Journal o f General Virology (1990), 71, 1887-1896.
1887
Printed in Great Britain
Nucleotide sequence and genomic organization of melon necrotic spot virus
C. J. Riviere 1 and D. M . Rochon z*
~Department of Plant Science, University of British Columbia, Vancouver, British Columbia V6T 2A2 and
2Research Station, Agriculture Canada, 6660 N.W. Marine Drive, Vancouver, British Columbia, Canada V6T 1X2
Cloned cDNA copies of the genomic RNA of melon
necrotic spot virus (MNSV) have been sequenced and
the sizes and locations of predicted viral proteins have
been deduced. The genome consists of at least 4262
nucleotides and the positive strand contains three to
five open reading frames (ORFs) which may be
expressed. The 5' proximal O R F encodes a 29K
protein (p29) and terminates with an amber codon
which may be read through to produce an 89K protein
(p89). Two small centrally located ORFs each encode a
7K protein (p7A and p7B). As p7A is in frame with
p7B, readthrough of the amber codon terminating p7A
may occur, producing a 14K protein (p14). The 3'
proximal O R F encodes the 42K coat protein. The
genomic organization of MNSV, its probable translation strategy and the amino acid sequences of its
putative proteins closely resemble those of known
carmoviruses, suggesting that M N S V should be classified as a member of the carmovirus group. Unusual
properties of the putative M N S V replicase (p89)
suggest that M N S V should be classified in a new virus
supergroup with several other viruses sharing these
properties.
Introduction
and tombusviruses. Detailed comparisons, however,
reveal that although MNSV is a carmovirus, its coat
protein resembles the coat proteins of tombusviruses
more closely than those of other carmoviruses (Riviere et
al., 1989). In this paper we report the nucleotide
sequence of the MNSV genome and compare the
organization and deduced amino acid sequences of its
major open reading frames (ORFs) with those of other
plant viruses.
Outbreaks of melon necrotic spot virus (MNSV) have
been reported mainly in greenhouse melons and cucumbers. All isolates are reported to have narrow experimental and natural host ranges limited almost
exclusively to members of the Cucurbitaceae. MNSV'is
transmitted in nature mainly by the soil-inhabiting
fungus Olpidium radicale, and can be effectively controlled by eradicating this vector (Hibi & Furuki, 1985).
MNSV shares physicochemical properties with viruses from two groups of small, isometric plant viruses,
the carmoviruses and the tombusviruses (Morris &
Carrington, 1988). Nucleotide sequence data have
shown that these two groups of viruses are closely related
(Rochon & Tremaine, 1989). Physicochemical properties
which MNSV shares with these viruses include a small
(about 30 nm) spherical particle (Hibi & Furuki, 1985)
with a single type of capsid protein of about 42K (Bos et
al., 1984; Riviere et al., 1989) and a monopartite, singlestranded (ss) RNA genome of Mr 1"5 × 106 (Hibi &
Furuki, 1985).
To characterize MNSV further and to determine its
relationships with other plant viruses, we have prepared
and sequenced cDNA clones representing most of the
MNSV genome. We have previously reported the
nucleotide sequence of the MNSV coat protein gene. Its
deduced amino acid sequence shares significant
sequence similarity with the coat proteins of both carmo0000-9430 © 1990 SGM
Methods
cDNA synthesis and cloning. Purification of MNSV and virion RNA
as well as synthesis and cloning of MNSV eDNA have been described
(Riviere et al., 1989).
Northern blots o f subgenomic RNAs. Total ssRNA from mockinoculated and MNSV-infected cucumber cotyledons was prepared as
described (Siegel et al., 1976). These RNAs plus MNSV virion RNA
and DNA size standards were denatured with glyoxal and electrophoresed as described (McMaster & Carmichael, 1977). Nucleic acids
were transferred to Zeta-Probe membranes (BRL) using alkaline
blotting conditions (Vrati et al., 1987). Blots were probed with selected
MNSV eDNA clones labelled with 3zp by nick translation (Rigby et
al., I977).
DNA sequencing. Subclones of the MNSV eDNA clone pMNS01A
(see Fig. 1) were generated by subcloning selected restriction fragments
of this clone into the phagemid vector Bluescribe (Stratagene).
Subclones of the MNSV cDNA clone pMNS17A (see Fig. 1) were
generated by subcloning the entire insert into Bluescript (Stratagene)
and generating ordered, nested deletion subclones of this insert
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1888
C. J. Riviere and D. M . Rochon
(Henikoff, 1984). The dideoxynucleotide chain termination method
(Sanger et al., 1977) was used to sequence double-stranded (ds) plasmid
DNA templates (Korneluk et al., 1985) using either the Klenow
fragment of DNA polymerase I under conditions recommended by
Stratagene or using modified T7 DNA polymerase (Sequenase, USB)
as described (Toneguzzo et al., 1988). Sequence ambiguities were
resolved by increasing the sequencing reaction temperature, using
dGTP analogues [7-deaza-dGTP with Klenow polymerase (Barr et al.,
1986) or dlTP with Sequenase (Tabor & Richardson, i987)] and/or
electrophoresing reaction products through 6% polyacrylamide gels
containing 40% formamide (Martin, 1987).
RNA sequencing. Selected portions of MNSV genomic RNA were
sequenced using specific oligonucleotide primers and genomic RNA as
template for dideoxynucleotide sequencing with avian myeloblastosis
virus reverse transcriptase. Conditions used were essentially those
recommended by Stratagene for sequencing synthetic RNA transcripts, except that annealing was performed at 42 to 45 °C rather than
at room temperature.
Storage and analysis of sequence data. Initial storage and analysis of
sequence data used either a microcomputer version of software
developed by Pustell (Pustell & Kafatos, 1984) or the Gene-Master
(Bio-Rad) system. Further analysis used programs and databases
available through the Canadian National Research Council's Scientific
Numeric Database Service, which operates on a DEC VAX running
VMS. Dot matrix comparisons of'amino acid sequences were made
with a version of DIAGON (Staden, 1982) using the proportional.
match method with a homology score of 350 and a span of 31 to 34.
Multiple sequence alignments were made by a progressive alignment
procedure which uses the Needleman-Wunsch algorithm (Needleman
& Wunsch, 1970) iteratively to generate a multiple sequence alignment
(Feng & Doolittle, 1987). The ensemble of programs required for this
method was kindly provided by Drs. D.-F. Feng and R. F. Doolittle of
the University of California-San Diego. The Needleman-Wunsch
algorithm used by the multiple sequence alignment program assesses
amino acid sequence similarity using the modified Protein Mutation
Matrix of Dayhoff (Dayhoff et al., 1978). The amino acid sequences of
probable MNSV proteins were compared to sequences in several
protein and nucleotide sequence databases using the FASTA and
TFASTA programs respectively (Pearson & Lipman, 1988). The RDF2
program (Pearson & Lipman, 1988) was used to help evaluate the
significance of these comparisons. Databases used included the protein
sequence libraries of the NBRF (Release 20), Swiss-Prot (Release 10)
and Pseqlp (Release 6) as well as the nucleotide sequence libraries of
the NBRF (Release 35), GenBank (Release 59) and EMBL (Release
19).
