Download Nucleotide sequence of the genomic RNA of pepper mild mottle

Journal o f General Virology (I991), 72, 2875-2884.
2875
Printed in Great Britain
Nucleotide sequence of the genomic RNA of pepper mild mottle virus, a
resistance-breaking tobamovirus in pepper
E. Alonso, I. Garcia-Luque, A. de la Cruz, B. Wicke, M. J. Avila-Rincbn, M. T. Serra,
C. Castresana and J. R. Diaz-Ruiz*
U.E.I. Fitopatologia, Centro de Investigaciones Biol6gicas, CSIC, Velhzquez, 144, 28006-Madrid, Spain
The entire genomic RNA of a Spanish isolate of pepper
mild mottle virus (PMMV-S), a resistance-breaking
virus in pepper, was cloned and sequenced and shown
to be similar to other tobamoviruses in its genomic
organization. It consisted of 6357 nucleotides (nt) and
contained four open reading frames (ORFs) which
encode a 126K protein and a readthrough 183K protein
(nt 70 to 4908), a 28K protein (nt 4909 to 5682) and a
17.5K coat protein (nt 5685 to 6158). This is the first
tobamovirus in which none of the ORFs overlap. Both
its nucleic acid and predicted protein sequences were
compared with the previously determined sequences of
other tobamoviruses. The variations and similarities
found and their relationship with the pathogenicity of
this virus are discussed.
Introduction
and ToMV (Wetter et al., 1984; Garcia-Luque et al.,
1990).
To develop an understanding of the mechanism(s)
involved in these biological properties, we have determined the nucleotide sequence of PMMV-S RNA, a
Spanish isolate of PMMV (Alonso et al., 1989). The
nucleotide sequences of its 5' and 3' non-coding regions
have previously been reported (Avila-Rinc6n et al.,
1989). In this paper, we present the cloning and complete
nucleotide sequence of PMMV-S and the analysis of its
deduced amino acid sequences.
Pepper mild mottle virus (PMMV) is a member of the
tobamovirus group of positive-strand RNA viruses. The
complete nucleotide sequence of three other tobamoviruses, tobacco mosaic virus (TMV) (Goelet et al., 1982),
tomato mosaic virus (ToMV) (Ohno et al., 1984) and
tobacco mild green mottle virus (TMGMV) (Solis &
Garcia-Arenal, 1990) have already been reported. The
tobamoviral RNA encodes four different proteins:
126K, 183K, 30K and 17.5K, in that order. The 126K
and 183K proteins are involved in the replication
processes (Young et al., 1987; Quadt & Jaspars, 1989),
the 30K protein participates in the cell-to-cell spread of
the virus (Deom et al., 1987; Meshi et al., 1987) and the
17-5K protein is the coat protein. The 126K and 183K
proteins are directly translated from the viral RNA,
whereas the 30K protein and coat protein are translated
from subgenomic RNAs (Palukaitis & Zaitlin, 1986).
PMMV is one of the most destructive pathogens of
protected pepper crops. It is found infecting pepper
cultivars with genetically incorporated resistance to
TMV and ToMV. The infection by this virus produces
important economic losses all over the world in crops
grown under plastic or glass (Wetter & Conti, 1988).
Additionally, PMMV is unable to infect tomato plants
and possesses a reduced capability to replicate and/or
accumulate in tobacco plants when compared to TMV
The nucleotide sequence data reported in this paper will appear in
the EMBL, GenBank and DDBJ nucleotide sequences databases under
the accession number M81413.
0001-0404 © 1991 SGM
Methods
Virus propagation, purification and RNA extraction. The origin of
PMMV-S has been reported previously (Alonso et al., 1989). The virus
was purified from Nicotiana clevelandii Gray plants as described
(Garcia-Luque et al., 1990). Virion RNA was prepared by conventional
SDS-phenol extraction after heating of the particles in 20 raM-sodium
phosphate buffer pH 7.0, 0.5,% SDS for 20 s at 100 °C.
