Download Complete nucleotide sequence of RNA 4 of rice stripe virus isolate T

Journal of General Virology(1992), 73, 1309-1312. Printedin Great Britain
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Complete nucleotide sequence of RNA 4 of rice stripe virus isolate T, and
comparison with another isolate and with maize stripe virus
Yafeng Zhu, 1. Takahiko Hayakawa 1 and Shigemitsu Toriyama 2
Plantech Research Institute, c/o M K C Research Center, 1000 Kamoshida-cho, Midori-ku, Yokohama-city 227 and
2National Institute of Agro-Environmental Sciences, 1-1, Kannondai, 3-chome, Tsukuba-city, Ibaraki 305, Japan
The complete nucleotide sequence of R N A 4 of rice
stripe virus isolate T (RSV-T) was determined and
found to consist of 2157 nucleotides, containing two
open reading frames (ORFs). One, deduced to be
present in the 5'-proximal region of the viral-sense
RNA, encodes the stripe disease-specific protein with
M, 20541, and the other ORF, in the 5'-proximal
region of the viral complementary sense RNA, encodes
an unknown protein with Mr 32 474. Between these two
ORFs there is an intergenic non-coding region that
could form a secondary structure with two base-paired
hairpin configurations. These characteristics indicate
that RSV-T RNA 4 has an ambisense coding strategy.
Comparison of the two ORFs of RSV-T with those of
another isolate revealed 97-2 % and 98.0 % identity for
the nucleotide sequences, and 98.3% and 98.2%
identity for the amino acid sequences. The leader
sequences of these two isolates were the same.
However, an insertion was found in the intergenic noncoding region of RSV-T. Furthermore, comparison of
the nucleotide and amino acid sequences of RSV-T
RNA 4 with those of R N A 4 of maize stripe virus,
which is another member of the tenuivirus group,
revealed greater identity, suggesting a close phylogenetic relationship between these two viruses.
Rice stripe virus (RSV) is a type member of the
delphacid planthopper-borne tenuivirus group of plant
viruses (Francki et al., 1991). Four species of ssRNA and
four species of dsRNA are found in the purified virus
preparation (Toriyama, 1982; Toriyama & Watanabe,
1989; Ishikawa et al., 1989). Recently, the complete
nucleotide sequences of R N A 3 and R N A 4 of RSV have
been determined and their ambisense coding strategy
identified (Kakutani et al., 1990, 1991 ; Zhu et al., 1991).
The virus particles contain a single coat protein with an
estimated Mr of 32K (Koganezawa et al., 1975;
Toriyama, 1982), which has been shown to be encoded
by the viral complementary (vc) sequence of the third
longest segment, RNA 3 (Kakutani et al., 1991 ; Zhu et
al., 1991). A large amount of a non-structural protein, the
stripe disease-specific protein (S protein), is produced in
RSV-infected rice plants (Kiso & Yamamoto, 1973;
Toriyama, 1986); this protein has been shown to be
encoded by the fourth longest segment, R N A 4 (Hayano
et al., 1990).
Toriyama (1986) observed that the S proteins isolated
from RSV-infected rice cultivars, and wheat and maize
plants produce peptides of different Mrs. Recently, three
isolates of RSV which produce different symptoms in
rice plants have been reported (Hayashi et al., 1990); the
M,s of their S proteins differ. However, differences
between these isolates at the molecular level have not yet
been identified. In this paper we report the complete
nucleotide sequence of R N A 4 of RSV isolate T
(RSV-T), and compare this sequence and the deduced
amino acid sequences with those of another isolate
(Kakutani et al., 1990), referred to as isolate M (RSV-M)
(personal communication), and R N A 4 of maize stripe
virus (MStV), which also encodes the non-structural
protein (Huiet et al., 1990).
Virus particles and viral R N A were prepared as
described previously (Zhu et al., 1991). Double-stranded
cDNA was synthesized according to Gubler & Hoffman
(1983) by using a 20-base oligonucleotide primer
(5' GGGCATATCTTTTGAGATTA 3')corresponding
to known parts of R N A 4 (Takahashi et al., 1990). The
double-stranded cDNA obtained was made blunt using
T4 DNA polymerase and inserted into the Sinai site of
pUC 19 (Yanisch-Perron et al., 1985). Transformation of
Escherichia coli strain DH5 was performed according to
Hanahan (1985).
Sequencing of cDNA and deletion mutants thereof
were carried out as described previously (Zhu et al.,
1991). The cDNA covering the full length of the R N A 4
The nucleotide sequencedata reported have been submitted to the
DNA Data Bankof Japan and assignedthe accessionnumberD01164.
0001-0664© 1992SGM
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Short communication
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Fig. 1. CompletenucleotidesequenceofRSV-TRNA 4andtwodeducedORFs(ORFl and ORF2). The numbers indicate the distance
~om the 5' terminus of vRNA. ORF1 (nucleotides 55 to 591) is predicted to be the S protein encoding region. The protein encoded by
the ORF within nucleotides 1246 to 2106 was deduced ~om the vcRNA sequence and contains 287 amino acids. The underlined
sequence indicates the 20-base inse~ion relative to this region of RSV-M.
segment was obtained by primer extension. The nucleotide sequence o f the c D N A was determined in both
directions. Parts o f the genomic c D N A were sequenced
from two or three independent clones. For confirmation,
the c D N A s obtained by polymerase chain r e a c t i o n
(PCR) amplification (Saiki et al., 1988) were sequenced
directly (Brow, 1990). Computer analysis was performed
using Genetyx (Software Development).
