Download The complete nucleotide sequence of cucumber green mottle

Journal of General Virology (1991), 72, 1487-1495. Printed in Great Britain
1487
The complete nucleotide sequence of cucumber green mottle mosaic virus
(SH strain) genomic RNA
M. Ugaki, ~ M. Tomiyama, 1 T. Kakutani, 1 S. Hidaka, l~ T. Kiguchi, 2 R. Nagata, 3 T. Sato, 2
F. Motoyoshil* and M. Nishiguchi 4.
1National Institute of Agrobiological Resources, 2-1-2 Kan-nondaL Tsukuba, Ibaraki 305, 2Hokkaido Central Agricultural
Experiment Station, Naganuma, Yu-ubari, Hokkaido 069-13, 3Miyazaki Prefectural Agricultural Experiment Station,
Sadowara, Miyazaki-gun, Miyazaki 880-02 and aKyushu National Agricultural Experiment Station, 2421 Suya,
Nishigoushi, Kikuchi-gun, Kumamoto 861-11, Japan
The complete nucleotide sequence of the genomic
RNA of cucumber green mottle mosaic virus watermelon strain SH (CGMMV-SH) was determined using
cloned cDNA. This sequence is 6421 nucleotides long
containing at least four open reading frames, which
correspond to 186K, 129K, 29K and 17.3K proteins.
The 17.3K protein is the coat protein. Sequence
analysis shows that C G M M V - S H is very closely
related to another watermelon strain, C G M M V - W ,
although three amino acid substitutions in the 29K
protein were found between these strains. The sequence
was also compared to those of other tobamoviruses,
tobacco mosaic virus (TMV) vulgare, TMV-L (a
tomato strain) and tobacco mild green mosaic virus
reported by other groups. It shows 55 to 56% identity
with these viruses. The size and location of the open
reading frames are very similar to those of T M V but the
129K and 186K proteins are composed of 1142 and
1646 amino acids, being larger than those of T M V by
27 and 31 amino acids, respectively. The deduced
amino acid sequences of these proteins are highly
homologous to those of TMV, especially in the
readthrough downstream region of the 186K protein.
Introduction
bottlegourd isolate, CGMMV-C (Vasudeva et al., 1949;
Vasudeva & Nariani, 1952), than to the cucumber strain,
the Yodo strain or two British isolates, E1 and E2.
Francki et al. (1986) pointed out that the CGMMV
watermelon strain is taxonomically different from the
cucumber strain (Inoue et al., 1967), shown by the lack of
molecular hybridization between these two strains, and
proposed a new virus name, 'kyuri green mottle mosaic
virus', for the cucumber strain.
The sequence of 1071 nucleotides from the 3' end of
the genomic RNA of a watermelon isolate (CGMMVW) of the watermelon strain was determined by Meshi et
al. (1983b) by analysing the cDNA of the genomic RNA.
The amino acid sequence of the coat protein of the same
isolate was determined by Nozu & Tsugita (1986) by the
conventional protein sequencing method. Furthermore
Saito et al. (1988) sequenced the region covering the 30K
protein gene and compared it with the known 30K
protein of other tobamoviruses.
A new type of mosaic disease was found in greenhouse-grown muskmelon crops in 1971, and the agent of
this disease was serologically identical with the watermelon strain (Furuki & Komuro, 1973). Necrotic lesions
surrounded by a water-soaked area are characteristic of
Cucumber green mottle mosaic virus (CGMMV) is a
member of the tobamovirus group. C G M M V causes diseases in cucurbitacea and is different in host range from
tobacco mosaic virus (TMV) whose main hosts are
members of the Solanaceae. Strains of CGMMV were
first described as cucumber virus 3 (CV3) and cucumber
virus 4 (CV4) by Ainsworth (1935). In Japan, three
different strains have been described: 'cucumber strain'
(Inoue et al., 1967), 'watermelon strain' (Komuro et al.,
1968) and 'Yodo strain' (Kitani et al., 1970). Among
them, the watermelon strain of CGMMV is the most
serious disease agent, causing severe disease symptoms
in infected watermelon plants, particularly the deterioration of fruit pulp which causes considerable economic
losses to watermelon growers (Komuro et al., 1971).
Tochihara & Komuro (1974) demonstrated that the
watermelon strain has a closer relationship to an Indian
t Present address: Tohoku National Agricultural Experiment
Station, Akahira 4, Shimokuriyagawa, Morioka, Iwate 020-01, Japan.
:[;Present address: Research Institute for Bioresources, Okayama
University, Chuo 2-20-1, Kurashiki 710, Japan.
0001-0044 © 1991 SGM
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1488
M. Ugaki and others
muskmelon fruit infected with the virus, and severely
affected the economic value of the products (I. Furuki &
T. Ohsawa, personal communication). However, the
disease of muskmelon caused by CGMMV can be
controlled by cross-protection, if the seedlings of
muskmelon are previously inoculated with an attenuated
CGMMV strain (Motoyoshi & Nishiguchi, 1988).
We have determined the complete nucleotide sequence of the genomic R N A of CGMMV-SH, a
muskmelon isolate of the CGMMV watermelon strain
derived from a diseased muskmelon, by preparing
cDNA clones covering almost the full-length of the R N A
and sequencing them with an automated fluorescent
D N A sequencer, and also, partially, by directly sequencing the genomic RNA with synthetic oligonucleotides.
We also discuss the difference at the level of nucleotide
sequence of this isolate from other tobamoviruses.
Methods
Virus purification and RNA isolation. CGMMV-SH is an isolate from
a muskmelon leaf that was supplied by Dr I. Furuki, Shizuoka
Agricultural Experiment Station. The virus sample for this study was
obtained after five passages through local lesions in the leaves of
Gomphrena globosa and propagated in muskmelon plants (Cucumis melo
L. cv. Earl's Favorite Natsukei-4).
Plants inoculated with the virus were grown in the greenhouse.