eDNA synthesis and cloning, cDNA was prepared as described by
Gubler & Hoffman (1983), using a commercial cDNA synthesis kit
(Boehringer-Mannheim). PMMV-S RNA was 3' polyadenylated in
vitro with Escherichia coti poly(A) polymerase (0-25 unit/~tg of RNA) for
7 min at 37 °C, under the conditions recommended by the manufacturer (Pharmacia LKB Biotechnology), and first-strand cDNA
synthesis was primed with oligo(dT). Double-stranded cDNA was sizefractionated in 0.8% agarose gels, and the cDNA was eluted and
ligated into plasmid pUC18 digested with HincII. In another
experiment, EcoRI linkers were added to ds cDNA that had previously
been treated with EcoRI methylase and EcoRI-digested prior to being
size-fractionated in agarose gels. After elution, the cDNA was cloned
into EcoRI-digested pUC18. Plasmids were tested for the presence of
viral cDNA inserts by colony hybridization, using randomly primed
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2876
E. Alonso and others
riP-labelled cDNA to PMMV-S RNA. Other cDNA clones were
prepared using a primer complementary to nucleotides (nt) 5211 to
5228, in which a BamHI restriction site was created by addition of an
extra G at its 5' end. Ds cDNA was restricted with BamHI and Bcil,
size-fractionated in agarose gels, and the 1200 nt fragment was eluted
and ligated to pU C 18 digested with BamHI. Another set of clones was
obtained after priming cDNA synthesis with an oligonucleotide
complementary to nt 4021 to 4036. Ds cDNA was restricted with Sail,
fractionated in a 6% polyacrylamide gel, and the 600 nt fragment was
eluted and cloned into the SalI-Smal sites of pUC 18. All recombinant
DNA techniques were as described by Maniatis et al. (1982), using E.
coli strains JM83 and DH5~.
Nucleotide sequence determination and analysis. The nucleotide
sequence ofcDNA clones was determined by the chemical degradation
procedure (Maxam & Gilbert, 1980). Subclones used for sequencing
were generated by deletions with nuclease Bal 31 or restriction enzyme
digestion. Sequences were analysed using the DNASTAR computer
programs.
Results and Discussion
Sequence determination and terminal non-coding regions
Fig. 1 shows the strategy used to determine the sequence
of the genomic RNA of PMMV-S from a set of
overlapping cDNA clones. They contain sequences
representing all but the first 34 nucleotides located at the
5' end of PMMV-S RNA. Most of the sequence was
obtained from at least two independent cDNA clones.
The nucleotides of the 5' and 3' non-coding regions of
PMMV-S RNA have previously been sequenced directly
on the viral RNA (Avila-Rinc6n et al., 1989). As with
other tobamoviruses, PMMV-S possesses a 69 nt leader
sequence, devoid of G residues, termed the ~) fragment
(Richards et al., 1977; Avila-Rinc6n et al., 1989). Its 3'
non-coding region is 199 nt long. It was previously
proposed that some structural features in the tRNA-like
conformation of PMMV-S RNA such as two unpaired
nucleotides connecting the aminoacyl and anticodon
arms could be related to its lower replicability observed
in tobacco plants (Avila-Rinc6n et al., 1989) as described
for certain chimeric tobamoviruses (Ishikawa et al.,
1988).
The determined nucleotide sequence of the cDNA
clones coincides with that of the RNA except for a t;ingle
base transition at position 6181 which would change the
C/G pair (6197/6181) situated at the beginning of the V
stem in the proposed secondary structure (Avila-Rinc6n
et al., 1989) to a C/A pair. This nucleotide substilution
was present in three of four sequenced cDNA clones. In
other parts of the genome no sequence heterogeneity was
found in the clones analysed. The only nucleotide
difference was found in clone 4, in which the insertion of
a T between nt 5385 and 5386 could lead to a truncated
protein.
Nucleotide sequence of P M M V - S and comparison with
other tobamoviruses
Fig. 2 shows the sequence of PMMV-S RNA and that of
its deduced amino acid. The genome of PMMV-S is 6357
nt long. It shares an overall sequence identity of 69.4%
with the RNA of ToMV (Ohno et al., 1984), 68.5% with
that of TMV (Goelet et al., 1982) and 64% with
TMGMV (Solis & Garcia-Arenal, 1990), the other
members of the tobamovirus group whose entire RNA
sequences are already known. As shown in Table 1, also,
PMMV-S shares a higher degree of amino acid sequence
identity with ToMV than with TMV and TMGMV.
