Journal of General Virology (1992), 73, 2483-2486. Printed in Great Britain
2483
A comparative study of the RNA-2 nucleotide sequences of two sweet
clover necrotic mosaic virus strains
Zhongming Ge, ~ Chuji Hiruki 1. and Kenneth L. Roy 2
Departments o f 1Plant Science and 2Microbiology, University o f Alberta, Edmonton, Alberta, Canada T6G 2P5
The nucleotide sequences of the RNA-2s of two strains
of sweet clover necrotic mosaic virus (SCNMV-38 and
-59) have been determined. The RNA-2s of SCNMV38 and -59 consist of 1446 and 1449 nucleotides,
respectively, and both contain one major open reading
frame (ORF) which potentially can encode polypeptides of 326 amino acid residues (about 36-5K),
designated SC38P2 and SC59P2, respectively. The
nucleotide sequences of SCNMV-38 and -59 RNA-2s
show 93-2% similarity, and the amino acid sequences
of SC38P2 and SC59P2 are 91-7 % identical, although
the identical nucleotides and amino acids are not
distributed uniformly in RNA-2 and the encoded
proteins. Two highly conserved regions (from positions
23 to 221 and 297 to 326) and a relatively divergent
region (from positions 222 to 296) are found in the P2
proteins of these strains. A similar pattern is apparent
on comparison of the nucleotide and deduced amino
acid sequences of RNA-2 of these S C N M V strains with
those of the Australian and Czechoslovakian isolates of
red clover necrotic mosaic virus.
Sweet clover necrotic mosaic virus (SCNMV), a member
of the dianthovirus group (Francki et al., 1991), is an
isometric particle with a diameter of 35 nm. A
comparative study has been made on two strains of
SCNMV, namely SCNMV-38 and -59, on the basis of
physicochemical properties, serology, host range and
symptomatology (Pappu et al., 1988). The viral genome
consists of two positive-sense R N A segments, RNA-1
and RNA-2, with Mrs of 1-35 x 106 and 0-55 x 10 6,
respectively (Hiruki et al., 1984). Both RNAs are
required for whole plant infection (Okuno et al., 1983).
RNA-I can replicate without RNA-2, but RNA-2 is not
capable of replicating in cowpea protoplasts in the
absence of RNA-1 (Pappu & Hiruki, 1988). Genetic
reassortant experiments have shown that there is
compatibility of RNA-1 and RNA-2, not only between
strains of the same virus, but also between certain
members of the dianthovirus group, with an exception
(Rao & Hiruki, 1987). It has been demonstrated that
physicochemical and serological features of progeny
virus are determined by the donor RNA-1 (Pappu &
Hiruki, 1989; Okuno et al., 1983). Similar work on red
clover necrotic mosaic virus (RCNMV) (Osman & Buck,
1991; Osman et al., 1986, 1991a; Paje-Manalo &
Lommel, 1989), another member of the dianthovirus
group, has demonstrated that RNA-2 is responsible for
cell-to-cell virus movement and that it plays a role in the
development of disease symptoms in infected plants. To
gain further understanding of the function of SCNMV
RNA-2, we have determined the primary structures of
RNA-2s of SCNMV-38 and -59.
Both strains were maintained and propagated in
Phaseolus vulgaris L. 'Red Kidney'. Virus purification
and R N A extraction were carried out as described by
Hiruki et al. (1984). Unfractionated viral RNAs were
polyadenylated in vitro (Ahlquist et al., 1981) and then
purified using an mRNA purification kit (Pharmacia
LKB) according to the supplier's instructions. Synthesis
of cDNA was performed using a cDNA synthesis kit
from Pharmacia LKB following the manufacturer's
manual. The double-stranded cDNA, tailed with E c o R I /
N o d adapters, was ligated with EcoRI-digested dephosphorylated pBluescript S K + / - . The RNA-2-specific
clones were characterized by Northern and Southern blot
hybridization (Maniatis et al., 1982) using 32p-labelled
viral RNA and nick-translated D N A probes (BRL). The
R N A probe was made using a 5' end-labelling method.