About 2 weeks after inoculation, the leaves were harvested and stored
at - 6 0 °C. Virus was purified according to the method reported by
Nozu et al. (1971). Viral RNA wag isolated from the purified virus
preparation using a phenol-SDS method (Gierer & Schramm, 1956;
Fraenkel-Conrat et al., 1957). The RNA was resuspended in H20 and
kept at - 7 0 °C until use.
Polyadenylation of virus RNA. Virus RNA was polyadenylated at the
3' end using poly(A) polymerase by the method of Meshi et al. (1982)
with a slight modification. Two-hundred lal of the reaction mixture
containing 50 mM-Tris-HCl pH 8.0, 0-2 M-NaCI, 10 m/~-MgCl~, 0-4
mM-EDTA, 1 mM-DTT, 30 lag RNA, 8.1 mM-[~-32p]ATP (Amersham,
50 Ci/mol) and poly(A) polymerase (Bethesda Research Laboratories,
20 units) was incubated at 37 °C for 13 rain. The resulting poly(A)tailed RNA was passed through a Sephadex G-50 column and collected
by ethanol precipitation.
Preparation of double-stranded eDNA W polyadenylated CGMMV
RNA, and construction and transformation of hybrid plasmids. Using the
cDNA synthesis system (Amersham) based on the method of Gubler &
Hoffman (1983), cDNA was synthesized by the manufacturer's
protocol. Larger sized cDNA was recovered by fractionation through a
Sephadex G-50 column. Addition of oligo(dC) to the double-stranded
cDNA at the 3' end was carried out using terminal deoxynucleotidyl
transferase (Takara Shuzo Company). The tailed cDNA (about 100 ng)
was annealed with 660 ng of the vector, oligo(dG)-tailed pUC9
(Pharmacia). The hybrid plasmid DNA was used to transform
competent cells of Escherichia coli JM101 prepared by the modified
RbCl/CaCl2-mediated method (Hanahan, 1985). White colonies on
the plates of selection medium were picked and used for colony
hybridization.
Preparation of probes and colony hybridization. Partially degraded
CGMMV-SH RNA or the 1"2fragment of TMV-OM RNA was used as
a probe after labelling at the 5' end with [y-3zp]ATP by the method
described by Meshi et al. (1982). The 12 fragment of TMV-OM RNA
was prepared as described by Mandeles (1968). Colony hybridization
was performed at 40 °C overnight using a probe of approximately 105
c.p.m, per filter (nitrocellulose filter BA85, Schleicher & Schiill) as
described by Ohshima (1981). For the SH RNA probe, the filter was
washed once with 4 x SSC, twice with 2 x SSC for 15 min each and
then treated with RNase A at 20 lag/ml for 30 min followed by washing
with 2 x SSC twice for 15 min. When the TMV-OM RNA f2 fragment
was used as a probe, the filter was washed only twice with 4 × SSC.
cDNA sequencing. Sequencing of eDNA was performed by the
dideoxynucleotide method (Sanger et al., 1977). The eDNA was cloned
into the pBluescript vector (Stratagene) and was used to make a series
of deletion clones as reported by Henikoff (1984). The deletion clones
were sequenced using an automated fluorescent DNA sequencer
(370A, Applied Biosystems) as described by Ugaki et al. (1988). The
DNA was sequenced in both directions.
RNA sequencing. The sequence of the coat protein-encoding and 3'
non-coding region of SH RNA was further confirmed by direct
sequencing of SH RNA using reverse transcriptase (Seikagaku Kogyo)
as described by Meshi et al. (1983a). The primer was a 5'-labelled
cDNA fragment which had been digested with the appropriate
restriction enzymes. The Y-terminal region of the genome RNA was
determined as follows: (i) SH-RNA was annealed to a synthetic primer
( G G C G T C A C G T T G G T T G T T G A T T , positions 81 to 102) which
had been labelled with [~,-32p]ATP and extended with reverse
transcriptase at the 5' end and (ii) the base at the 5' end was identified
by the methods described by Moss (1977) and Konarska (1984). The 3'
end of SH-RNA was determined as described by Miura et al. (1974)
after labelling of the RNA with T4 RNA ligase (England et al., 1980).
Sequence analysis. Sequence data were analysed using DNASIS
(Hitachi).
Results and Discussion
Cloning
For cDNA synthesis, virus R N A was extracted from
purified virus particles and subjected to agarose gel
electrophoresis. Two main R N A bands were detected
(data not shown). The main larger one corresponded to
full-length genomic RNA. The smaller R N A band was
presumed to be derived from short virus particles as
shown by Okada et al. (1980) and Fukuda et al. (1981).
Some of the cDNA clones are shown in Fig. 1. All of
these were selected using fragmented SH-RNA probes.
The largest cDNA clone, pSH-K-3, covers almost the
whole length of the genomic RNA, but lacks only 33
nucleotides at the 5' end, proved by sequencing using a
cDNA primer. This and two other clones (pSH-G-6 and
pSH-J-44) were found to hybridize with the probe of
TMV-OM R N A f2 fragments. Clones of pSH-G-6, pSHJ-44 and pSH-P-11 were thought to be derived from the
fragments which lost large 3' portions of SH-RNA prior
to the addition of poly(A).
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Genome sequence of C G M M V
I
I
I
III
t
I
I ]3'
pSH-A-6
pSH-G-6
pSH-J-44
pSH-K-3
pSH-O-9
pSH-P-II
Fig. 1. cDNA clones obtained from genome RNA of CGMMV-SH.
5"
I
30K
I
-4CP
t 30/lg0K
3"
CGMMV'SH "~
(1142)
[
0646) ~
6421nt
TMVvulgare ~
(1115)
j
(1615, ~
6395nt
TMVL q
(1.5,
J (16.S, ~
63~4n,
Fig. 2. Comparison of ORFs of three tobamoviruses deduced from the
nucleotide sequences. Figures in parentheses represent the number of
amino acid residues. Data are from Goelet et al. (1982) (TMV vulgate),
Ohno et aL (1984) (TMV-L) and Soils & Garcia-Arenal (1990)
(TMGMV).