Results and Discussion
Sequencing strategy
B o t h s t r a n d s o f two c D N A clones r e p r e s e n t i n g a b o u t
9 5 % o f the g e n o m e ( p M N S I 7 A a n d p M N S 0 1 A ; Fig. 1)
were s e q u e n c e d at least o n c e a n d e a c h n u c l e o t i d e was
s e q u e n c e d a n a v e r a g e o f six times, T h e s e clones o v e r l a p
for a b o u t 390 nucleotides. A single n u c l e o t i d e difference
(A or U) was f o u n d b e t w e e n these clones in this o v e r l a p
region at n u c l e o t i d e 2389. T h i s p o l y m o r p h i s m w o u l d
result in a single c o n s e r v a t i v e a m i n o a c i d s u b s t i t u t i o n
(see Fig. 2). A s i n d i c a t e d in Fig. 1,178 n u c l e o t i d e s at the
5' end o f the g e n o m e were d e t e r m i n e d using v i r i o n R N A
MNSV RNA
4262
I
5'1
J3'
Primer 2
~ Primer 3
Primer 4
Primer 1
~
~
pMNS40A
~ pMNS40A
pMNSO6F
Hincll
Sac I
Eco R I
I
J
I
Hin c ] ]
EcoRI
pMNSO1A I
[
I
500 bases
pMNS17A
Sinai
I
I
HindIIl
BamHI EcoRV
BamHI
[Sinai
I
,|
I
I
I HinclI
Hincll
SalI
I
Fig. 1. Strategy used to sequence the MNSV genome. Solid bars
represent the two MNSV cDNA clones (pMNS01A and pMNS17A)
used to sequence most of the genome. Arrows indicate the length and
direction of regions of the genome sequenced using either virion RNA
as template and specific oligonucleotide primers (primers 1 to 4) or
other MNSV cDNA clones (pMNS40A and pMNS06F).
as t e m p l a t e for d i d e o x y n u c l e o t i d e s e q u e n c i n g b e c a u s e
the c D N A clone p M N S 1 7 A does not c o v e r this area. T h e
a u t o r a d i o g r a m showing the sequence at the 5' e n d
c o n t a i n e d one m a j o r stop point, i n d i c a t i n g t h a t only the
u l t i m a t e 5' n u c l e o t i d e h a d n o t b e e n d e t e r m i n e d . T h e
central region o f the g e n o m e was initially s e q u e n c e d
f r o m c D N A clones. B a s e d on the s e q u e n c e o f these
clones, the o r g a n i z a t i o n o f the O R F s in this a r e a differed
from that o f related viruses (see Fig. 9). T h e sequence o f
418 nucleotides in this a r e a (nucleotides 2416 to 2833)
was therefore c o n f i r m e d using o l i g o n u c l e o t i d e p r i m e r s
a n d virion R N A as t e m p l a t e for d i d e o x y n u c l e o t i d e
sequencing.
T h r e e different c D N A clones ( p M N S 0 1 A, p M N S 4 0 A
a n d p M N S 0 6 F ) m a p p i n g to the 3' t e r m i n u s o f the
g e n o m e were used to d e t e r m i n e the Y - t e r m i n a l sequence
o f M N S V . T h e sequence o f e a c h could be r e a d up to the
p o l y ( A ) tail a d d e d d u r i n g the cloning p r o c e d u r e a n d the
sequence o f p M N S 4 0 A could also be r e a d in the o p p o s i t e
direction. T h e e x t r e m e Y - t e r m i n a l s e q u e n c e o f e a c h
clone is different as shown in Fig. 2 a n d as discussed
below. T h e u l t i m a t e 3' n u c l e o t i d e o f e a c h clone is
d e s i g n a t e d ( A , ) b e c a u s e it could not be d e t e r m i n e d by
the sequencing m e t h o d used how m a n y , if any, o f the A
residues on the a u t o r a d i o g r a m at this p o i n t were p a r t o f
the viral sequence r a t h e r t h a n the p o l y ( A ) tail.
Nucleotide sequence and organization o f the M N S V
genome
Fig. 2 shows the n u c l e o t i d e sequence o f the M N S V
g e n o m e a n d the d e d u c e d a m i n o a c i d sequences o f its
p r o b a b l e proteins. T h e g e n o m e consists o f at least 4262
nucleotides. T h i s agrees well w i t h p r e v i o u s e s t i m a t e s o f
the Mr o f M N S V virion R N A ( H i b i & F u r u k i , 1985).
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M N S V genomic sequence
1889
1 NGAUUACUCUAG CCG GAUCCC CGACUCUCUUAUUUCCUUAAGUUAGUUCGUGUAUUGAUUAUCUGUCUUGAUCAG
M
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S
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A
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76 UAUAGGUUAGCA AUGGAUACUGGUUUGAAAUUUCUUGUUUCUGGGGGUUUAG C CACCUC AUCUGUUAUUAGGA A A
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151 GUGAGUGCUGUGAGUUCAUUGGAUUCGUCCCUUCCUUCAUCAUCUAUAUUAUCUGCCAUCCAUGGGUCUUGGACU
S A I S H D C S K I A g V A A I V G I G Y L G V K
226 AGUG CUAUCAG CCACGAUUGUAGUAAGAUUG CCAAGGUUGC CGCCAUA GUUGGGAUUGGUUAUCUUGGG GUUAGG
I G A A W C R R T P G I T N S I I T Y G E R V V E
301 AUUGGUGCCGCUUGGUGCCG CCGUA CUC CCGGA AUAACGAAUUCC AUAAUCACCUAUGGGGAAGAAGUG GUUGAG
O V K V D I D R D A E E E S D I G E E I V V G T I
376 CAAGUGAAGGUAGAUADUGAUGAA G AUG CUGAA GAG GAGUCCGA U AUUGGUGAG GAAAUUGUG GUUGGUACGAUA
G I G I H T N %' N P E V R A K R R H R S R P F I K
481 GGUAUUGGUAUACACACAAACGUCAACCCUGAAGUUCGAGCUAAGCGCAGACAUAGAUCGAG G CCAUUC AUCAA G
K I V N L T K N E F G G C P D S S K S N V M A V S
626 AAGAUCGUGAAUt~A ACGAAGAAUCACUUCGGUGGAUGCCCCGACUCUAGUA A AUCCAACGUCAUGGCUG UA AGU
K F V Y E q C K Q H N C L P H Q T R L I M S I A V
601 AAAUUCGUIK/AUGAA CAAUGUAAACAGCACA AUUCUCUUCCACAUCAAACCAG AUUGAUCAUGAGUAUUG CAGUU
P L V L S P D M Y D I S S K A L L N 5 E I L T E N
676 CCAUUGGUGUUAAGUCCCGACAUGUACGACAUUUCCAGCAAAGCUCUGCUAAACAGCGAGAUAUUGACAGAAAAC
R
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A
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751 AGAGCCACGCUGGACCGCCUCAAAACUCUCGACCGGUGGCUAACACACUUGGUGUGCCACCCCCUUAGCGCGAAG
A
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N
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A
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826 G CUUGGAGGCGGGCAADUGACAACUUGUGUG GUCUUCCAGAD"JGGAAGGCDUUC AAGUUG GUCA A CUAGG GGUGC
L E E L A G F C T S V K R G T H P D M T E F P Q D