The gel-purified RNA-2 was partially hydrolysed in 50 ~tl
of 50 mM-carbonate/bicarbonate buffer pH 9-0 at 90 °C
for 15 min, followed by neutralization with 0.3 volumes
of Tris-HCl pH 7.5. The hydrolysed R N A fragments
(0.5 to 1 mg) were then labelled in 10 ~tl of a reaction
mixture containing 1 x kinase buffer (50 mM-Tris-HCl
pH 9-0, 10 mM-MgC12, 1 mM-spermidine and 5 mMDTT), 10 mCi of [y-32p]ATP (>4000 Ci/mmol; NEN/
Dupont) and 2 units of polynucleotide kinase (Pharma-
0001-0988 © 1992SGM
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2484
I
Short communication
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CTGTTCAAGTG GAAAACG
TAAATGATTTGGCTAAGACAAATG~ CGGAGTAGCGGTGCTATTGAACCGTTATACTGATTGGAAATGCAGGTCCGGAGTGACGGAAGCTCCTCTCATACC
II I II
II
III
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-i00
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TACAGGATTTGGCCAAGACAAATAAAGGAGAAGAGGTCCTTAAGAACCGTTATACTGATTGGAAATGCAGGTCTGGAGTGACTGAAGCTCCTCTCATACC
TGCCAGTATAATGAGTAAGATAACCGACTATGCCAGGACAACTGCCAAAG GTAATAGTGTCGCGCTTAATTACACCCACGTAATCCTTTCATTAGCACCT
I111111
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TGCCAGTATAATGAGTAAAATAACcGACTATGCCAAGACAACTGCCAAAGGC-AACAGTGTTGCGCTTAATTATACACACGT~TC~FFL~CA~A~ACCA
ACAATTGGTGTGG CAAT CCCAGGACACGTTAC CGTAGAACTGGTAAACCC AAACGTGGATGGACt-FFFrCAAGTTATG ~ A~CAGACCTTGTCATGGT
II11111
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ACAATTGGTGTGGCAATTCCAGGACACGTTACCGTAGAACTGGTAAACCCAAACGTGGATGGACCt-F1-rCAAGTCATGAGCGGACAGACCTTGTCATGGT
-400
CGCCAGGAGCAGG AAAAC CCTG CCTCATGATCTTCTCAGTCCACCACCAA CTCAACTCGGATCACGAGCCTTTTAGGGTG CGTATCACTAATTCCGGAAT
--499
II11 11 II 11
III
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lltllllll 111
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ACCTACCAAGAAGTCGTATGCAAGATGCCATGCGTA~TACGACG
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ACCTACCAAAAAATCGTATGCAAGATGTCATGCGTA~TACGACG
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T T G G A A G T A G G T T A C C A A CC4~AC G T T A C T G T C G A G C A T C A A A G C A G T G G A A C 4 C T T A C G T G C . A A T T T A C C T T T G A C A C A A G C A G G A T G G A G A G C A A T C C T C --699
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A A T T G T G T A C C A A A T C T A A G G T G A A C A T C G T C C CTCCCAAAGC.AGAGACG " F F I ' C C A A T C A G A ~ A T C G C C C C G C C T C T C A G T G T T G G G A A G A A T C A A G
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CACCCAATCAGAGGTATCG CCCCGCCTCTCAGTGTTGCd~AAAAG CCAAAG -800
AATTGTGCACCAAATCCAAAGTGAACATCGTCCCTCCTAAGGTAGAGACA
ACAACAGTCCGAGGTGTCGAAGCAGCAAGGAGGTAATGGCAACGAATCCATTAAG~F~M~TCATCGT~ACCCACTCTCAGGCCCGGTTCCGt-FF~.GAGT
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III I
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GGAGGGGTTGGACTCTCCTGGTGTGAGAAGAAAACACATCAAAACAAAAAACAGGGTTCTGTAACCC4~TGCGACAATATGCCCTTCCAAGGCACTGACCG
II II I~
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GCACGGATAAAAt-FF~'ATTTGCCTAGTCGTATAACGGCTAGGCACCCC
- 1446
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Fig. h Alignment of SCNMV-38 (upper line) and -59 RNA-2 (lower line) sequences. Start codons and stop codons are underlined.
cia LKB) at 37 °C for 30 min. The R N A probes were
purified by passage through a 3 ml Sephadex G-50
column.
