1999) which showed that the deletion of one stem-loop did not interfere the formation of the
other two stem-loops leaving the secondary structure intact as wild type.
Overall of these results indicated
that stem-loops B and C might play an important role in viral RNA replication, whereas stem-loop A was not
the major determinant of BaMV RNA replication in protoplasts
Stem-loop A is possibly involved in long distance movement.
To evaluate the replication efficiency of these mutants in plants, the altered sequences were transferred
from strain O to strain S which is derived from BaMV-O (Lin et al., 1994) and had a better replication rate and
efficient systemic movement in N. benthamiana plants than the strain O (Liao, 2000).
Similar relative
accumulations of coat protein were found for the same mutations in strain S as in O (Fig. 3A).
Therefore,
transcripts of BaMV-S and its derivatives were inoculated onto N. benthamiana plants with 5 æg RNA per leaf.
The ratio of coat protein accumulation of mutants on the inoculated leaves to that of wild type analyzed on
Western blot (Fig. 3B) showed a similar ratio to those observed in protoplasts.
However, none of the
mutants could move systemically except BaMV-S/A which showed little coat protein accumulation in the third
systemic leaf (Fig. 3C). In contrast, the coat protein could be detected in each systemic leaf when inoculated
with BaMV-S.
Since, mutants BaMV-S/ABC, -/B, and -/C have a very low RNA replication efficiency in
protoplasts (Fig. 3A) and inoculated leaves (Fig. 3B) this could be the reason for the undetectable viral
proteins are in systemic leaves,
whereas for BaMV-S/A the reason is likely due to a failure in long distance
movement rather than a defect in viral RNA replication.
Helicase-like domain of BaMV ORF1 interacts with the cloverleaf-like structure.
We are curious to know if the co-linearity of the domains of the BaMV ORF1 polypeptide corresponds to
the co-linearity of the domains of the 3' UTR.
Since, it was shown that the recombinant polymerase domain
of BaMV ORF 1 specifically interacts with D and E domains of the BaMV 3' UTR (Huang et al., 2001), it
seemed possible for the helicase-like domain to interact with ABC domain owing to its co-linearity to the
polymerase domain and the resultant steric proximity to ABC domain of the 3' UTR.
To investigate this we
have used 50 fmole of 32P-labeled 100-nt transcript rABC (Fig. 1, nt 39 to 138) for UV cross-linking with
purified recombinant helicase-like domain of BaMV ORF1 (Li et al., 2001).
In the presence of 50 ng of the E.
coli-expressed BaMV helicase, a single radio-labeled protein was observed on the SDS polyacrylamide gel
(Fig. 4, lane 1).
No signal was detected when the radio-labeled rABC was incubated with 5 or 10 æg of BSA
(Fig. 4, lanes 2 and 3).
To investigate the specificity of the interaction between the helicase-like protein and rABC, unlabeled
RNAs were used as competitors during the UV cross-linking reaction.
Unlabeled rABC was a good
competitor needing only 2-fold molar excess to compete out 50% of labeled probe (Fig. 5).
In contrast the
r84/40A (Fig. 1, nt 1 to 84 with 40 adenylate residues at the 3' end), satRNA (satellite RNA derived from pSF4;
Hsu et al., 1998), and bovine liver tRNA could not compete out 50% of the labeled probe efficiently even at
10-fold molar excess (Fig. 5).
These results indicated that the helicase-like domain of BaMV ORF1 could
specifically bind to the cloverleaf-like domain of the 3' UTR.
Stem-loop B of the 3' UTR is the interaction site with the BaMV helicase-like domain of ORF1.
Footprinting analysis was used to locate the sites of interaction of ABC domain with the helicase-like
domain of ORF1.
digestion.
The condition used for footprinting was optimized for protein binding and RNase T2
Transcript rABC was 5' end-labeled, and was treated with RNase T2 in the absence or presence
of 50 ng of the E. coli-expressed BaMV helicase-like protein after UV cross-linking (Fig. 6).
Nucleotides G92
to G88 in loop C showed equal density of banding with or without the addition helicase-like protein (lanes 5
and 6), indicating that they were not protected by the helicase-like protein.
By contrast, nucleotides A108 in
loop B and the sequence at the part of the stem B including U109 showed weaker banding signals in the
presence of helicase-like protein (Fig. 6, lane 6) than without helicase-like protein (Fig. 6, lane 5), whereas
the sequence in loop A showed a minor protection in the presence of helicase-like protein as compared to
banding density of G126 and U125.