901 CUGGAGGAGCUCGCUGGGUUCUCUACUUCCGUACGGAGAGGGACACACCCAGACAUGACCGAGUUUCCUCAGGAU
R P I K T K K L Y C L G G V G T S V K F N V H N N
976 CGUCCCAUUAAGACACGCAAACUGUAUUGUUUAGGGGGAGUUGGAACUAGCGUGAAGUUCAACGUGCACAAUAAC
S L A N L R R G L V E R V F F V E N D K K E L E P
1961 UCUCUAGCUAACCUUCGGCGCGGUCUAGUUGAGCGCGUUUUCUUUGUUGAAAAUGAUAAGAAGGAACUGGAGCCU
A
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A
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I]26 GC CC CUAAACCUCUUAGUG GUGC GUUUGAUCGCUUAACUUG GUUUCGUC GGA AACUCCAUAGUAUUGUG G GUACU
H
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1201 CAUUCCAGUAUUAGUCCAGGUCAGUUCUUGGACUUCUAUACUGGCAGGAGGCGCACGAUUUAUGAAGGUGCUGUG
K
S
L
E
G
L
S
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Q
R
R
D
A
Y
L
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F
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A
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1276 AAAUCGUUGGAGGGGUUAAGUGUUCAACGAAGGGAUGCCUAUCUGAAAACGUUUGUUAAAGCGGAGAAGAUUAAU
T T K K P D P A P R V I O P R N V R Y N V E V G R
]351 ACCACUA AG A AACCUGACC CAGCUCCGC GGGUUAUACAAC C GAGGAAC GUAAGAUACA ACGUUGA GGUUGGUC GU
Y L R R F E H Y L Y R G I D E I W N G P T I I K G
1426 UAUCUACGUAGGUUUCAGCAUUACCUCUAUCGAGGAAUUGACGAAAUCUGGAAUGGCCCCACCAUAAUAAAAGGA
Y T V E q I G K I A R D A W D S F V S P V A I G F
1501 UACACUGUCGAGCAAAUUGGGAAAAUCGCCCGUGACGCAUGGGACUCCUUCGUUAGUCCUGUAGCAAUCGGAUUU
D M K R F D ~ H V S S D A L K W E H S V Y L D A F
]576 GACAUGAAAAGGUUCGACCAACAUGUAUCCUCCGACGCUCUUAAAUCGGAACAUAGUGUUUAUCUUCACGCUUUU
C E D S Y L
A E L L K W Q L V N K G V G Y A S D G
1661 UGCCACGACUCAUAUCUUGCAGAAUUGUUGAAGUGGCAAUUAGUUAAUAAGGGUGUUGGGUAUGCUAGUGAUGGA
M
I
K
Y
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V
D
G
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G
D
M
N
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A
M
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A
1726 AUGAUUAAAUAUAAGGUUGAUGGGUGCCGGAUGAGUGGUGACAUGAAUACAGCUAUGGGUAACUGUUUGAUUGCC
C A I T H D F F R $ R G I R A R L M N N G D D C V
1801 UGUGCCAUCACGCAUGAUUUCUUCCGUAGUCGUGGUAUCAGGGCGCGUUUGAUGAACAAUGGUGAUGACUGUGUC
V I C E K E C A A V V K A D M V R H W R q F G F Q
1876 GUAAUAUGCGA A A A AGAAUGUGCCGCGGUGGUUAAAGCCGACAUGGUAAGGCACUGGAGACAAUUCGGGUUUCAA
C E L R C D A E I F E ~ I E F C ~ M R P V Y D G E
1951 UGCGAACUCGA AUGCGAUGCAGAAAUCUUCGAGCAAAUUGAGUUUUGUCA AAUGCGGCCUGUGUACGA C G G GGA A
K
Y
V
M
V
K
N
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L
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D
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$
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2026 AA AUAUGUGAUG GUACGGA AUCCCUffGGUUAGCCUAUCCAAAGAUUC C U ACUCAGUC G G CCCUUG GAAUGGAAUC
N
H
A
R
K
W
V
N
A
V
G
L
C
G
L
S
L
T
G
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P
V
V
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2101 AACCAUGCACGCAAGUGGGUCAAUGCAGUUGGCUUGUGUGGCUUAUCCCUCACUGGUGGAAUUCCUGUUGUCCAA
S Y Y N M M I R N T Q S V N $ S G I L R D V S F A
2176 AG[NJAUUAUA AUAUGAUGAUC CGCAACACUCAGUCCGUG AACAGUUCUGGCAUACUUC GCGAUGUCA GUUUUG CU
S G F R E L A R L G N R K S G A I S E D A R F S F
2251 AGUGGAU~CGGGAGUUAfiCGCGAUUGGGUAACAGGAAAAGUGGUGCCAUAUCUGAAGACGCCCG~UUAGCUUU
S
Y L A F G I T P D L q R A M E S D Y D A H T I E W
2326 UAUCUCGCAUUUGGCA~ACUCCAGA~UACAACGUGCCAUGGAAAGUGACUAUGAUGCUCAU~CUAUAGAGUGG
G F V P Q G N P R I Q P I SMW
TsL
_N R E
LV*
D
Q
T
E L T N P
2401 GG~UCGUGCCCCAGGGAAAUCCUAGAAUACAGCCAAUCUCAUGGACUCUCAACGAACUGUAGAA~AACUAAUC
R G R S K E R G D S G G K Q K N S M G R K I A N D
2476 CUCGGGGAAGAAGUAAAGAACGUGGUGACAGCGGGGGAAAACAGAAGAACUCAAUGGGGCGAAAGAUAGCCAAUG
A I S E S K ~ G V K G A S T Y I A D K I E V T I N
2551AUGCUAU~CUGAAUCGAAGCAAGGAG~AUGGGUGCCAGCACAUACAUUGCUGAUAAAA~AAGGUGACUAUUA
F N F * C M A C C R C D S S P G D Y S G A L L I L
2626 A C U U U A A U U U ~ A G U G U A U C C C U U G ~ G C C G ~ G U G A C U C C A G C C C C G G G G A U U A C U C U G G A G C A ~ G C U U A U A U
F I S F V F F Y I T S L S P Q G N T Y V H H F D S
2701 U A ~ U A U C U C A U ~ G U U ~ C U ~ U A U A ~ A C C U C G C ~ A G C C C G C A A G G A A A U A C ~ A C G ~ C A U C A C U U C G A U A
S S V K T q Y V G I S T NMGADMGv*K
I N N L P T
2776 G ~ C U U C C G U U A A A A C A C A A U A C G U U G G C A U C U C U A C A A A U G G C G A U G G U U A A A C G C A U U A A U A A ~ U A C C C A C A
V K L A K O A L P L L A N P K L V N K A I D V V P
2881 GUGAAGC~GCUAAGCAGGCUCUACCCCUGC~GCGAAUCCUAAACUUGUAAAUAAAGCUAUAGAUGUGG~CCU
L V V Q G G R K L S K A A K R L L G A Y G G N I S
2926 U U G G U C G U C C A A G G U G G U C G G A A A U U G U C C A A G G C U G C U A A G C G G ~ G C U U G G C G C U U A U G G A G G C A A C A ~ U C G
Y T E G A K P G A I S A P V A I S R R V A G M K P
3001 UACACUGAGGGUGCCAAACCGGGUGCAAUAUCAGCUCCUGUCGCUAUUAGUCGGCGAGUGGCUGGUAUGAAGCCU
K F V R S E G S V K I V H K E F I A S V L P S S D
3076 AGGUUUGUCAGAUCUGAAGGAUCUGUGAAGAUAGUUCAUAGGGAGUUUAUUGCCUCUGUUCUUCC~CGAGUGAU
L T V N N G D V N I G K Y R V N P S N N A L F T W
3181 CUCACUGUCAAUAAUGGUGAUGUCAAUAUCGGUAAGUAUACAGUCAAUCCUAGUAAUAACGCUUUAUUCACCUGG
L Q G Q A Q L Y D M Y R F T R L R I T Y I P T T G
3226 CUUCAGGGACAAGCUCAACUAUAUGAUAUGUACAGA~UACUCGGCUCCGUAUCACCUACAUUCCUACUACCGGA
S T S T G R V S L L W D K D S Q D P L P I D K A A
3301 UCCACUUCCACGGGUCGUGUCUCUCUCCUCUGGGAUAGAGA~CUCAGGACCCCCUCCCUAUAGACCGUGCUGCC
I S S Y A H S A D S A P W A E N V L V V P C D N T
3376 AUUAGCUCUUAUGCUCAUUCCGCUGA~CAGCGCCUUGGGCUGAGAAUGUUCUAGUGGUCCCAUGUGACAAUACG
W R Y M N D T N A V D R K L V D F G Q F L F A T Y
3451 U G G A G G U A C A U G A A U G A U A C C A A U G C U G U C G A C C G G A A G ~ G G U U G A U U U U G G G C A G U U C ~ A U U C G C U A C U U A U
S G A G S T A H G D L Y V E Y A V E F K D P O P I