The nucleotide sequences of the c D N A clones of
RNA-2 (pBSC3802 and pBSC3803 for SCNMV-38;
pBSC5906, -08 and -13 for SCNMV-59) were determined
by the dideoxynucleotide chain termination method
(Sanger et al., 1980) with Sequenase (United States
Biochemical) and site-specific primers complementary
to known parts of the templates. The ssDNA templates
were recovered with the aid of VCS-M13 interferenceresistant helper phage according to the supplier's
instructions (Stratagene). The primers were synthesized
using an ABI 391 D N A synthesizer. The compression of
some nucleotide sequences was overcome by replacing
dGTP with dlTP in the sequencing reaction, pBSC3802
and pBSC3803 were 1418 (nucleotides 29 to 1446) and
1375 (nucleotides 64 to 1438) nucleotides long respectively. The sequences of pBSC5906, -08 and -13 covered
nucleotides 529 to 1449, 205 to 1449 and 126 to 1439,
respectively. Nucleotides 1 to 624 ofSCNMV-38 RNA-2
and 1 to 296 of SCNMV-59 RNA-2 were analysed by the
primer extension method using viral RNA-2 templates
and specific primers as described by Chang et al. (1989).
Since the cDNA inserts carrying the sequences CCCC
(SCNMV-38) or CCCCC (SCNMV-59) at their 3'
termini had an artificial poly(A) tail at their 3' termini
linked directly to the 3' C residue, it was deduced that
RNA-2 segments of both SCNMV strains terminated at
the sequence CCCC or CCCCC. Sequence data were
analysed by a PC/GENE software package which
predicted ORFs, and aligned and predicted the secondary structures of the nucleic acid and amino acid
sequences.
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Short communication
The complete nucleotide sequences of SCNMV-38
RNA-2 and SCNMV-59 RNA-2 were 1446 and 1449
nucleotides in length, respectively (Fig. 1). They contained a 5' non-coding leader sequence (nearly 80
nucleotides), a major open reading frame (ORF) and a 3'
non-coding trailing sequence (about 390 nucleotides).
The structural features of the 5' non-coding and 3' noncoding sequences, as well as the gene organization of
SCNMV RNA-2, were similar to those found for
RCNMV RNA-2 (Lommel et al., 1988; Osman et al.,
1991b). An alignment analysis revealed that the
sequence similarity between the RNA-2s was about
93-2~; however, the distribution of identical nucleotide
sequences was not uniform (Fig. 1). For example, the
similarity between the regions from position 790 to 850
was only 70~, whereas that between the regions from
nucleotide 1293 to 1448 was as high as 98"3~o.
The major ORF identified for both strains was capable
of directing the synthesis of a protein of 326 amino acids
(36-5K), designated SC38P2 and SC59P2 (Fig. 2). The
size of this proposed protein is in agreement with that of
the in vitro translation product of native RNA-2 of both
strains (data not shown). The amino acid sequences of
SC38P2 and SC592P2 shared 93 ~ amino acid sequence
identity, but this was not distributed uniformly along the
proteins. Two conserved regions (from positions 23 to
221 and 297 to 326 at the N terminus, designated Cons 1
and Cons 2, respectively) and a variable region (from
positions 222 to 296, designated V) were found. The
identity of amino acid residues in Cons I was nearly 99 ~o
and in Cons 2 100~, but in V was only 77-3~.