Besides, nucleotides U80, A79, and C72 in the single-stranded region
was shown to be well protected by the helicase-like protein.
These results suggested that the helicase-like
domain of the ORF1 of BaMV could bind to loop B, part of stem B, and the single-stranded region
downstream of the cloverleaf-like structure of the 3' UTR of BaMV RNA.
Discussion
The 3' UTR of positive-sense viral RNA could serve as a cis-acting element in the initiation of
minus-strand RNA synthesis and play a regulatory role during viral RNA replication.
We had demonstrated
that the accumulation levels of coat protein and viral RNAs were reduced significantly when the cloverleaf-like
structure (ABC domain) in the 3' UTR of BaMV RNA was removed (Cheng and Tsai, 1999).
Further analysis
by deleting the stem-loop B or C of ABC domain in the 3' UTR of BaMV RNA showed a similar replication
defect as that of ABC domain deletion.
Since the truncated polymerase domain of BaMV ORF1 was
identified to interact with D and E domains of the 3'UTR (Huang et al., 2001), it is reasonable to correlate the
co-linearity of polymerase domain with helicase-like domain of ORF1 to the co-linearity of D and E domains
with cloverleaf-like domain of the 3' UTR.
Indeed UV cross-linking experiments have shown that the E.
coli-expressed BaMV helicase-like protein could preferentially bind to the cloverleaf-like domain of the 3' UTR
when compared to the nonspecific competitor RNAs (Fig. 5).
Further analysis by footprinting study
revealed that loop region of stem-loop B was preferred to interact with the over-expressed helicase-like
protein.
It is likely that the replication of viral genome required the 155 kDa RNA-dependent RNA
polymerase derived from ORF1 of BaMV interact with the 3'UTR.
The helicase-like domain interacts with
stem-loop B and the polymerase domain interacts with stem-loop D and poly (A) (Huang, et al., 2001).
These interactions could be the major determinants of
minus-strand RNA synthesis.
the efficiency of BaMV RNA replication especially the
Although stem-loop C does not interact with helicase-like protein directly as
the results shown in footprinting experiment, the deletion of stem-loop C may cause a steric change which
may interfere with the interaction between stem-loop B and the helicase-like protein.
The similar level of viral products accumulating in protoplasts and leaves inoculated with BaMV-S/A and
BaMV-S suggested that stem-loop A was not a determinant of BaMV RNA replication.
However, the
accumulation of viral products in mutant BaMV-S/A was negligible or absent in systemic leaves when
compared to inoculated leaves and protoplasts (Fig. 3).
This interesting observation led to recognizing a
defect in systemic accumulation for stem-loop A deletion mutant.
Long distance movement is required for
plant viruses to establish their infection through the vascular tissue, and infiltrate the entire plant.
Thus
systemic accumulation of the long distance movement for BaMV is attributed to stem-loop A of ABC domain
in the 3' UTR.
To inquire if stem-loop A could be involved in the process of BaMV RNA encapsidation, viral
particles were extracted from inoculated leaves and examined under electron microscopy.
and mutant showed the same particle size and morphology (data not shown).
stem-loop A is not involved in RNA encapsidation.
Both wild type
These results suggested that
In many instances, encapsidation is required for efficient
movement of plant viruses through the vascular tissue (Andersen and Johansen, 1998; Spitsin et al., 1999;
Okinaka et al., 2001).
Plant viruses failing to accomplish the long distance movement are defective either in their coat protein
or movement protein (Osman et al., 1990; Saito et al., 1990; Okinaka et al., 2001).
Here, we report, for the
first time, a non-coding region of a viral RNA could be responsible for a long distance movement.
It is
possible that stem-loop A or the entire ABC domain interacts with host or viral proteins that are involved in
long distance movement.
If such an interaction exists as mentioned above, it must be independent of the
process of encapsidation since stem-loop A deletion mutant can be encapsidated.
If encapsidation is
required for the long distance movement of BaMV, then there must be more than one type of protein on the
virion.
There are a few reports showed that a virion may not be homogenously encapsidated with only a
single type of protein.
There is a fixed ratio of gag/pol fusion protein over gag in the life cycle of retrovirus
that play an important role for virion formation (Dinman and Wickner, 1992).
There is one copy of
ubiquitinated coat protein of the viral particle of TMV with unclear function (Collmer and Zaitlin, 1983; Collmer
et al., 1983; Dunigan et al., 1998).