3626 U C U G G U G C U G G U A G C A C C G C C C A U G G U G A U C ~ U A U G U U G A G U A U G C U G U A G A A U ~ A A G G A C C C C C A G C C U A U C
A G M V C M F D R L V S L S E V G S T I K G V N Y
860] GCUGGGAUGGUAUGUAUGUUUGAUCGCUUGGUCUCUCUUUCCGAAGUUGGAUCCACUAUCAAGGGUGUCAAUUAC
I A D R D V I T T G G N I G V N I N I P G T Y L V
8676 A U U G C U G A U C G U G A U G U G A U A A C U A C U G G G G G U A A U A U U G G U G U U A A C A U C A A U A ~ C C C G C G A C ~ A U C U C G U C
T I V L N A T S I G P L T F T G N S K L V G N S L
3781 A C G A U U G ~ C U U A A U G C U A C A U C G A U U G G U C C C C U C A C C U U C A C U G G U A A ~ C U A A A C U U G U A G G C A A C A G U C U U
N L T S S G A S A L T F T L N S T G V P N S S D S
3826 AAUCUUACCAGCAGUGGUGCAUCUGCUCUUACGUUCACCC~AACUCCACCGGUGUGCCCAACAGUAGCGAUUCU
S F S V G T V V A L T R V R M T I T R C S P R T A
3901 UCAUUCUCUGUGGGUACCGUUG~GCCUUGACUAGGGUGCGUAUGACGAUCACUCGCUGCUCUCCAGAAACUGCU
Y L A *
3976 U A C C U C G C C U A A ~ U G A U ~ A ~ G C A C U C C A A A U C C G G U C U C C C U U G ~ C C U A C C U G U U C U C A G C C U G A U A U C U G
4081 UUCUGGUGUCCUAUAGGCGUCCUUGUCCUGUGUAGUGCGGUCUGCCUAACCGUAAUGGCGUAUCGGCUUGGA~U
4126 CCGAUGA~UGGCUCCGGGAUGUACGACAUAGCUGAAGAUGGUUGGAGUUUGGUGGACCACCGCUAGCAAAAUAC
4201 ACUCUGUGUGGGGCGUGCUAGUCGAUAGUCAUGUAUGU~GAGAUGGGUUAUAGGCCCAUCC(An)
4262
CU~(An)
4266
CGCCU(An)
4267
Fig. 2. Nucleotide sequence of the MNSV genome. Deduced amino acid sequences of probable proteins are shown above the
corresponding nucleotide sequence. Asterisks indicate termination codons. Nucleotide number one is represented by an N because its
identity is unknown. Polymorphisms are indicated at nucleotide 2389 and at the 3' terminus.
Fig. 3 shows Northern blots of total ssRNA from
MNSV-infected cucumber probed with MNSV cDNA
clones corresponding approximately to either the 5' half
of the MNSV genome (pMNS17A) or the 3' half of the
genome (pMNS01A; see Fig. 1). As shown in Fig. 3,
pMNS17A hybridizes only to genomic length RNA (a),
but pMNS01A hybridizes to genomic length RNA and
to two prominent, shorter RNAs of about 1.9 kb and 1-6
kb (b). These shorter MNSV-specific RNAs are probably Y-coterminal subgenomic RNAs. The genomic
positions of these RNAs were compared with the
locations of ORFs deduced from the MNSV sequence in
all three reading frames, from both the positive and
negative sense of the sequence. In addition, the genomic
organization and amino acid sequences of proteins
encoded by these ORFs were compared with those of
related viruses. Both comparisons indicate that the
ORFs shown in Fig. 4, which are all translated from the
positive sense of the MNSV sequence, are those most
likely to be expressed in vivo.
(a)
(b)
1
2
3
!
4
1
2
3
4
--4-3
--1"9
--1'6
Fig. 3. Northern blots showing MNSV-specific ssRNAs generated
during infection of cucumber. Blots were probed with either
pMNS17A (a) or pMNS01A (b). Both blots contain MNSV virion
RNA (lane 1), total ssRNA from uninfected cucumber cotyledons (lane
2), total ssRNA from MNSV-infected cucumber cotyledons (lane 3),
and MNSV virion RNA plus total ssRNA from uninfected cucumber
cotyledons (lane 4). Sizes of MNSV-specific ssRNA species are
indicated on the right in kb.
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1890
C. J. Riviere and D. M. Rochon
UAG
"~
I
88
p29
I
I
892
089
I
I
2460
UAG
4.3 kb/F1
1.9 kb/F3
I
I
2442 I 2825
2637
MNSV
"-t
p42 (coat) ~ - - 1.6 kb/F1
I
I
2815
3984
UAG
p27
]
CarMV
(Carmovirus)
UAG
p86
[p98 I
[]
4"0 k b / F l
1.7 kb/F3
---[ p38 (coat) ~--1-5 kb/F2
UAG
4.7 kb/Fl
p41 (coat)
CNV
(Tombusvirus)
I
2.1 kb/F2
0.9 kb/Fl
0.9 kb/F3
Fig. 4. Comparison of the genomic organization and probable
translation strategy of MNSV with those of CarMV and CNV. The
genomicorganizationof the carmovirusTCV is not shown, but closely
resembles that of CarMV (Carrington et al., 1989). Numbers beneath
the coding regions of MNSV indicate nucleotide positions. The
approximate sizes of virus-specific ssRNA species which serve as
templates for translation are indicated on the right in kb. F1 to F3
indicate the positive-sense reading frame from which proteins are
translated. Arrows indicate termination codons proposed to be read
through.
The genomic organization and probable translation
strategy of MNSV therefore appears to be as follows (see
Fig. 4). The identity of the ultimate 5' nucleotide of the
genome was not determined, and it is not known whether
the 5' end of MNSV is modified. A Y-proximal noncoding region of 87 nucleotides precedes the first A U G
codon found in the sequence. This initiator is in reading
frame 1 and is followed by an open region which encodes
a protein of 29 228 Mr (p29). This O R F terminates with
an amber codon, but the frame remains open so that if
readthrough occurs, a protein of 88 683 Mr (p89) would
be produced. Overlapping the p89 ORF at the 3' end by
19 nucleotides is an O R F in reading frame 3 encoding a
7104 Mr protein (p7A). This ORF also terminates in an
amber codon and the frame remains open so that if
readthrough occurs, a protein of 13 915 Mr (p 14) would
be produced. This readthrough area also has an in-frame
initiation codon three nucleotides 3' to the p7A terminator raising the possibility that this ORF is expressed as an
independent protein of 6589 Mr (p7B). The ORFs
encoding p14 and p7B terminate with an ochre codon.
Overlapping the 3' ends of these ORFs by 11 nucleotides
is an ORF in reading frame 1, which encodes the 41840
Mr coat protein (p42) which has been previously
described (Riviere et al., 1989). The ochre terminator of
the p42 ORF is followed by a Y-proximal non-coding
region of at least 276 nucleotides.