Genome organization
Sequencing was performed using a series of deletion
clones from pSH-K-3 in both directions. The region not
covered by the clone was directly sequenced for SHRNA as described above. The sequences of SH-RNA
and the c D N A clone were identical for both the coat
protein gene and the 3' non-coding region. The genome
organization of CGMMV-SH was compared to three
other sequenced tobamoviruses, TMV vulgare (Goelet et
al., 1982), TMV-L (Ohno et al., 1984) and tobacco mild
green mosaic virus (TMGMV, TMV-U2) (Solis &
Garcia-Arenal, 1990) (Fig. 2), The genome of CGMMVSH is 6421 nucleotides long. At least four open reading
frames (ORFs) were found in the positive strand,
encoding putative proteins of 186K, 129K, 29K and
17-3K. The proteins encoded correspond to the 180K,
130K, 30K and 17.5K proteins ofTMV vulgare or TMVL. Each ORF of the CGMMV-SH genome is slightly
larger than the corresponding ORF of the other
tobamoviruses in the number of nucleotides except for
the ORF encoding the 30K protein of TMV vulgare and
TMV-L. Some ORF-like structures, at the largest 324
nucleotides long, were found in the negative strand (data
1489
not shown). It is apparent, however, that they are rather
short to encode proteins although we have not yet tested
this by in vitro or in vivo translation assays.
The complete nucleotide sequence of CGMMV-SH is
shown in Fig. 3. The first AUG initiation codon was
found at residues 61 to 63, which starts an ORF encoding
a protein composed of 1142 amino acids (129K). This
ORF terminates at an amber codon, UAG, positioned at
residues 3490 to 3492. The ORF encoding the readthrough protein composed of 1646 amino acids (186K)
terminates at residues 5002 to 5004. The terminal 14
bases of the ORF encoding the 186K protein overlap the
ORF encoding the 29K protein, which terminates with
an amber codon. The coat protein gene initiates at 25
bases upstream from the terminal nucleotide of the
amber codon for the 29K protein. The number ofnucleotides encoding the 29K protein was exactly the same as
that of another CGMMV isolate (CGMMV-W) (Meshi
et al., 1983b; Saito et al., 1988). The coat protein gene of
our CGMMV isolate was also found to be composed of
the same number of nucleotides as that found in
CGMMV-W (Meshi et al., 1983b). In total, 27 nucleotide
substitutions (six in the 186K protein, 14 in the 29K
protein and seven in the coat protein) were found
between CGMMV-SH and CGMMV-W, when the
sequenced region (1878 nucleotides from the 3' end) of
CGMMV-W was compared (Fig. 4). They include 25
transitions (15 between T and C, 10 between A and G)
and two transversions (one between T and A, one
between A and C). Twenty-five are in the third codon
position and only two are in the first. Three resulted in
amino acid substitutions which were all found in the 29K
protein. The nucleotide substitutions at 5375 (CCTTCT), 5675 (GGT-AGT) and 5758 (GAA-GAC)
correspond to P in CGMMV-SH for S in CGMMV-W
(at position 128), G for S (228) and E for D (255),
respectively. The number of T residues in a cluster (6401
to 6404) is one nucleotide less in CGMMV-SH than that
in the corresponding T cluster of CGMMV-W.
CGMMV-SH was isolated originally in Shizuoka
Prefecture which is more than 200 km distant from
Chiba Prefecture where CGMMV-W was isolated from
watermelon. The number of nucleotide substitutions
between these two strains was, however, very small,
suggesting that CGMMV-SH is a variant of the
CGMMV watermelon strain. It is likely that the three
amino acid substitutions found in the 29K protein are
responsible for adaptation of these strains to different
host species. The 30K protein of tobamoviruses is known
to be necessary for the movement of virus from cell to cell
(Deom et al., 1987; Meshi et al., 1987). However, it
remains to be determined whether CGMMV-SH moves