Organization of the 126K/183K gene sequence
The first open reading frame (ORF) of PMMV-S RNA
begins at nt 70, in the first AUG encountered from the 5'
end, and extends to nt 3423, encoding a protein of 1117
amino acids (126K), with a calculated Mr of 126 304. The
readthrough of the amber codon (UAG), possibly by
insertion of tyrosine (Beier et al., 1984), results in a 183K
protein which terminates at position 4908. It is composed
of 1612 amino acids with a predicted Mr of 183340. The
nucleotide and amino acid sequences in the readthrough
part of the 183K protein (nt 3421 to 4908, amino acids
1118 to 1612) (Fig. 2 and 3) are the most highlyconserved in all the genome, with only 14 and 15 nonconservative amino acid substitutions with respect to the
corresponding proteins of ToMV and TMV,
respectively.
The 126K and 183K proteins are thought to be
involved in viral replication because they have been
detected in partially purified preparations of the viral
polymerase complex and because they contain several
sequence motifs which are conserved in proteins known
to act in replicative processes of plant and animal viruses
(Young et al., 1987; Goldbach & Wellink, 1988; Strauss
& Strauss, 1988; Quadt & Jaspars, 1989). The alignment
of the 126K/183K proteins of PMMV-S with those from
the more closely related tobamoviruses (ToMV and
TMV) shows that the sequence is well conserved along all
the protein (Fig. 3), except for three stretches (amino
acids 155 to 191,623 to 669 and 768 to 791) in which nonconservative substitutions as well as deletions and
insertions occur. Other regions of weaker amino acid
sequence identity correspond to positions 382 to 388,537
to 555 and 991 to 1001 (Fig. 3).
Based upon the existence of conserved motifs between
the tobamoviral 126K protein and those from other
RNA viruses, two functional domains have been
defined. The first one, in the amino part of the protein,
has homology with the nsP1 protein of alphaviruses and
with the amino part of other proteins implicated in the
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PMMV-S
126K
5r
EH
A
II
I
I
NsBABSa K
f'¢ ff (
,,
I
20'00
1000
AE A
It I
183K
3oK
B
K
H .Bs N
II
l
I
,1If
I
I'll
3000
III
d
D j
IV-we-1
Sc
f
,
5000
4000
2877
sequence
P
BS BcNsBc
EC-8
~
RNA
r3,
6000
85
t
75
174
H-92
U-11;17
BKB-8;3
Fig. 1. Genomic organization, partial restriction map and sequencing strategy for PMMV-S cDNA. Open boxes drawn approximately
to scale represent the coding regions for the 126K, 183K, 30K and coat protein (CP) gene products. Arrows represent the strategy
followed to determine the sequence of the overlapping cDNA clones used (EC-8, 75, H-92, U-I 1, U-17, BKB-8, BKB-3, 85, 4 and 174).
Abbreviations for restriction sites are : A, AvaI ; B, Bgll I ; Bc, BclI ; E, EcoR V ; H, HindlII ; K, KpnI ; N, NsiI ; S, SalI; Sa, SacI ; Sc, SaclI.
Table 1. P e r c e n t a g e s o f s e q u e n c e identity b e t w e e n the P M M V - S
other t o b a m o v i r u s e s
g e n e s a n d those f r o m
Gene
126K
183Kt
30K
CP
Virus*
N:~
A~
N
A
N
A
N
A
ToMV
TMV
TMGMV a
TMGMVb
CGMMV-W
SHMV
68.8
67.9
62.4
-§
.
.
74.7
73.3
62.0
-
73-7
73.0
68.4
.
.
82.0
80.0
73-2
-
65.8
63.4
63-1
64.7
46.0
38.5
64-5
67.4
61.5
65.0
33-5
26-7
67.5
65.6
65.6
65.6
48.1
46-4
73.9
72.0
70.1
70.1
36.5
40.8
.