However, if the fully encapsidated viral particle along is incapable for
long distance movement, then the coat protein, movement protein, and the unidentified protein in
combination with viral RNA could form a ribonucleic particle capable of passing through the vascular tissue.
Overall, the stem-loop A deletion would be a good tool for further explore the mechanism involved in long
distance movement.
Hunting for the stem-loop A binding protein and investigating its role in long distance
movement would be the goal of our future research.
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Figure legend
Fig. 1.
Secondary structure of the 3'UTR of BaMV RNA.
Nucleotides are numbered from the 3'-end
cytosine just upstream of the poly(A) tail.
Fig. 2. Amplification of BaMV40A and its derivatives in N. benthamiana protoplasts.
Protoplasts (4x105
cells) were inoculated with 5μg transcripts of BaMV40A, BaMV40A/ΔABC, -/ΔA, -/ΔB, and -/ΔC. (A) Total
proteins were separated on a 14% SDS-polyacrylamide gel, blotted, and probed with an antiserum against
BaMV coat protein.
The amount of total proteins loaded in lanes 2, 4, and 5 corresponded to 3 folds that
loaded in lanes 1 and 3.
(B) Representative experiments of Northern blot analysis of viral genomic RNA (6.4
kb) and two subgenomic RNAs (2.0 and 1.0 kb) and the density of genomic RNAs is provided as the
quantitative data in panel C.
The level of genomic RNA from each inoculated sample was compared to that
of BaMV40A at 48 h time point.
RNAs were probed with a 32P-labeled RNA transcript complementary to
0.6 kb of the 3'-end of genomic RNA; total RNAs below the blot were the loading control.
Data presented
were the average of at least four independent protoplast inoculation with at least two Northern blot analysis of
each inoculation.
Fig. 3.
Amplification of BaMV-S and its derivatives in N. benthamiana protoplasts (A) and plants (B and C).
Protoplasts (4x105 cells) and plants (each leaf) were inoculated with 5æg transcripts of BaMV-S,
BaMV-S/ΔABC, -/ΔA, -/ΔB, and -Δ/C and harvested 48 h and 10 d post-inoculation, respectively.
(A)
Northern blot analysis of viral genomic RNA (6.4 kb) and two subgenomic RNAs (2.0 and 1.0 kb).
RNA were
probed with a 32P-labeled RNA transcript complementary to 0.6 kb of the 3'-end of genomic RNA.
The level
of genomic RNA from each inoculated sample was compared to that of BaMV40A at 48 h time point.
Total
proteins were extracted from inoculated leaves (B) and systemic leaves (C) and separated on a 14%
SDS-polyacrylamide gel, blotted, and probed with an antiserum against BaMV coat protein.
The numbers
shown under the blots were the protein loaded with 5 folds (in B, lanes 2, 4, 5) or 3 folds (in C, lanes 2 to 5) to
that of BaMV-S (in B and C, lane 1).
Fig. 4.
UV cross-linking experiment of the purified helicase-like protein with ABC domain RNA.
The
purified helicase-like protein (lane 1) and bovine serum albumin (BAS; lanes 2 and 3) were UV cross-linked to
32P-labeled rABC.
After treatment with RNase A, the labeled proteins were separated on a 14%
SDS-polyacrylamide gel and analyzed with PhosphrImager.
The arrow indicates the position of
helicase-like protein.
Fig. 5.
Interaction between helicase-like protein and ABC domain RNA, investigated in competitive UV
cross-linking assay using different RNAs.
Purified helicase-like protein (50 ng) was incubated with the
indicated molar excesses of unlabeled RNAs and 32P-labeled rABC (50 fmole) for 10 min.
activities were analyzed by UV cross-linking assay.
The competition
(A) Representative experiments of UV cross-linking
assay that have contributed to the quantitative data in (B).
All assays were done at least three times.
Fig. 6.
RNase T2 footprint of the helicase-like protein binding site on the rABC.
Data presented is an
autoradiograph of RNase T2 cleavage products on a 10% polyacrylamide gel containing 7M urea.
Lanes: 1,
rABC probe as a control, labeled C; 2, Alkaline ladders, labeled Alk; 3, RNase A digest ladders, labeled A; 4,
RNase T1 digest ladders, labeled T1; 5 and 6, RNase T2 cleavage (labeled T2) without and with helicase-like
protein, respectively.
The positions of selected nucleotides in the rABC sequence are indicated on the right
for reference.
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Cheng, C. -P., and Tsai, C. -H. (1999). Structural and functional analysis of the 3' untranslated region of
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