from cell to cell more efficiently in muskmelon plants
than does CGMMV-W.
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M. Ugaki and others
> I 2 9 K . 186K
~A
N I N E Q I N N
O R D A A A S G R N
GTTTTAATTTTTATAATTAAACAAACAACAACAACAACAACAAACAATTTTAAAACAACA ATGGCAAACATTAATGAACAAATCAACAACCAACGTGACGCCGCGGCTAGCGGGAGAAAC 1 2 0
N L V S Q L A S K
AATCTCGTTAGCCAATTGGCGTCAAAAAGG
R V Y D E A V R S
GTGTATGACGAGGCTGTTCGCTCGTTGGAT
L D H O D R R P K
CATCAAGACAGACGCCCGAAAATGAATTTT
M N F S R V V S T E H T
TCTCGTGTGGTCAGCACAGAGCACACCAGG
240
L V T D A Y P E F
CTTOTAACTGACGCGTATCCGGAGTTTTCG
S I S F T A T K N S V
H S L A G G L R L L E L E Y M M M Q V P
ATTAGCTTTACCGCCACCAAGAACTCTGTA
C~CTCCC~GCGGG%GG~C~GAGGC~C~
GAA~TGGAA~A~ATGATGATGCAGGTGCCC
360
Y O S P C Y D I G G N Y T Q H L F K G R S Y V H C C N P C L D L K D V A R N V M
TACGGCTCACCTTGTTATGACATCGGCGGT AACTATACGCAGCACTTGTTCAAAGGTAGA TCATATGTGCATTGCTGCAATCCGTGCCTA GATCTTAAGGATGTTOCGAGGAATGTGATG
480
Y N D M I T Q H V Q
R H K G S C G C H P
L F T F Q I D A F R R Y D S S P C A V T
TACAACGATATOATTACGCAACATGTACAG AGGCACAAGGGATCTTGCGGGTGCAGACCT CTTCCAACTTTCCAGATAGA~GCATTCAGG AGGTACGATAGTTCTCCCTGTGCGGTCACC
600
C S D V F Q E C S Y D F G S G R D N H A
V S L H S I Y D I P Y S S I G P A L H R
TGTTCAGACGTTTTCCAAGAGTGTTCCTAT GATTTTGGGAGTGGTAGGGATAATCATGCA GTCTCGTTGCATTCAATCTACGATATCCCT TATTCTTCGATCGGACCTGCTCTTCATAGG
720
K N V R V C Y A A F H F S E A L L L G S
P V G N L N S I G A Q F R V D G D D V H
AAGAATGTGCGAGTTTGTTATGCAGCCTTT CATTTCTCGGAGGCA~TGCTTTTAGGTTCG CCTGTAGGTAATTTAAATAGTATTGGCGCT CAGTTTAGGGTCGATGGTGATGATGTGCAT
840
F L F S E E S T L H Y T H S L E N I K L I V M R T Y F P A D D R F V Y I K E F M
TTTCTTTTTAGTGAAGAGTCTACTTTGCAT TATACTCATAGTTTAGAAAATATCAAGTTA ATCGTGATGCGTACTTACTTTCCTGCTGAT GATAGGTTTGTATATATTAAGGAGTTCATG
960
R
V K R V D T F F F R L V R A D T H M L H K S V G H Y S K S K
S E Y F A L N T P P
GTTAAGCGTGTGGATACTTTTTTCTTTAGG TTGGTCAGAGCAGATACACACATGCTTCAT AAATC~GTGGGGCACTATTUGAAATGGAAG ~C¢GAGTACTTCGCGCTGAATACCCCTCCO 1080
I F Q D K A T F S V W F P E A K K V L I P K F E L S R F L S G N V K I S R M L V
ATCTTCCAAGATAAAGCCACGTTTTCTGTG TGGTTTCCTGAAGCGAAGAAGGTGTTGATA CCCAAGTTTGAACTTTCGAGATTCCTTTCT GGGAATGTGAAAATCTCTAGGATGCTTGTC 1200
O A D F V H T I I N H I S T Y D N K A L V W K N V Q S F V E S I R S R V I V N
GATGCTGATTTCGTCCATACCATTATTAAT CACATTAGCACGTATGATAACAAGGCCTTA GTGTGGAAGAATGTTCAGTCCTTTGTGGAA TCCATACGTTCAAGAGTAATTGTAAACGGA 1320
V S V K S E W N V P V D Q L T D I S F S
I F P L V K V R K V Q I E L M S D K V V
GTTTCCGTGAAATCTGAGTGGAACGTACCG GTTGATCAGCTCACTGATATCTCGTTCTCG ATATTCCCTCTCGTGAAGGTTAGGAAGGTA O~OA~CGAGTTAATGTCTGATAAAGTTGTA 1440
I E A R G L L R R F A D S L K S A V E G
L C D C V Y D A L V Q T G W F D T S S D
ATCGAGGCGAGGGGTTTGCTTCGGAGGTTC GCAGACAGTCTTAAATCTGCCGTAGAAGGA CTAGGTGATTGCGTCTATGATGCTCTAGTT CAAACCGGCTGGTTTGACACCTCTAGCGAC
1560
E L K V L L P E P F M T F S D Y L E G M Y E A D A K I E H E S V S E L L A S G D
GAACTGAAAGTATTGCTACCTGAACCGTTT ATGACCTTTTCGGATTATCTTGAAGGGATG TACGAGCCAGATGCAAAGATCGAGAGAGAG AGTGTCTCTGAGTTGCTCGCTTCCGGTGAT
1680
D L F K K I D E I R N N Y S G V E F D V
E K F Q E F C K E L N V N P M L I G H V
GATTTGTTCAAGAAAATCGATGAGATAAGA AACAATTACAGTGGAGTCGAETTTGATGTA GAGAAATTCCAAGAA~TTGCAAGGAACTG AATGTTAATCCTATGCTAATTGGCCATGTC 1800
I E A I F S Q K A G V T V T G L G T L S P E M G A S V A L $ S T S V D T C E D M
ATCGAAGCTATTTTTTCGCAGAAGGCTGGG GTAACAGTAACGGGTCTGGGCACGCTCTCT CCTGAGATGGGCGCTTCTGTTGCGTTATCC AGTACCTCTGTAGATACATGTGAAGATATG 1920
D V T E D M E D I V L M A D K S H S Y M S p E M A R W A D V K Y G N N K G A L V
GATGTAACTGAAGATATGGAGGATATAGTG TTqATGGCGGACAAGAGTCATTCTTACATG TCCCCTGAAATGGCGAGATGGGCTGATGTT AAATATGGCAAOAATAAAGGGGCTCTAGTC 2040
E Y K V G T S M T L P A T W A E K V K A V L P L S G I C V R K P Q F S K P L D E