.
.
.
* Data are from Ohno et al. (1984), Goelet et at. (1982), Meshi et at. (1981, 1982), Saito et aL (1988) and
Meshi et al. (1983), for ToMV, TMV, SHMV (30K and coat protein), CGMMV 30K and CGMMV-W CP,
respectively. Data reported for two isolates of TMGMV (TMGMVa and TMGMVb) are from Solis &
Garcia-Arenal (1990) and Nejidat et al. (1991), respectively.
t The sequences analysed correspond only to the readthrough part of the 183K protein.
:~N and A, nucleotide and amino acid sequence homologies, respectively.
§ No sequence data are available for comparison.
replication of R N A viruses with a m o n o p a r t i t e or
d i v i d e d g e n o m e (Ahlquist e t al., 1985). I n this region,
R o z a n o v e t al. (1990) have identified two conserved
sequence motifs defined by the presence of an i n v a r i a n t
His in the first m o t i f a n d the sequence AspoX-X-Arg in
the second one, that are located at a m i n o acid positions
76 to 81 a n d 134 to 138 in the 126K/183K p r o t e i n of
T M V (Fig. 3), respectively. By analogy with the nsP1
p r o t e i n of S i n d b i s virus (Mi e t al., 1989), this d o m a i n
m a y be responsible for the m e t h y l t r a n s f e r a s e activity
necessary for the cap f o r m a t i o n of the g e n o m i c a n d
s u b g e n o m i c R N A s . O f the two a m i n o acid sequence
motifs described by R o z a n o v e t al. (1990), the predicted
126K/183K p r o t e i n from P M M V - S possesses both,
except for a c o n s e r v a t i v e s u b s t i t u t i o n lie to Val at
position 135 (Fig. 3). This d o m a i n is the best conserved
with respect to T M G M V .
T h e second f u n c t i o n a l d o m a i n in the 126K/183K
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2878
E, Alonso and others
~12~183K
C~ CAAAATAC~CTAC~T GGC~ACACAC~C~GCTACC~C GCCGCA~AGC~GTACTCTCC~
M A Y T Q Q A T N A A L A S T L R
•
1 GT~.I.~.I.I.I
CA C ~ C ~ C ~ C ~ C A C A A A C ~ C ~ C ~ C A ~ A
•
121 GG~T~CCCC~GGTG~C~TC~GCT~TCGGA~CTGTACG~ TCAGCGGTCG~c~TGC~TGcACATGACCGCAGGCCC~GG~I'I'I'I~GGTC~T~GC~G
G N N P L V N D L A N R R L Y E S A V E Q C N A H D R R P K V N F L R S I S E E
241 CA~CGC~ATCGC~CT~GGCCTACCCTGAG~CC~TCACG~CTAC~CACGCAG~CGCTGTGCACAGTCTCGCAGGTGGAC~CGGTC~GG~CTAG~TAC~GATGATG
QTL
A T K A Y P E F Q
T F Y N T Q N A V H S L A G G L R S L E L E Y L M M
361 CAGATCCCCTACGG~C~CGACATATGATATCGGGGG~TTTTGCTGCTCACATG~GGTCGTGACTACG~CA~GCTGCATGCCT~CATG~C~ACGT~CGT~TGCGT
Q I P Y G S T T Y D I G G
F A A H M F K G B D Y V H C C M P N M D L R D V M R