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Short communication
The complete nucleotide sequence of RSV-T R N A 4 is
given in Fig. 1. RSV-T R N A 4 consists of 2157
nucleotides, and two open reading frames (ORFs) were
predicted from the nucleotide sequence. One putative
O R F , located in the Y-proximal region of the viral-sense
R N A (vRNA), encoded a protein of 178 amino acids
with an Mr of 20541 (20K protein) (Fig. 1, ORF1). Its
amino acid composition was more than 98 % identical to
that of purified S protein (data not shown), confirming
that the 20K protein is the S protein. The other O R F , in
the Y-proximal region of the v c R N A , encoded a protein
of 286 amino acids with an Mr of 32474 (32K protein)
(Fig. 1, ORF2). No O R F of significant length was
deduced to exist in the Y-proximal regions of either
v R N A or v c R N A . These observations confirmed the
previous report that RSV-M R N A 4 shows an ambisense
coding strategy (Kakutani et al., 1990).
Between the two O R F s there is an intergenic noncoding region of 634 nucleotides. Computer analysis
showed that this intergenic region can form two basepaired hairpin configurations. One involves 36 nucleotides (918 to 953) with a calculated free energy of - 36 kJ
(Tinoco et al., 1973), and the other involves 46
nucleotides (1010 to 1055) with a calculated free energy
of - 160 kJ (Fig. 2). These configurations might function
in termination of the subgenomic R N A , as suggested
previously (Auperin et al., 1984; Zhu et al., 1991).
We compared the nucleotide and deduced amino acid
sequences of RSV-T R N A 4 with those of RSV-M R N A
4 (Kakutani et al., 1990). A schematic summary of the
comparison is shown in Fig. 3. The nucleotide sequences
of ORF1 and O R F 2 were 97.2% and 98.0% identical,
respectively; at the amino acid level, 93.3 % and 98.2 % of
residues were identical. The intergenic non-coding
region of RSV-T, however, showed 86.9% identity with
that of RSV-M, this region of RSV-T containing a
20-base insertion (Fig. 1, underline). This was confirmed
by direct sequencing of the c D N A synthesized by PCR.
The insertion does not alter the stable hairpin structure
described above. This kind of insertion or deletion has
been observed in the intergenic non-coding region of the
S R N A of two isolates of tomato spotted wilt virus,
which also has an ambisense coding strategy (de H a a n et
al., 1990; Maiss et al., 1991). These observations
suggested that the intergenic non-coding region, which is
predicted to have a complex higher structure, is
susceptible to mutation, and that the secondary structure
rather than the nucleotide sequence is important for viral
activity. It remains to be elucidated whether the
sequence divergence between the two isolates of RSV
relates to their biological characteristics, including
disease symptoms. In addition to the O R F s and the
intergenic non-coding region, the leader sequences from
each viral strand were also compared and shown to be
1311
AG = -160 kJ
AG = -36 kJ
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Fig. 2. The two predicted hairpin structures in the intergenic noncoding region. Dashes and asterisks show base-pairing in the stem
configuration. The numbers indicate the distance from the 5' terminus
of the viral ssRNA 4 (see Fig. 1).
Nucleotides
Intergenic
Leader ORF1 non-coding
sequence
region
(a)
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Fig. 3. Schematic representation of the identities between the
nucleotide and deduced amino acid sequences of RSV-T and RSV-M.
The percentage identity in each region (leader sequence, ORF and
intergenic non-coding region) is shown in the corresponding box. The
upper bar (a) shows a comparison of the nucleotide sequences. The
middle and lower bars (b and c) represent comparisons of the amino
acid sequences encoded by v- and vcRNAs, respectively.The numbers
in (a) indicate the distance from the 5' terminus of the viral ssRNA. In
(b) and (c), the numbers indicate the amino acid positions relative to the
first methionine of each ORF encoded by the v- and vcRNA 4,
respectively.
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Short communication
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Fig. 4. Comparison of the amino acid sequence of the S protein
deduced from the RSV-T RNA 4 nucleotide sequence with that of
NCP encoded by MStV RNA 4. Identical amino acids are shown by
dashed lines. The upper line is the RSV-T-eneoded sequence and the
lower line is that of MStV. Maximum matching analysis was performed
using Genetyx.
identical. This observation suggests that the leader
sequence could be an important factor in R N A replication and/or protein translation.
Huiet et al. (1990) partially sequenced the RNA 4
segment of MStV, another member of the tenuivirus
group, and reported that it encodes a major non-capsid
protein (NCP). Comparison of the ORFs encoding NCP
and the S protein of RSV-T revealed 69.6~o identity at
the nucleotide sequence level and 74.1 ~o identity for the
deduced amino acid sequence (Fig. 4). Recently, the
complete nucleotide sequence of MStV RNA 3 has been
reported (Huiet et al., 1991), and comparison of R N A 3
of RSV-T and MStV revealed more than 65 ~o identity in
the two ORFs, and about 53~o identity in the intergenic
non-coding region. These results demonstrate the close
phylogenetic relationships between RSV and MStV. The
function of the S protein is not known; however, the high
degree of identity between the S protein of RSV and the
NCP of MStV indicates that they should have similar
functions.
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(Received 3 October 1991; Accepted 2 January 1992)
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