GAGTACAAAGTCGGAACCTCGATGACTTTA CCTGCCACCTGGGCAGAGAAAGTTAAGGCT GTCTTACCGTTGTCGGGGATCTGTGTGAGG AAACCCCAATTTTCGAAQCCGCTTGATGAG 2160
E D D L R L S N M N F F K V S D L K L K
K T I T P V V Y T G
T I R E R Q M K N Y
GAAGATGACTTGAGGTTATCAAACATGAAT TTCTTTAAGGTGAGCGATCTAAAGTTG~AG AAGACTATCACTCCAGTCGTTTACACTGGG AOCATTCGAGAGAGGCAAATGAAGAATTAT
2280
I D Y L S A S L G S T L G N L E R I V R S D W N G T E E S M Q T F G L Y D C E K
ATTGATTACTTATCGGCCTCTCTTGGTTCC ACGCTGGGTAATCTGGAGAGAATCGTGCGG AGTGATTGGAATGGTACTGAGGAGAGTATG CAAACGTTCGGGTTGTATGACTGCGAAAAG 2400
C K W L L L P A E K K H A W A V V L A S
D D T T R I I F L S
TGCAAGTGGTTATTGTTGCCAGCCGAGAAG AAGCACGCATGGGCCGTGGTTCTGGCAAGT GACGATACCACTCGCATAATCTTCCTTTCA
Y D E S G S P I I D
TATGACGAA~CTGGTTCTCCTATAA~TGAT 2520
K K N W K R F A V C S E T K V Y S V I R S L E V L N K E A I V D P G V H I T L V
AAGAAAAACTGGAAGCGATTTGCTGTCTGT TCCGAGACCAAAGTCTATAGTGTAATTCGT AGCTTAGAGGTTCTAAATAAGGAAGCAATA GTCGACCCCGGGGTTCACATAACATTAGTT 2 6 4 0
D G V P G C G K T A E I I A R V N W K T D L V L T P G R E A A A M I R R R A C A
GACGGAGTGCCGGGTTGTGGAAAGACCGCC GAGKTTATAGCGAGGGTCAATTGGAAAACT GATCTAGTATTGACTCCCGGAAGGGAGGCA GCTGCTATGATTAGGCGGAGAGCCTGCGCC
2760
L H K S P V A T N D N V R T F D S F V M N R K I F K F D A V Y V D E G L M V H T
CTOCACAAGTCACCTGTGGCAACCAATGAC AACGTCAGAACTTTCGATTCTTTTGTGATG AATAGGAAAATCTTCAAGTTTGACGCTGTG TATGTTGACGAGGGTCTGATGGTCCATACG 2880
G L L N F A L K I S G C K K A F V F G D A K Q I P F I N R V M N F D Y P K E L R
GGATTACTT&~TTTTGCGTT~K~TCTCA GGT~GTA~AAGCCTTCGTCTT~GGTGAT GCTAAGCAAATCCCGTTTATAAACAOAGTC ATGAATTTCGATTATCCTAAGGAGTTAAGA 3000
T L I V D N V E R R Y V T H R C P R D V
T S F L N T I Y K A A V A T T S P V V H
ACTTTAATAGTCGATAATGTAGAGCGTAGG TATGTCACCCATAGGTGTCCTAGAGATGTC ACTAGTTTTCTTAATACTATCTATAAAGCC GCTGTCGCTACTACTAGTCCGOTTGTACAT
3120
S V K A I K V S G A G I L R P E L T K I K G K I I T F T O S D K Q S L I K S G Y
TC~AAGGCARTTAA~TCAG~GCC
GGTATTCTGAGGCCTGAGTTDACAAAGATC AAAGGAAAGATAATAACGTTTACTCAATCT GATAAGCAGTCCTTGATCAAGAGTGGGTAC
3240
N D V N T V H E I Q G E T F E E T A V V
R A T P T P I G L I
A R D S P H V L V A
A A ~ A ~ A A T A C T G ~ C A ~ A A A T T C A G GGAGAAACCTT~AGGAGACG~CAGT~TG CGTGCCACCCCGACTCCAATAGGTTTGATT GCCCGTGATTCACCACATGTACTAGTGGCC
3360
L T R H T K A M V Y Y T V V F D A V T S
TT~CTAGGCACACTA~GCA&~GTGTAT TATAC~TTGTATTCGATGCAGT~ACAAGT
3480
I I A D V E K V D O
S I L T M F A T T V
ATAATAGCGGATGTGGAAAAGGTCGATCAG TCGATCTTGACCATGTTTGCTACCACTGTG
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G
Genome sequence o f C G M M V
129K <
P T K * ] Q L M O N S L Y V H R N I F L P V S K T G F Y T D M Q E
CCTACCAAATAGCAATTAATGC~AATTCG CTGTATGTCCATCGTAATATTTTCCTCCCT GTTAGTAAAACGGGGTTTTATACAGACATG
>186K
N S F V L N D F D A V T M R L R D N E F N L ' Q P C R L T L S N L
AATTCCTTCGTACTAAA~ATTTCGATGCC GTAACCATGCGGTTGAGGGACAACGAATTT AACTTACAACCTTCTAGGCTAACCTTGAGT
1491
F Y D R C L P G
C~G~TTCTACGATAGATGCCTTCCTGGG 3600
D P V P A L I K
AATTTAGATC~GTACCCGCTTTGATTAAG
3720
N E A Q N F L I P V L R T A C E R ' P R I P G L L E N L V A M I K R N M N T P D L
AATGAAGCGCAGAATTTTCTGATCCCCGTT TTGCGTACGGCCTGTGAA~GCCGCGCATT CCGGGTCTTCTTGAGAATCTTGTAGCTATG ATAAAGAGGAATATGAATACTCCTGATTTA 3840
A G T V D I T N M $
I S I V D N F F S S
F V R D E V L L D H
L D C V R A S S I Q
GCTGGGACCGTAGATATA~TAACA~TCG ATTTCTATAGTAGATAACTTCTTTTCTTCT TTTGTTAGGGACGAGGTTTTGCTTGATCAC TTAGATTG~TTAGGGCTAGTTCCATTCAA
3960
S F S D W F $ C Q P T S A V G Q L A N F N F I D L P A F D T Y M H M I K R Q P K
AGTTTTTCTGATTGGTTTTCGTGTCAACCA ACCTCAGCGGTTGGCCAGTTAGCTAATTTC AATTTCATAGATTTGCCTGCCTTTGATACT TATATGCATATGATTAAGAGGCAACCCA~
4080
S R L D T S I Q S E Y P A L Q T I V Y H
P K V V N A V F G P V F K Y L T T K F L
AGTCGGTT~ATACTTCGATTCAGTCTGAA TATCCGGCCTTGCAAACTATTGTTTATCAC CCTAAAGTGGTAAATGCAGTTTTTGGTCCG GTTTTCAAGTATTTAACCACCAAGTTTCTT 4200
$ M V D S S K F F F Y T R K K P E D L Q
E F F S D L S S H S
D Y E I L E L D V S