Exactly where the 3' end of the MNSV genome
terminates and the identity of the Y-terminal nucleotides
is uncertain even though, as noted previously, three
different c D N A clones corresponding to this region were
sequenced. The sequence of these three clones was
identical up to and including nucleotides 4261 to 4262.
All three clones have the sequence CC at these two
positions. The sequence of pMNS06F terminates at
nucleotide 4262. pMNS01A contains four more nucleotides [CUUU(An)] and pMNS40A contains five more
nucleotides [CGCCU(An)]. The only matching nucleotide in the extensions of these two clones is the C at
nucleotide 4263. It is unclear why the 3' termini of these
three clones differ, pMNS06F may have been synthesized from a molecule of viral R N A which was slightly
degraded before or during polyadenylation. The different length and sequence of the 3' ends of pMNS01A and
pMNS40A are more difficult to explain. The differences
may be artefacts occurring during c D N A synthesis and
cloning or through errors in replicating these sequences
after cloning. Alternatively, the observed variability may
reflect natural variability in the 3' end of MNSV RNA.
This could arise through errors in replication or through
post-transcriptional, non-templated additions (Rao et
al., 1989) of short, variable, 3' extensions. Viral RNAs
with such variable 3' ends would have to be able to be
efficiently replicated or edited in order to build up to
levels in the population where they would be detected in
cDNAs synthesized from viral RNA,
Probable translation strategy
The translation strategy illustrated in Fig. 4 is proposed
for MNSV based on the corresponding genomic locations of ORFs deduced from the MNSV nucleotide
sequence (see Fig. 2) and of MNSV-specific ssRNAs
produced during infection (see Fig. 3) as well as on
comparisons with the translation strategies of related
carmoviruses. It is suggested that p29 and its readthrough protein p89 are expressed from genomic-length
RNA, whereas p7A and its readthrough protein p14 are
expressed from the 1-9 kb MNSV subgenomic RNA.
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MNSV genomic sequence
The 1-6 kb subgenomic R N A would serve as the
template for expression of the MNSV coat protein. A
separate subgenomic R N A for p7B was not detected. If
such a subgenomic R N A does not exist, p7B could be
expressed via some unusual mechanism operating at the
level of RNA processing or translation.
1891
(Hillman et al., 1989). Fig. 4 also shows that the proposed
translation strategy of MNSV closely resembles that of
CarMV and TCV but is distinct from that of the
tombusvirus CNV. These comparisons suggest that
MNSV should be classified as a carmovirus.
Amino acid sequence comparisons
Comparison of the genomic organization and translation
strategy of MNSV with those of similar viruses
Fig. 4 compares the genomic organization of MNSV to
those of the carmovirus, carnation mottle (CarMV;
Guilley et al., 1985) and the tombusvirus cucumber
necrosis (CNV; Rochon & Tremaine, 1989). The
genomic organization of the carmovirus turnip crinkle
(TCV) is not shown in Fig. 4, but closely resembles that
of CarMV (Carrington et al., 1989). This comparison
shows that MNSV shares similarities in genomic
organization with all three viruses but resembles the
carmoviruses more closely overall. In particular, the
central genomic region of MNSV, like those of CarMV
and TCV, encodes at least one small protein not present
in CNV and lacks ORFs analogous to the two nested
ORFs found near the 3' termini of the tombusviruses
CNV and the cherry strain of tomato bushy stunt virus
CarMV (247/
TCV
MCMV
(252) - (440) -
C
C
CNV
(298) - (340) L C
G
BYDV
F
RE
V RE
qlS
E EWL
VQSR~T
VR~P
VV~QTN
EG~CTASGFESPF
TCV
(283) -- VE~ .R.I. .F } { IQC ~ i M][G. N. .G. .L D
CarMV (278'
MCMV (471) - T R V F R I A
LGNLYE
CNV
BYDV
MCMV
CNV
BYDV
(322) A Q
Q
(610) D K G E L
(375) - N G Q
(416) G D ~ A I
KPA
Q IP
V S C ~IEI: I S ~
TRT
O TK
Y P-~R~E
HD
MNSV (379) Q. . . .
CarMV (355)
TCV
(361)
CNV
G
N
~
R
i,~:l,~,l:l,~I~:l,!ii
G
~ AV
E
~T~K~I~E
DLVDC
R
K Y P S F YIK]G RR ~ T
q
A~-D R[~
W~
LLT
......
~ ........
S
KEMGYL
RRFHRVC
NHTRISAN
I -GK~SGI
RRLVRLA
NHTPVPRE
IIFFW K N
K ...............
A -GR
5P
KAVCEK
VAHRLGYD
I ~ T D T ~ D Y ~ K S I I E EV ~ Y C K T Y P A ~
G~V K~
. . . . . QRR~G Y~
I EN
hA
LD]~AIERK
D
I Y]~ K AIL D L Y M T C R L S R K D~EIL K T F ~ K A E[
(413)
BYDV
MNSV
(419) ~ T ~ K ~ P [ D
TCV
MCMV
(401)
(590)
F
L
h KDPAPRVIQPRD
K A D P~ P RV I QP A
BYDV
(495)
M
K--
TCV
(441)
AM
CNV
(492)
GV
BYDV
(533)
(499)
~S
K
P A P R V I ~ P RIg V
AP
~
K
K F~Y
S
K IM$
RKTC
NKPVAIG
AC
(532)
(573)
Q
K
V LDA -C
C LAC E-GDAH
~
Kg
. . .E. - -~ ~A ~ KT ~ V
V~ N A G~ N C P
V S R I Y GY
GF
KAMYP-GNKL
SK
D
I N G I[~ - - G O S E
L
E
M
V
TI
T
MNSV
CarMV
TCV
MCMV
(078) F F R S R
(553)
HLHK
(658) - Y L M K
(749) F V T K L
RA-R
M
V C~KRCAAV
K .....
RS-R
I
C RTDIDY
VSNLTTG
KC-K
I
F ADEVDR
R-ERL8
P A - RILIII~ N G D DINIV L IIC P A V E V G R ~ R Q E L Y R
DYDV (651) YLKKL
E A- E ~ C ~ I ~ T
]~INSV (617) ~ F ~ y ~ . . . . . . . . . ~
~. . .
Car'{V (590) DF
. . . . . . (~C ~ E ~ m ~
KI ~
TCV
(695)
I a E
E
KV
MCMV (708) N Y
EV
PVY~
CNV
(651) N L
YTMKV
PPVFQ
EV
B'~'DV
(689) q Y
CarMV (629)
TCV
(634)
M CivIV (827)
CNV
(690)
BYDV (727)
N I( V T
g
E
QL
V MSKDSHSLVHWNNETN
KQ
V L
YSTT~WANEKD
AR
T T MIS K D ~ Y A V T P F N T P T AL~R R
TAMSKDVHCVNNIRDLATRKA
D~I
TTLLSMLN~SDVKSYMS
S
I
L
D RA N- E K L F D G MYD~F L
.
~KYV~
A~-~
s
AH
g
L
LA
MR
SN
[~F
S
E
E
QHHG
Q
]{~L
1. . . . .
E Gw v
G-GWK
-KYR
AIA
S T
VA
TS
q N
N R
D R
NVR
R~-
V
L
I
V
CNV
BYDV
SY
NMMIK,TQSVNS--SGILR
EF
~KYVETAGNVRE--~KNITEK
-S
FFMMADRAK
SY
SCLKR)~FGPLAGDYKKKMQDV
FD
FYRLSKNGM
EY
TALVKH--GLDP
- K N IK Q GK D F D S [ ~ L Y Y L S K L S ~
(730) E K F ~ S R F T L Y
.......