481 CAC~TGCTCAAAAGGATAGCA~G~CTGTACC~C~GC~GCGCAAAAGAAAAAGGT~TACCGCCATATCAAAAGCCATGC~GAT~TACACGGAC~TCCGC~T~A
H N A Q K D S I E L Y L S K L A Q K K K V I P P Y Q K P C F
D K Y T D D P Q S V
•
°
601 GTGTGCTCG~CC.~-~-~.CAGCACTGCG~GG~G~CGcACTG~ACGGAT~GTATACGCTGT~GC~GCACAG~ATAC~CA~CcAGCAGATG~GGGG~G~C~TG
V C S K P F Q H C E G V S H C T D K V Y A V A
H S L Y D I P A D
F G A A L L
721 AGGAG~TG~CATGTCTGCTATGCTGCC~CCACTTTTCTGAG~TC~CTTTTAG~GA~CGTATGTCAGTC~GACGACATAGGCGC~C~CTCGA~GAGGGCGATATG~G
R R N V H V C Y A A F H F S E N L L L E D S Y V S L D D I G A F F
R E G D M L
841 ~C.F~-~CTTTTGTAGCAGAGAGTAC~ATACTCA~CCTATAGT~TGTGC~GTATGTGTGT~GAC~AC~CCCCGC~CTAGTAGAG~GTGTACATG~GGAG~
NFS
V A E S T L N Y T H S y S N V L K Y V
K T Y F P A S S R
v Y M K E F
961 ~GGT~CTAGGGT~TAC~GGTTTTGT~G~TC~GG~AGATACC~TGTACTATATAGAGGTGTATACCACAGAGGTGTAGAC~GGAGCAA~ACAGTGC~TGG~AT
L V T R V N T W F C K F S R L D T F V L Y R G V Y H R G V D K E Q F Y S A M E D
1081 GC~GGCA~ACAAAAAGAC~GGC~TGATG~TAGCG~G~TCCTC~AGAGGA~CATcGTCTG~A~GG~CC~G~TATGGT~TAGTACC~G~C
A W H Y K K T L A M M N S E R I L L E D S S S V N Y W F P K M K D M V I V P L F
1201 GACGTATC~ACAG~CGAGGGGAAAAGG~AGC~G~GGAGGTCATGGTCAGC~GGAC~CG~ATACTGTGC~TCATA~CGCACATAcCAGTCG~GCGc~AC~AC
D V S L Q N E G K R L A R K E V M V S K D F V Y T V L N H I R T Y Q S K A L T Y
1321 GC~TGTA~ATCG~CG~GAGTC~AT~GATC~GAGTGAT~TC~TGGGGTGACTGCGcGCTCAGAGTGGGATGTGGAT~GGC~G~GCAGTCCCTGTC~TGAc~zT~.~C
A N V L S F V E S I R S R V I I N G V T A R S E W D V D K A
L Q S L S M T F F
1441
~GCAGACc~GGCCATGCTC~GGATGACcTCGTGGTTCAG~CC~GTGCATTCC~TCGCTCACTG~TATGTCTGGGATGAGA~ACTGCTGCTTTT~c~G~i~i~i.
L Q T K L A M L K D D L V V Q K F Q V H S K S L T E Y V W D E I T A A F H N C F
1561 C C T A ~ T C ~ G G A G A G G ~ C ~ G ~ C T C A T ~ C T G ~ T C G G A A A A G G C T C ~ G ~ G T A C C T G A ~ G T A T G T ~ C ~ C C A C G A T A ~ G G ~ G G A G T A C ~ G
P T I K E R L I N K K L I T V S E K A L E I K V P D L Y V T F H D R L V K E Y K
1681 Tc~CGGTGG~TGCcGGTACTGGACG~AAAAAGAGC~GG~G~GCAG~GTGATGTAC~TGCT~GTCAG~TCTC~C~GAcAGTGAC~G~GATG~TG~
SSV
M P V L D V K K S L E E A E V M Y N A L S E I S I L K D S
K F D V D V
1801
.~.~.~WCCCG~TGTGT~TACA~AGGCGTAGATcCA~GGTGGcAGC~GGT~TGGTAGCTGTGG~C~TGAGAGTGG~GACC~cG~GA~GGcCTACC~GCAAAT
F S R M C N T L G V D P L V A A K V M V A V V
N E S G L T L T F
R P T E A N
1921 GTCGCAC~GCA~GC~CCGAC~ACATC~GGAGG~GG~cG~G~GA~GTGTCGTCAGACGTAG~TGAGTCCTC~Tc~GG~GTGG~CGAAAATCA~GA~CTATG
V A L A L Q P T I T S K E E G S L K I V S S D V G E S S I K E V V R K S E I S M
°
.
.
.