AGTATGGTAGATAGTTCTAAGTTTTTCTTT TACACTAGGAAAAAACCAGAAGATCTGCAG GAATTTTTCTCAGATCTCTCTTCCCATTCT GATTATGAGATTCTTGAGCTTGATGTTTCT 4320
K Y D K S O S D F H F S I E M A I W E K
L G L D D I L A W M W S M G H K R T I L
AAATATCACAAGTCGCAATCCGATTTCCAC TTCTCTATTGAGATGGCAATTTGGGAAAAA TTAGGGCTTGACGATATTTTGGCTTGGATG TGGTCTATGGGTCACAAAAGAACTATACTG 4440
Q D F Q A G I K T L I Y Y Q R K S G D V
T T F I G N T F I I
A A C V A S M L P L
CAAGATTTCCAAGCCGCGATAAAGACGCTC ATTTACTATCAACGGAAGTCTGGTGATGTA ACTACTTTTATAGGTAATACCTTTATTATC GCAGCGTGTGTGGCTAGTATGTTGCCGTTA 4560
D K C F K A S F C G
D D S L I Y L P K G
L E Y P D I Q A T A N L V W N F E A K L
GATAAGTGTTTTAAAGCTAGTTTTTGTGGT GATGATTCGCTGATCTACCTTCCTAAGGGT TTGGAGTATCCTGATATACAGGCTACTGCC AACCTTGTTTGGAATTTTGAGGCGAAACTT
C
A
F R K K Y G Y F C G K Y I I H H A N G C I V Y P D P L K L I S K L G N K 6 L V G
TTCCGAAAGAAGTATGGTTACTTCTGCGGG AAGTATATAATTCACCATGCCAACGGCTGT ATTGTTTACCCTGACCCTTTAAAATTAATT AGTAAATTAGGTAATAAGACTCTTGTAGGG
T
Y E H V E E F 6 1 S L L D V A H S L F N G A Y F H L L D D A I H E L F P N A G G
TATGAGCATGTTGAGCAGTTTCGTATATCT CTCCTCGACGTTGCTCATAGTTTGTTTAAT GGTGCTTATTTCCATTTACTCGACCATGCA ATCCACGAATTATTTCCTAATGCTGGGGGT
C
C
186K (
C S F V I N C L C K Y L S D K R L F R S L Y I D V S K ' ]
TGCAGTTTTGTAATTAATTGTTTGTCTAAG TATTTGAGTGATAAGCGCCTTTTCCGTAGT CTTTACATAGATGTCTCTAAGTAAGGTGTC AGTCGAGAACTCGTTGAAACCTGAGAAGTT
T
T
S L S K vAsGv
E N SAL K P E K F
I
>29K
TGTCAAAATCTCTTGGGTCCATAAGTTGCT CCCTAACTATTTTTCCATTCTCAAGTATTT ATCTATAACTGACTTTAGTGTAGTTAAAGC TCAGAGCTATGAATCCCTCGTGCCTGTCAA
V K I S W V D K L L P N y F S I L K Y L S I T D F S V V K A Q S Y E S L V P V K
4680
4800
4920
5040
5160
GTTGTTGCGTGGTGTTGATCTTACAAAACA CCTTTATGTCACATTGTTGGGCGTTGTCGT TTCTGGTGTATGGAACGTACCGGAATCCTG TAGGGGTGGTGCTACTGTTGCTCTGGTTGA 5280
L L R G V D L T K H
L Y V T L L G V V V
5 G V W N V P E S C
R G G A T V A L V D
S
CACAAGGATGCATTCTGT~CAGAGGGAAC TATATGCAAATTTTCAGCTCCCCCCACCCT CCGCGAATTCTCTCTTAGGTTCATACCTAA TTATCCTGTGGTGGCTGCGGATCCCCTTCG 5aO0
T R M H S V A E G T I C K F S A P A T V
R E F S V R F I P
N Y T P V V A A D A L R
CGATCCTTCGTCTTTATTTGTGAGACTCTC TAATGTGGGTATTAAAGATGGTTTCCATCC TTTGACTTTAGAGGTCGCTTGTTTAGTCGC TACAACTAAOTCTATTATCAAAAAGGGTCT 5520
D P W S L F V R L S N V A G I K D G F H P L T C L E V A C L V A T T N S I I K K G L
TAGAGCTTCTGTAGTCGAGTCTGTCGTCTC TTCCGATCAGTCTAT~TCCTAGATTCCTT ATCCGAGAAAGTTGAACCTT2CTTTGACAA AGTTCCTATTTCAGCGGCTGTAATGGCAAG
R A S V V E S V V S S D Q S C I V L D S T L
$ E K V E P F F D T K
V P I S G A A V M A R
S
D
AGATCCCAGTTATAGGTCTAGGTCGCAGTC TGTCGGTGGTCGTGGTAAGCGGCATTCTAA ACCTCCAAATCGGAGGTTGGACTCTGCTTC TGAAGAGTCCAGTTCTGTTTCTTTTGAAGA
C o a t ~ S Y p R S R S Q S V A G G R G K R H S K P P N R R L D S A S E E S S S V S F C E C D
N
I T P S K L I A F S A S Y V P V R T L L N F L V A S Q G T A F Q T Q A
~GCTTACAATCCGATCACACCTAGCAAAC TTATTGCGTTTAGTGCTTCTTATGTTCCCG TCAGGACTTTACTTAATTTTCTAGTTGCTT CACAAGGTACCGCTTTCCAGACTCAAGCGG
G L o s D ~ T ,129K
c
G R D S F R E S L S A L P S S V V D I N S R F P D A G F Y A F L N G P V L R P I
GAAGAGATTCTTTCCGCGAGTCCCTGTCTG CCTTACCCTCGTCTGTCGTAGATATTAATT CTAGATTCCCAGATGCCGGTTTTTACGCTT TCCTCAACCGTCCTGTGTTGAGGCCTATCT
G
F V S L L S S T D T R N R V I E V V D P S N P T T A E S L N A V K R T D D A S T
TCGTTTCGCTTCTCAGCTCCACGGATACGC GTAATAGGGTCATTGAGGTTGTAGATCCTA GCAATCCTACGACTGCTGAGTCGCTTAACG CTGTAAAGCGTACTGATGACGCGTCTACAG
A
G
A A R A E I D N L I E S I S K G F D V Y
D R A S F E A A F S
V V W S E A T T S K
CCGCTAGGGCCGAGATACATAATTTAATAG AGTCTATTTCTAAGGGTTTTGATGTTTACG ATAGGGCTTCATTTGAACCCGCGTTTTCGG TAGTCTGGTCAGAGGCTACCACCTCGAAAG
<
TA
T
A .]Coat
CTTAGTTTCGAGGGTCT~TGATGGTGGTG CACACCAAAGTGCATAGTCCTTTCCCGTTC ACTTAAATCGAACGGTTTGCTCATTGGTTT GCGGAAACCTCTCACGTGTGACGTTGAAGT
5640
5760
5880
6000
6120
62a0
6360
TTCTATGGCCAGTAATTCTGCAAGGGGTTC CAATCCCCCCTTTTCCCCGGGTAGGGGCCC A 6421
Fig. 3. The complete nucleotide sequence of the CGMMV-SH genome RNA and the deduced amino acid sequences of ORFs. The