EVPKKH~RI~TVT~V~K~IRGSG
(767) E S F ~ K C L Y K S S G Y K K V S E E F I K N V I ~ Y G T D E R L Q G R R T Y N
CarMV
(706) G Y S A ~ S E V C
TCV
(714) ~ s ~ v s ~
MCMV
DA
A
E
A~A I~g D
(571) , ~ K ~ Y ~ D
G T I T ~ R K EIG C R ' S G D]I ~ T S~L G NIYIL~M C[A ~ I Y G
(611) N I K M F V E D ~ M e R F K ~ R ~ G ~
T~S
MG ~ K ~
I M~G M M H A
MNS¥
CarMV
TCV
R
H
KFN
DR
OK
BYDV
L~R G
HL
PI
DE
K
EF
R~
R
QF
F V K A EI
~
KPY
RP V
D V G EL[~N[~M SNT
CNV
MCMV (670)
V
Cl
TADEV
~FRDK
V FLUS P L ~ V L SG ~ D ~ F K ~[~RL~I A K K
VL
SD
A
D~
g
g
(475)
(481) ~
R]
~
BYDV
CNV
,
G F L S Y N S N I KL~LR T N T R N V Z S L E I T V V S E R I D ~ E ~ N T
(453) e e A N S ~ S A GE ~
M~g g~l I~K
T V P Y H ~ KD ~
VQ
MNSV
CarMV
TCV
,5i
'614)
,
MCMV I709) RIGITAYIASI) 61AF NIYIQLy]DIG[~KHSGDIMIN~SI,LG~ C ~ L
•
M
Y
D
M
Y KD
(336) - D
NIFMVA
CPSQAR
L
K
K
K
V
C
K
(376) V S S N I R Y L S Q T H h G L V Y K A PLeA S[~HL~A L 7 A V E RR L ~ T L ~ G K
MNSV ( 3 4 0 ) D K ~ E~EP A~ K ~ L S ~
CarMV (317) - N E v s c O E P
TCV
--GE SY/EVDGqCPL
AMRFFPLAN
G ..... --EN
PP
.% . . . . . - - R ~ K L R S H F K D T
DIP $GV LLP qEVLEVRA
GPPNA
.... PILGL~EIAVTDGARLR
(i) MNSV RNA-dependent RNA polymerase
The readthrough domain of MNSV p89 is identified as
the putative RNA-dependent RNA polymerase as it
contains (see Fig. 5) the G D D motif and surrounding
conserved amino acid residues characteristic of most
viral RNA-dependent R N A polymerases (Kamer &
Argos, 1984; Argos, 1988). As shown in Fig. 5 and Table
1, the readthrough domain of MNSV shares very high
amino acid sequence identity with the putative polymerases of the carmoviruses CarMV (50 ~o) and TCV (53 ~o),
the tombusvirus CNV (39~o), the unclassified virus
(Nutter et al., 1989) maize chlorotic mottle (MCMV)
(51~o) and the luteovirus (Miller et al., 1988) barley
yellow dwarf (BYDV) (33~).
It has previously been reported that the polymerase
domains of several of these viruses share a high degree of
amino acid sequence similarity with each other (Rochon
(696)
(669)
(674)
(867)
F
g ~[A Y GII[T P "[QI~[IIA L EIG E I R S LL~I N T g
~
~IYI~P~I~I~I~I~I~YF~L~LC~
MCMV
(903) K~mv~ss
CNV
(762) . . . . . . . . . . . . . .
BYDV
(807) g - T p I T ~ H SL~ML~Y W E S~ L ~ v ~
MNSV (772)
CarMV
TCV
MCMV
CNV
BYDV
L
K
V
L
Y
~LIA~I~ITPDIEIQI~I~LSlEYF~S~[~PTFXW
G F v P Q S N P It I ~ P I s W
D ~ K E~L ~L~vL~lD R L~ . . . . . . . .
I~ Q~
T~NE- - L . . . .
(746) - - - V G P ~ C E A A D S L WI
(764) D - - P T G Y K E E L S D R
WI
(943) S - - T T G I L A E I P E C L L
(800)
-FGEEGVDAHEPSI
(846) Q S V K V T T P H
LQS ILLSI
RYY ~ GL~VSAQL
gRK - Y Q
~E- -FPTTL
KHNPLPPTDSAVA
PE~ HSQNEY
Fig. 5. Amino acid sequence alignment of the putative polymerase domains of the replicases MNSV p89, CarMV p86, TCV p88,
MCMV pl 11, CNV p92 and BYDV p60. Amino acid sequences are numbered from the replicase start codon. Dashes indicate gaps
introduced to optimize the alignment. Amino acids shared by four or more of the proteins are boxed.
Downloaded from www.microbiologyresearch.org by
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1892
C. J. Riviere and D. M. Rochon
Table 1. Amino acid sequence identity (%)* in pairwise
comparisons~f of the putative polymerase domains of M N S V
p89, CarMV p86, TCV p88, M C M V p l l l , CNV p92 and
B YD V p60
MNSV
CarMV
TCV
MCMV
CNV
CarMV
TCV
MCMV
CNV
BYDV
49-7
-
53.2
50.0
-
51.0
44.1
51.0
38-7
38'5
38-9
40.3
-
33.2
32.2
32.4
31.8
31.3
-
-
* Sequence identity (~) = [number of identical residues shared by
the two sequences/average length of the two sequences without
gaps] × 100.
~"Pairwise comparisons were based on the multiple alignment in
Fig. 5.
CarMV
TCV
MCMV
MNSV
M T
KF V S G
TSSVIRKV
SSLDSS
PSSSIL
(1)
S
VE V I G C T
VG~L
AVG~AA
AVRATI
(I)
NT
VG
G A R Y - - Y P E V q T F ~ G ~ P D YVlGI
(171) A R ~ L S N ~ G ~ N
M S T R N K~T R T~G~I
0 q T[]R Y Y RrL[AV R F[]
(40)
J%W C ~
vvE
......
F.REIc~R~[~I~S~GG~clL~v[C-SP[~
..z
.....
. v v R~V]F q G S G[i" V l V [ ~ S D WV ~ V ~ - [ ~ T[YJSIN~G
MNSV
CarMV
"rCV
MCMV
I -(80) TP I T N S I [ ] T Y G [ ] E V ~ V K
[]
.K
.A
.[
.]
.V
..DG
(69) ~ L[~R[~ED E g I D ~~EV~ S T P ~ T
. . .... ~. I. . ~ V
(69) ~ I ~ S S L[]C~L[~V P DS ~ D
I EI ~ L D~. . . . R ~ V 6 T~] EIEIA- T S
(246) E q S Q ~ S Q VE Q~M[]q NG ~ M M V M K ~ G P T A[P~Ie S e q D C L TK H
CarMV (103) TC¥
(104) C
MCMV (286) V
......
K
VN HL
-......
T
PR
V
K
D E E G[~ V T 0 [ ~ S T F L IR[~EF ~
A
E
S
H
NL
~
MNSV
(156) [ ~ C [ ~ D ~ V ~ Y
E ~ H ~ L ~ r ~ I [ ~
TCV
(138)
V
R
qY
MCMV
(325)
I
E
R
MNSV (196) V
CarMV (173) MV
TCV
MCMV
LS
M
(~12)
( 2 3 5 )~
~T~
-
(217) -
DS
NT
T
M
K[~L[~N[~ll L T E I~[~ATIeD R L KT L D
~I~
- ~ M M[A]G]LIA]S[D A~] Y G I K I I ~ ] A I S I L I~ R K
EDI]- 0 ....