•
2041 ~GGTCT~CAGGC~CACAGTGTCCGATGAG~CC~G~GTACAG~TCGAGTCG~GCAGCAG~CATATGGTATCCACAGAGACGA~ATCCGT~CAGATGCATGCGATG
L G L T G N T V S D E F Q R S T E I E S L Q Q F H M V S T E T I I R K Q M H A M
2161
2281
GTGTATACTGGTCCGCTAAAAG~C~c~Gc~G~cTA~TAGAcAGccTGGTAGCCTcGCTcTcTGcTGcGGTATc~ccTG~GAT~Tc~GAcACAGCTGCTATA~T
CTcGA~C~GGAAAAA~GGAGTCTACGACGTGTGCC~G~TGGTTGGTG~ccTcTATcA~AGGACATGC~GGGGTGTGGTGATGGACT~GACTAT~GTGC~G~
L E T K E K F G V Y D V C L K K W L V K P L S K G H A W G V V M D S D Y K C F V
V Y T G P L K V Q Q C K N Y L D S L V A S L S A A V S N L K K I I K D T A A I D
2401 GCGC~CTCACATAC~TGGC~G~CA~GTGTGCGGAGAGACATGGCGTAGAGTCGCAGTGAGcTCCG~TC~GGTGTA~CAGATATGGGG~GAT~GAGcTATACGCTcTGTG
A L L T Y D G E N I V C G E T W R R V A V S S E S L V Y S D M G K I R A I R S V
2521 C~GACGGTG~CCCCATAT~GCAGTGC~GG~ACAC~GTTGATGGTG~CCTGG~GCGG~GAC~GGAGA~C~CGAGGGTC~C~GACG~GATCTAG~CTG
L K D G E P H I S S A K V T L V D G V P G C G K T K E I L S R V N F D E D L V L
•
.
.
•
•
2641 GTACCAGGAAAACAGGCTGCTG~TGAT~G~G~GGGC~CAG~TGG~TCGTGG~GACC~GGAG~TGT~GGACGGTAGACTC~C~TG~ACGGTC~GGT
V P G K Q A A E M I R R R A N S S G L I V A T K E N V R T V D S F L M N Y G R G
2761 CCGTGCC~TACAAAAGGCTG~CTGGAT~GGTCT~TG~ACAC C~TGG~GTG~TTTTCTGG~GGCATGTCTCTATGCTCCGAGGC'I"F~G~ATG~cCCAG~G
p C Q Y K R L F L D E G L M L H P G C V N F L V G M S L C S E A F V Y G D T Q Q
2881 A~CC~ACATC~CA~G~G~CTTTTCCCTATCCT~GCA~GAGTC~CTCGAGGTCGATGCTG~G~CTCGCAG~C~CG~GCGGTGTC~GCT~TATCACC~C~C
IPY
N R V A T F P Y P
H L S Q L E V D A V E T R R T T L R C P A D I T F F
3001 ~TCAG~ACG~GGGC~G~ATGTGCACATC~GTG~ACACGCTCGGTGTCACACGAGGTCATCC~GGTGCAGCGGT~T~TCCAGTGTCT~CCAC~GGG~G
L N Q K Y E G Q V M C T S
V T R S V S H E V
Q G A A V M N P V
K P L K G K
3121 GTGA~ACA~CACTCAGT~C~GTCA~GCTGCTCTCGAGGGG~ACG~GATGTGCATACCG~CATGAGGTGC~GGGG~CG~G~GACGTCTCAcTAGT~GG~CG
VIT
T Q S D K S L L L
R G Y E D V H T V
E V Q G E T F E D V S L V R L T
3241 CC~CACCCGTGGG~T~CAAAGCAGAGTCCGCACCTG~GGTCTCA~GTCTAGGCATAC~GGTCGATC~TA~ACACAG~GTGCTAGATGCAGTCG~GTG~A~
P T P V G I I S K Q S P H L L V S L S R H T R
I K Y Y T V V L D A V V S V L R
3361 ~T~G~GTGTGT~GTAG~ACCTG~AGATATGTAC~G~GATGTGTCGACTC~TAG~ACAGATAG~TCGGTGTAC~GGTG~CC.~.~.~.~.CGTCGCAG~c~AAAA
DLE
V S S Y L L D M Y K V D V S T Q * Q L
I E S V Y K G V N L F V A A P K
3481 ACAGGAGATG~CTGACATGC~TA~A~ACGAC~GTGT~GCCGGG~CAGTACTATACTC~TGAGTATGATGCTGT~CTATGcAAATACG~AG~TAG~G~TGTC~G
T G D V S D M Q Y Y Y D K C L P G N S T I L N E Y D A V T M Q I R E N S L N V K
•
•
•
.