numbering refers to the nucleotide sequence. The amino acid sequences are given in the one-letter code. The nucleotide sequence within
1878 nucleotides from the 3' end determined by Meshi et al. (1983 b) and saito et al. (1988), and the deduced amino acid sequences of the
CGMMV-W genome RNA are shown only in the positions where they are different from those of CGMMV-SH. The wavy line marks a
T cluster where the number of Ts is three in CGMMV-W.
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1492
M. Ugaki and others
CGMMV-SII
T M V vulgare
TMV-L
PMMV-S
TMV-U2
U
....
A
- - -CAAC-
C
-
- -UUUUAAA . . . .
I~1o. . . . . . .
-~~ -ff~,~, o,,~~]
- '..]'A'~I'~'~ ~; ~l ~ a n ~ ' A a cA]- tt ¢~AAC;A'~AAA CAAC - .n~ACA~CJA~ U~ CAb- - - A ACAAAA U~ACA~ACU~CAA O . ~
~ U G U U U U G ~ C G A C A ~ -' - ' A ~ A ~
........
--~
U~IJ~
- - AACAA CA C/~_C~ACA~CAAIJ G G~A~g't C al
Fig. 4. Alignment of the nucleotide sequence of the 5' non-coding regions of tobamoviruses. Common nucleotides among them are
boxed. The initiation codon of translation is underlined. A base indicated by the symbol * is not determined. The wavy line shows a
ribosome-binding site (Tyc et al., 1984). Gaps indicated by slots have been introduced to obtain maximal alignment. The sequences are
from Richards et al. (1978) (TMV vulgate), Ohno et al. (1984) (TMV-L), Avila-Rincon et al. (1989) (PMMV-S) and Solis & GarciaArenal (1990) (TMGMV).
180K
The identity of the entire nucleotide sequences is
approximately 55 to 56~ between CGMMV-SH and
TMV vulgare, between CGMMV-SH and TMV-L or
between CGMMV-SH and TMGMV.
TMV
161
I
I
I
5' and 3" Non-coding regions
The 5' non-coding region of the ordinary TMV (such as
vulgate) is referred to as f2 and is characteristically free of
G residues (Mandeles, 1968), The 5' non-coding regions
of several tobamoviruses are compared in Fig. 4. The
deletion of the Y-terminal eight nucleotides
(GUAUUUUU) of TMV-L caused the complete loss of
infectivity and the other sequence of the region seemed
not to be crucial although a large deletion caused
considerable effects (Takamatsu et al., 1991). The role of
the sequence of eight nucleotides is however not known.
This region is well conserved among five tobamoviruses.
The ribosome-binding site is thought to be AUU (Tyc et
al., 1984), being present in all the five viruses. The three
nucleotides (ACA) upstream and the three (GCA)
downstream to the initiation codon are also well
conserved. The 5' non-coding region of CGMMV-SH
has 60 nucleotides and is 67 to 6 8 ~ similar to that of
TMV vulgare, TMV-L and a Spanish isolate of pepper
mild mottle virus (PMMV-S; Avila-Rincon et al., 1989)
or TMGMV. The first nucleotide of the CGMMV-SH
genome was found to be G, as are those of the other three
(TMV vulgate, TMV-L and TMGMV).
Although the nature of the cap was not determined, we
assume it to be mTGppp. The 3' non-coding region of
CGMMV-SH genome is composed of 176 nucleotides.
The T-terminal sequence of CGMMV-SH fits the
pseudoknot structure model which was proposed for
tobamovirus genomes by Van Belkum et al. (1985) (data
not shown).
Open reading frames
No CGMMV-encoded proteins except for the coat
protein have yet been identified in vivo (Okada, 1986).
However by analogy with other tobamoviruses, at least
four ORFs can be identified on the genome sequence.
>
.\
\
\
164~
I
I
1
I
30K
TMV
I
\
267
I
-,
>
O
263
m
Fig. 5. Homology plot comparison of 180K and 30K proteins between
CGMMV-SH and TMV vulgare. Amino acid sequences were analysed
using a DNASIS program based on the theory of Needleman &
Wunsch (1970). Each dot marks where at least six of 10 amino acids are
identical between the two viruses.
The putative 129K and 186K proteins correspond to the
130K and 180K proteins of TMV vulgare and TM¥-L.