]LIHS ~ R~j tl D lt[~ N ~ ' ]. . . . . .
RT
G ~ A S I P ~ R D ~ S I E I C ~ Y R ~ F VE E T P -
(178)
(356)
MCMV (405)
(ii) M N S V p29
As shown in Fig. 6 and Table 2, the pre-readthrough
domain of p89, MNSV p29, shares a high degree of
amino acid sequence identity with similarly positioned
proteins of the carmoviruses CarMV (21~) and TCV
(23~) and the carboxy-terminal half of p50 of the
unclassified virus MCMV (18%). It has previously been
reported that this domain shares amino acid sequence
similarity in several of these viruses (Carrington et al.,
1989; Nutter et al., 1989). MNSV p29 shows no
discernible amino acid sequence similarity with the
analogous proteins of either the tombusvirus CNV p33 or
the luteovirus BYDV p39. This provides further support
for classifying MNSV as a carmovirus.
The actual function of MNSV p29, either as a separate
protein or as a domain of MNSV p89, is unknown. Its
amino acid sequence showed no obvious similarities with
A A I [ ~ I G YGL~ I [ ] A
(361
(36)
C
M ~NMSVV
TCV
& Tremaine, 1989; Carrington et al., 1989; Nutter et al.,
1989; Miller et al., 1988). It has also been reported that
the polymerases of BYDV (Miller et al., 1988) and
CNVand CarMV (Rochon & Tremaine, 1989) show no
discernible amino acid sequence similarity (outside of
the GDD motif and surrounding amino acid residues)
with the polymerases of viruses in either the alpha- or
picornavirus supergroups proposed by Goldbach (1987).
On this basis, it has been suggested that the carmoviruses, tombusviruses and BYDV should be classified in a
third supergroup (Rochon & Tremaine, 1989). The
polymerase domains of MNSV, TCV and MCMV share
high amino acid sequence similarity with the polymerases of CNV, CarMV and BYDV but, like these viruses,
show very little similarity with the polymerases of viruses
in the two established supergroups (unpublished observations). It seems, therefore, that MNSV, TCV and
MCMV should also be included in the new supergroup
suggested for CNV, CarMV and BYDV.
A I H G S W T S A I S H D~S K I~V
C~MV
TCV
V~
R
~~
~
V
D R.h~RKR~AS
.HNHPP
.LM~S
. N .N ~
IM
MT RS
FN
C V V~N
W
R
H
S~
P V IV
T KLE~L
A~Lp
~
DQ IS
R
P
Q
Fig. 6. Amino acid sequence alignment of MNSV p29, CarMV p27,
TCV p28 and MCMV p50. Amino acids are numbered from the start
codon of each protein. Dashes indicate gaps introduced to optimize the
alignment. Amino acids shared by two or more of the proteins
compared are boxed. Only the carboxy-terminal 268 amino acids of
MCMV p50 are included in the alignment.
Table 2. Amino acid sequence identity (%)* in pairwise
comparisonst of MNS V p29, CarM V p27, TC V p28 and
M C M V p50
MNSV
CarMV
TCV
CarMV
TCV
MCMV
21.4
-
23.6
27.5
-
18.3
17-9
18.9
* Amino acid sequence identity (~) was calculated as described for
Table 1.
t Pairwise comparisons were based on the multiple alignment in
Fig. 6.
proteins in the databases searched and therefore no clues
as to its function were obtained in this way.
(iii) Nucleotide-binding proteins
MNSV lacks a protein containing the complete amino
acid sequence motif characteristic of nucleotide-binding
proteins (Gorbalenya et al., 1988, 1989; Hodgman, 1988)
that are found in regions N-terminal to the polymerase
domains of nearly all positive-sense, monopartite,
ssRNA viruses (Gorbalenya et al., 1989) including
viruses from both of the proposed supergroups. TCV and
CarMV (Carrington et al., 1989) as well as CNV,
MCMV and BYDV (unpublished observations) also lack
a protein containing a complete nucleotide-binding
motif. It has been suggested that the lack of a nucleotidebinding domain may mean that the enzymol0gy of R N A
replication is unique in these viruses, or that host
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M N S V genomic sequence
proteins have been adapted to carry out the required
activities (Carrington et al., 1989). The fact that the
amino acid sequence of the polymerases of these viruses
is so different from those of viruses in the two proposed
supergroups may also indicate these viruses have a
unique replication strategy. Alternatively, they may use a
similar replication strategy but encode replicative proteins that, although they carry out the same function,
differ significantly in amino acid sequence from the
analogous proteins of viruses in the two supergroups.
However, the unusual features of these viruses' putative
replicative proteins, i.e. absence of a protein containing
a typical nucleotide-binding domain and an unusual
polymerase amino acid sequence, would still seem to
support the idea of classifying MNSV and the other five
viruses compared in a new supergroup.
(iv) Differences in the degree of amino acid sequence
conservation between the two replicase domains
The fact that the pre-readthrough domain of the putative
viral replicase is detectably similar in only four of the six
viruses compared although the polymerase domain is
similar in all six may provide an example of two different
functional domains of the same protein evolving at
different rates. Alternatively, the two functional
domains may have had independent evolutionary origins
and have become part of the same protein via RNA
recombination (Zimmern, 1988).
(v) Central genomic region
Fig. 9 compares the central regions of MNSV, CarMV,
TCV and MCMV showing regions that share amino acid
sequence similarity. As shown in Fig. 7 and Table 3,
MNSV p7A shows significant amino acid sequence
identity with the analogous proteins of the carmoviruses,
CarMV p7 (32~) and TCV p8 (26~) and the unclassified virus MCMV p9 (15~). Such similarity has
previously been reported among the latter three viruses
(Nutter et al., 1989) and between CarMV p7 and TCV p8
(Carrington et al., 1989). The high degree of amino acid
sequence identity between MNSV p7A and analogous
carmovirus proteins again supports classifying MNSV as
a carmovirus. The function of these proteins is unknown.
No obvious sequence similarities that might suggest the
function of MNSV p7A were found in the databases
searched. It has been suggested that CarMV p7 and TCV
p8 may be involved in virus transport (Guilley et al.,
1985; Carrington et al., 1989).
As shown in Fig. 8 and Table 4, MNSV p7B (or the
readthrough portion of MNSV p14) shows amino acid
sequence similarity with the deduced amino acid
sequences of analogous regions of the CarMV, TCV and
MCMV genomes (see Fig. 9). Such similarity has
previously been reported between TCV and CarMV
N,v
CarMV
TCV
MCMV
TCV
,1,
(i)
(i)
P
(1) M V It N G Y F G R N SIaM
(34) ~ B ~ A
1893
. . . .
....
- .~ S E V.~ V V[G
.
. K ~ M L AGINR
G~KKq K T R
P Y N SL ~ D S D A T G K RIK K G GIEK S A K R L V
S S S q T Q S~T
DAV~ l ~ q
QT ~ T A V R S E D N
S S V L N K~RN~G
S A S H~TIWV
I~VIAD E[VlEVlS[I~F ~ F I-
Fig. 7. Aminoacid sequencealignmentof MNSV p7A, CarMV p7,
TCV p8 and MCMV p9. Aminoacids are numbered from the start
codonof eachprotein.Dashesindicategapsintroducedto optimizethe
alignment. Amino acids shared by two or more of the proteins
compared are boxed.
Table 3. Amino acid sequence identity (%)* in pairwise
comparisons% of M N S V p7A, CarMV p7, TCV p8 and
M C M V p9
MNSV
CarMV
TCV
CarMV
TCV
MCMV
31.7
-
26.3
36-1
-
15.2
12.8
13-2
* Amino acid sequence identity (~) was calculated as described for
Table l.