The RNA-2s of two RCNMV isolates, Aus (Australian) and TpM-34, both 1448 nucleotides in length
(Lommel et al., 1988; Osman et al., 1991 b), showed great
similarity with those of the two SCNMV strains, 84.2~o
and 94.8~ with SCNMV-38 RNA-2, and 82"7~o and
94-3~ with SCNMV-59 RNA-2. An alignment of the
four RNA-2-encoded proteins from these two SCNMV
strains and two RCNMV isolates is shown in Fig. 2. The
P2 of SCNMV consists of the same number of amino
acids as that of RCNMV-TpM-34, but is nine amino
acids longer than the predicted P2 of RCNMV-Aus. It is
worth noting that the four P2 proteins show a highly
conserved region (Cons 1, 92-9~ identity) and have a
relatively variable region (V, 48~ identity) (Fig. 2).
In addition, the C-terminal 20 residues of RCNMVTpM-34 and SCNMV-38 and -59 P2 are 100~ identical
except for the last amino acid. It is interesting that the
similarity between SCNMV RNA-2 and P2 and
RCNMV-TpM-34 RNA-2 and P2 is higher than that
with RCNMV-Aus, and even higher than that between
RCNMV-TpM-34 and -Aus, suggesting that the two
strains of SCNMV are more closely related to RCNMVTpM-34 than to RCNMV-Aus.
2485
M A V H V E N L S DLAA'TNDGVAVS L N R Y T D W K C R S G V S E A P L I P A S M M S K I T D
M A I H V E S V Q D L A I C / N D G V A V L L N R Y T D W K C R S G V T E A P L IPAS IMS K I T D
M A V H V E S V N D L A A ' T N D G V A V L L N R Y T D W K C R S G V T E A P L I P A S IMS K I T D
M A V Q V E N V Q D L A K T N KGEESrLKNRYT D W K C R S G V T E A P L I PAS IMS K I T D
**..**.. * * * * * * . , .* * * * * * * * * * * * * * * * * * * * * * * * * * * * *
50
50
50
50
Y A K T T A K G N S V A I ~ N Y T H V V L SL A P T I G V A I P G H V T V E L I N P N V E G P F Q V M
Y A R T T A K G N S VAI~NYTHV I LS L A P T I G V A I P G H V T V E L V N P N V D G p F Q V M
Y A R T T A K G N S V A L N Y T H V I LS L A P T I G V A I P G H V T V E L V N p N V D G P F Q V M
Y A K T T A K G N S V A L N Y T H V I LS L A P T I G V A I P G H V T V E L V N P N V D G p F Q V M
** •* * * * * * * * * * * * * * * .* * * * * * * * * * * * * * * * * * * , * * * * °* * * * * *
i00
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100
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SGQTLSWSPGAGKPCLMI FSVHHQLNS DHEPFRVRITNTGI PTKKSYARC
SGQTLSWS PGAGKPCLMI FSVHHQLNS DHEPFRVRITNSG IPTKKSYARC
N G Q T L S W S PGAGF,P C L M I F S V H H Q L N S D H E P F R V R I T N S G I P T K K S Y A R C
SGQTLSWS PGAGRPCLMI FSVHHQLNS DHE PFRVRITNSGIPTKKSYARC
•* * * * * * * * * * * , * * * * * * * * * * * * * * * * * * * * * * * * * . * * * * * * * * * * *
150
150
150
150
H A YWG F D V G T R H R Y Y K S E P A R L I E L E V G Y Q R T L L S S I K A V E A Y V Q F T F D T
HAYWGY DVGTRHRyyKs E PARLIELEVGYQRTLLS S IKAVEAYVQF TFDT
HAI'WGYDVGTRHRyyKSE PARLIELEVGYQRTLLS S IKAVEAYVQFTFDT
HAYWGYDVGTRHRYYKS EPARLIELEVGYQRTLI~S IKAVEAYVQFTFDT
-*****. * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *
200
200
200
200
SRMEKNPQLCTKSNVN I IPPKAETGS IRGIAPPLSVVPNQGRES KVLKQK
SP/4ESNPQLCTKS K'VNT I P P K V E T H P I R G I A P P L S V G K S Q R L S S E V S K P E
SRMES NPQLCTKS KVNIVPPKAETFPIRGIAPPLSVGKNQGQQS EVSKQQ
SRM~SNPQLCTKSKVN IVPPKVETHPIRGIAPPLSVGKSQRRKS EVSKPK
250
250
250
250
* * * * ' * * * * * * * * ' * * " "***AA** " * * * * * * * * * *
"*
*'* *" "