The former protein of CGMMV-SH has 1142 amino
acids which is 27 amino acids more than those of TMV
vulgare and TMV-L. The 186K readthrough product of
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Genome sequence of C G M M V
1493
(a)
I
860 t ' J , I ~ ' J ~ t ~ ; ~ I / X ~ ; ' ~ ' ~ / K T I . ~ ' I ' ~ ' ~ , ~ E ~ . ~ C A L H K ~ P - - ~ N ~ R ~
917
831 ~J'I~'t;R'I~',~KF~LS~;~FDEI~'~Ii~V~KQ~E~NS---~GII~K~K~%/~'~885
II
III
918 ~V~'~R-~I----~--4"~DAVYV;~'~'@~V~LL~KI-~C~KK~F~F~AK~ 969
886 ~ F O . ~ C ~ L ~ - - - - ~ , ~ L , . ~ a ~ . C V ~ - ~ V ~ L ~ E ~ y ~ y ~ T Q ~ V ~ r ~
940
IV
970 ~ N ~ D ~ - - - - ~ . ~ E , ~ T L ~ N ~ - ~ V V ~ , ~ , , ~ S m ~ Z ~ A ~ X ~ U ~
1024
941 ~ S C ~ P ~ A H F ~ - ~ E - - - t ~ [ ~ a ~ ' ~ T - ~ L ~ : ' ~ I ~ A I . ~ Y ~ ] R R ~ E G F ~ M S ~ ' ~ - - - - ~ - 990
V
1025 L~-~----~-~G-~]LR~ELTK~-~.~---~I~"~S~IK~-~Ni%~N~T~'~I~ 1072
991 - 4 ~ S ~ T ~ Q E I ~ C ~ A V ~ N - ~ - - - - ~ S ~ P L H ~ L ~ ' ~ E / ~ L - ~ R ~ S ~ % / ~ ] ~ , ' g r ~
1043
VI
1073 ~FEETA~--~A~--~GL~Ri,);I~;t,~,I;~;'~---4.~AMV~1110
(b)
I
1415
r~~r~SD~FSIL~MA~EKII~.LD~I~AWM~SM~R~I~Q~FQ~y~
1383 m ' ~ m ~ m i m t ' ~ N E ~ C m ~ E ~ R , ~ F E , ~ 2 D E V ~ Q ~ , 2 ~ : ~ , ~ Y ~ r ~ ' ~ C ~
II
1474
1442
1473
1441
III
~e};I:~'Ieor/~U~'(~b~F~"]~I)~C~.~-~S~I~L~r.~L~Yr~I~AT~
~,)8'~.'i~,r~i,~(~kf~1~L~"%"~I~II~?~-9~~F~C~S~
IV
1533 V ~ R ~ . ' ~ L 4 ~ I ~ A N ~ I ' ~
1501 ~ - ~ , v ~ O ~ R i ~ [ ~ R ~ r ~
1532
1500
1560
1527
Fig. 6. Comparison of partial amino acid sequences of the 186K protein of CGMMV-SH with those of the 180K protein of TMV
vulgare (Goelet et al., 1982). These sequences contain motifs indicated by Habili & Symons (1989). (a) The region contains nucleic acid
helicase motifs; (b) the region contains RNA polymerase motifs. Upper and lower lines are the amino acid sequences of CGMMV-SH
and TMV vulgate, respectively. Identical amino acids are written in white letters. Motifs are overlined. Roman letters represent
numbers of motifs used by Habili & Symons (1989). Gaps have been introduced to obtain the closest match.
CGMMV-SH is longer by 31 amino acids than those of
TMV vulgare and TMV-L. A homology plot of the amino
acids of the 180K (/130K) protein is shown in Fig. 5.
Amino acid sequence identity of the CGMMV-SH 186K
protein is approximately 48% with the corresponding
180K protein of TMV vulgare.
The N-terminal one-third, the C-terminal one-third
and the remainder of the CGMMV-SH 129K protein
have identities of 48%, 47% and 33% with those of TMV
vulgate respectively. These values are the same for TMVL except for that of the C-terminal one-third of the 130K
protein (49% identity). The level of similarity of the
amino acid sequence of the middle one-third portion of
the CGMMV-SH 129K protein is not as high as those of
the other portions. The readthrough part of CGMMVSH 186K has 58% identity with the corresponding
regions of TMV vulgare and TMV-L, which is higher
than those of any other regions of the protein.
Rozanov et al. (1990) indicated that the N-terminal
portions of large putative NTPases of'Sindbis-like' plant
viruses including tobamoviruses might be methyltransferases. It is of interest that the 130K protein of TMV-U1
(vulgate) was found to have guanylytransferase-
like activity (Dunigan & Zaitlin, 1990). Although the
capping mechanism of plant virus R N A is not known, it
might be possible that motifs for capping-related enzyme
activities are located in the N-terminal one-third portion
of the tobamovirus 130K protein.
Habili & Symons (1989) showed that positive-strand
viruses could be grouped based on amino acid sequence
motifs of nucleic acid helicases and R N A polymerases,
and proposed a new luteovirus supergroup. The amino
acid sequences were compared in the presumed motifs in
Fig. 6. These motifs in the 186K protein of CGMMV-SH
are characteristic for tobamoviruses. The amino acid
sequence identities in the helicase motifs region (a) were
56% and 53% with TMV-L and TMV vulgare and those
in the polymerase motifs region (b) were 70% and 67%,
respectively.
A homology plot of amino acids of the 30K protein
between CGMMV-SH and TMV vulgate is also shown in
Fig. 5. Saito et al. (1988) showed local high homology in
the 30K protein among tobamoviruses and a tobravirus.
The 30K protein is thought to have at least two functions,
binding to single-stranded nucleic acid (Citovsky et al.,
1990) and interacting with plasmodesmata of host cells
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1494
M . Ugaki ~and others
(Tomenius et al., 1987). A closely similar region
exhibited in Fig. 5 may be important in the binding of
the 30K protein to single-stranded nucleic acid, which
was indicated by Citovsky et al. (1990).
The authors are very grateful to Drs I. Furuki and T. Ohsawa for
providing them with the sample of CGMMV-SH and the muskmelon
seeds.
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