~ Pairwise comparisons were based on the multiple alignment in
Fig. 7.
TCV
MCMV
PTSPWVIY
M K V L L V ~ T G V ] L~
L~
K~W K S~Q S ~ S T ~ S N Q T C Q C
p N R T* S ~ R K R W ~ S I T T S I S T E L E ~ J V ~ V D S ~ W ~ Q W L R N L I ~ G I L ~ S
TCV
MCMV
Y N S L S L VL ~ L ~ H ~ I ~ E
I K P~T
L .......
~ I L ~ E T Q D T V A VY ~ E
A
I
S ~ N TDH ~ Q
Q ~ i 5 i N~GIN G~K
P S V Y ~ I D qT ~ K F q K ~ D I HN ~ G K ~
Fig. 8. Amino acid sequence alignment of MNSV p7B and analogous
regions of CarMV, TCV and MCMV. Dashes indicate gaps introduced
to optimize the alignment. Amino acids shared by two or more of the
proteins compared are boxed. Asterisks indicate termination codons.
Table 4. Amino acid sequence identity (%)* in pairwise
comparisons~f of MNS V p7B and regions of amino acid
sequence similarity in CarMV, TCV and M C M V
MNSV
CarMV
TCV
CarMV
TCV
MCMV
17.8
12.3
35-3
-
21.9
12-9
8.2
-
* Amino acid sequence identity (~) was calculated as described for
Table 1.
t Pairwise comparisons were based on the multiple alignment in
Fig. 8.
(Carrington et al., 1989). MNSV p7B shows no discernible amino acid sequence similarity with any regions of
CNV or BYDV. The selection pressure for conservation
in these areas among the first four viruses appears to be
at the amino acid sequence level rather than at the
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1894
C. J. Riviere a n d D. M . R o c h o n
UAG
UAG
p29 ~
p89
i
p42 (coat)
d\ \ \ \ \ \ ~ / / / / / / / / / / / ' / / / / / / / / / / / / ~
p7A~p7B
pa41
I
MNSV
UAG
p27 i
CarMV
(Carmovirus)
p7E=~
UAG
p28 1
d\ \ \ \ \ \
p33
-[
UAG
~
p88
~
/
p38 (coat)
/
/
/
/
p8~,~
t392
~
p41 (coat)
p20
[ - - ]
p2I
p50
MCMV
(Unclassified) 1
UAG
l
p ll l
p25 (coat)
\ \ ~\ \ \ ~/////////////////////////A-~;~:~F-------V- p31"6
BYDV
(Luteovirus)
UAG
1p98 p38 (coat)
p86
\ \ \ \ \ \V////////////////////////,{~.~d!:i:i::!::~:!::~::::::::!:~:i:i:i:F
--
TCV
(Carmovirus)
CNV
(Tombusvirus)
Expression of the analogous region in MCMV is even
more difficult to explain. It cannot be expressed by
readthrough of the replicase because of intervening stop
codons. Expression as a separate small protein seems
impossible because no in-frame start codons exist in this
area. This region could be expressed as an extension of
M C M V p9 via a frameshift mechanism.
Given that expression of this region as protein is
questionable in all four viruses and that the observed
amino acid sequence does not appear to arise from
conservation at the nucleotide sequence level, it is
difficult to explain why the amino acid sequence is so
conserved. If this region is expressed in one or more of
these viruses it would most likely form part of a different
protein in each virus (see Fig. 9).
No obvious amino acid sequence similarity was found
between MNSV p7B and proteins in the databases
searched which might suggest its function.
I p32"7
UGA
Frameshift
p39 l
p60 (p99)
-~
Ft/##/#l#///#//////b-{
[
pl7 UAG
Ii
p50 (p72)
J
p6.7 p6
} ~
Fig. 9. Summary of the amino acid sequence similarities between
MNSV and several other plant viruses. Regions sharing amino acid
sequence similarity are indicated by similar shading. Dashed rectangles in TCV and MCMV indicate areas which exhibit amino acid
sequence similarity with MNSV but lack an obvious means of
expression. Arrows indicate termination codons thought to be read
through and in BYDV indicate a proposedframeshiftsite. The BYDV
p99 and p72 proteins would result from framesbift or readthrough
translation, respectively.
(vi) C o a t protein
The nucleotide sequence of the ORF encoding the coat
protein of MNSV, and its deduced amino acid sequence
have previously been described (Riviere et al., 1989).
Detailed comparisons showed that although the MNSV
coat protein shares a high degree of similarity with the
coat proteins of both the carmo- and the tombusviruses,
it resembles the coat proteins of tombusviruses more
closely.
nucleotide sequence level. Fig. 9 shows these regions are
arranged differently in each virus, and some viruses have
more plausible translation strategies than others for
expressing the conserved region.
In MNSV this region could be expressed as part o f p l 4
by suppression of the p7A amber terminator, or as a
separate protein, p7B. However, a separate subgenomic
R N A enabling expression of MNSV p7B has not been
detected. Similarly, this region in CarMV could be
expressed as the carboxy-terminal portion of p98 via
suppression of the two amber terminators of CarMV p27
and p86, respectively. Readthrough of both these
terminators has been shown to occur in vitro (Harbison et
al., 1985). This region of CarMV also contains two inframe start codons, either of which could initiate
translation of a small protein from the conserved region,
but a separate subgenomic R N A for expression of such a
protein has not been reported (Carrington & Morris,
1986). Similarly, expression of this region in TCV could
occur as a small, separate protein, but a corresponding
subgenomic R N A has not been reported (Carrington et
al., 1987).
Fig. 9 compares the genomic organizations of MNSV,
the carmoviruses CarMV and TCV, the tombusvirus
CNV, the unclassified virus M C M V and the luteovirus
BYDV. It also indicates regions of amino acid sequence
similarity between MNSV and these viruses. Although
MNSV shares some amino acid sequence similarity with
each of these viruses, its non-structural proteins share the
greatest sequence similarity with those of the carmoviruses. MNSV also closely resembles these viruses in the
number, size and genomic organization of its probable
proteins, as well as in its likely translation strategy. Based
on these molecular similarities, as well as on previously
reported physical, chemical and biological similarities to
various carmoviruses, we propose that MNSV be
classified as a member of the carmovirus group.
During the final stages of our preparing this manuscript, Habili & Symons (1989) published an exhaustive
analysis of the nucleic acid helicase and RNA-dependent
R N A polymerase motifs found in positive-sense R N A
plant viruses. They propose that these motifs serve as a
new basis for classifying these viruses into supergroups.
Their analysis supports the conclusion in this paper that
the carmoviruses MNSV, CarMV and TCV, the tombusvirus CNV, the unclassified virus M C M V and the
p22 (coat)
Conclusions
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MNSVgenom~sequence
luteovirus BYDV should be classified in a new virus
supergroup. In addition, the putative polymerases of the
dianthovirus red clover necrotic mosaic (RCNMV) and
the human pathogen hepatitis C virus (HCV) have
recently been reported to share extensive sequence
similarity with the polymerases of some of the viruses in
the proposed new supergroup (Xiong & Lommel, 1989;
Miller & Purcell, 1990). RCNMV may therefore become
the first multipartite member and HCV the first animal
virus member of this new supergroup. The addition of
these viruses would suggest that the members of this new
viral supergroup are as diverse in genome type and host
range as members of the established alpha- and
picornavirus supergroups.
The sequence reported has been entered into the
EMBL and GenBank databases under accession number
M29671.
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