G G T G S K T T K L P S L E pS S G S S S G L S M S R R S H R N V L N S S I P I K R N Q D G N W L G
GGNGNETTKLPAS GPTLRPGSALSMKRGS RRNIAYS SNPIKRNQDDNWLG
GGNGNES IKLS S S GPTLRPGSALSMKRGS RRNIAYTSNPIKRNQDGNWLG
G G N D N E T T K L S A S G P S L R P G S A L S M K Q G S R R N T A Y T S T P I KI%NQDDNWLG
** ...... **.. .*.
..*.***.. *•**.
.* * * * * * * * . * * * *
~A
DHLS DKGRVTDPN ....... PERL-DHLSNKGRDIRPKAGETLDE PVKLDT
DHLSNKGRDIRPKAGETLDEPVKLDA
DHLSNKGRDIRPKAGETLDEPVKLDA
****. *** . *.
* .* •
300
300
300
300
317
326
326
326
Fig. 2. Alignmentof the aminoacid sequencesof the P2 proteinsof
RCNMV-Aus,RCNMV-TpM-34,SCNMV-38and SCNMV-59(from
top to bottom in each section). Asterisks and black points indicate
perfectly conserved and well conserved residues, respectively. The
limitsof conservedregionsare denotedby solidtrianglesand thoseof
the variable region by open triangles.
Some potentially functional modules of RNA-2 have
been highly conserved during their evolution. The stable
secondary structures generated by RNA-2 seem to be a
common feature for SCNMV and RCNMV, and may be
involved in viral replicase recognition during replication, as suggested by Dreher et al. (1984) and Bujarski et
al. (1986) for brome mosaic virus. The existence of the
Cons 1 region may enable these viruses to complete their
life cycle. On the other hand, alteration of the amino acid
sequence in the C terminus of P2 may promote better
survival of these viruses under different environmental
pressures, probably contributing to the creation of a new
strain of the same virus or a new virus. A recent example
is a mutant of RCNMV-TpM-34, which was produced
spontanously by deleting an A residue in a sequence of
four A residues (positions 790 to 793) located in the
supposed variable region of RCNMV RNA-2 (Osman et
al., 1991 b). This mutant, which is capable of directing
synthesis of a polypeptide truncated in the C terminus of
the protein encoded by the P 2 0 R F of RCNMV-TpM-34
RNA-2, causes symptoms in cowpea plants different
from those produced by the parent virus.
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2486
Short communication
More recently, an extensive degree of sequence
identity between the N-terminal 230 amino acids of P2 of
R C N M V and those of carnation ringspot virus, a type
member of the dianthoviruses (64-3% and 62-6% with
RCNMV-Aus and -TpM-34, compared with 59~6% and
55.7 % sequence identity of the whole P2), has been found
(Kendall & Lommel, 1992). The current evidence
suggests that the gene organization of RNA-2 and the
functional domains of P2 may be similar among the
dianthoviruses.
Combined with previous work on pseudorecombination of R C N M V and SCNMV (Okuno et al., 1983; Rao
& Hiruki, 1987), the high degree of similarity of
the RNA-2 nucleotide sequences and P2 amino acid
sequences, not only between SCNMV-38 and -59, but
also between these SCNMV strains, RCNMV-Aus and
-TpM-34, suggests closely related evolution of the RNA2s of these viruses. However, full consideration of the
classification of dianthoviruses must wait until the
complete nucleotide sequences of the member viruses are
known.
We thank Steven A. Lommel for critically reviewing the manuscript,
and Thomas Tribe and Gina Figueiredo for technical assistance. This
study was supported by a Strategic Grant (G1450) and an Operating
Grant (A3843) from the Natural Sciences and Engineering Research
Council of Canada.
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(Received 19 March 1992; Accepted 3 June 1992)
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