.
.
3601 GA~GT~G~GGATATGTCG~TCGGTGCCTC~CCGAGAG~TCTGAGACGACA~G~CCTGTGATCAGGACTGCTGCTGAAAAACCTCGAAAACCTG~G~GGAAAA~G
D C V L D M S K S V P L P R E S E T T L K P V
R T A A E K P R K P G L L E N L
•
•
.
.
•
3721 GTCGCGATGATCAAAAG~C~CTCTCCCG~AGTAGGGGTTG~GACATCG~GACACCGC~CTCTAGTAGTAGAT~G~GATGCATAC~GAAAAGAAA
V A M I K R N F N S P E L V G V V D I E D T A S L V V D K F F D A Y L I K E K K
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P M M V - S R N A sequence
3 8 4 1
2879
AAACCAAAAAATATA~CT~TG~TTTC~GGGCGAG~GGAAAGATGGATCGAAAAG~GAG~GT~C~GGCCA~GGCTGATTTTGACTTTA~GA~ACC~GCCG~GAT
K P K N I P L L S R A S L E R W I E K Q E K S T I G Q L A D F D F
D L P A V D
3961
~TACAGGCACATGATC~GCAGCAGC~GAAACAG~G~GGAT~AGTA~AAACTG~TACCcGGC~G~AAACTA~GTGTATCATAGC~GAAAATC~TGCGC.~-~-~.~.~.GGT
Q Y R H M I K Q Q P K Q R L D L S I Q T E Y P A L Q T I V Y H S K K I N A L F G
4081
CCTGTA'I'I'I'I'CA~6~CAGCTGCTA~GAC~GACAG~6~GA~CATGTTTTATAC~GG
AAAACGCCTACACAGATCG~G~'I'I~£'I~CTCA~TCTGGACTCT~T
PVF
E L T R Q L L E T
D S S R F M F Y T R K T P T Q I E E F F S D L D S N
.
.
°
4201 G~CCTATGGACATA~A~GCTAGACA~CC~GTATGAcAAATCACAG~CG~CA~GTGCAGTCGAGTATGAGA~GGAAAAGG~AGGC~AGACGA~C~GGCTG~
V P M D I L E L D I S K Y
K S Q N E F H C A V E Y E I W K R L G L D D F L A E
4321
G~GGAAACACGGGCAT~GG~GAC~CG~GAAAGACTACACAGCCGG~TAAAAACGTG~GTGGTA~CAGAGGAAAAGCGGTGATGTCAC~ACA~GGAAACACGATCA~
V W K H G H R K T T L K D
T A G I K T C L W
Q R K S G D V T T
I G N T I I
4441 A~GCTGCATGTCTGTCCTCTATGCTACCGATGGAGAGA~G~AAAGGTGCCTTTTGTGGTGATGATAGTATA~ATAC~CCAAAGGGCACTGA~CCCC~TA~C~CAGGGC
IAA
L S S M L P M E R
I K G A F C G D D
I L Y F P K G T D
P D I Q Q G
4561
GCAAACC~CTCTGG~TTT~G~GCC~G~G~AGG~GAGATATGG~ACTTTTGCGGTAGGTACAT~CACCATGAcAGAGGCTGTA~GTATA~ATGAcC6TCTAAAA~G
ANL
W N F E A K L F R K R Y G Y F C G R Y
I H H D R G C I V
y D p L K L
4681 A T C T C G A A A C T C G G T G C A A A A C A C A T C ~ G ~ T A G A G ~ C A ~ A G A G G ~ A G G A C C T C T C ~ G T G A T G ~ G C T G G G T C G ~ G ~ C ~ G T G C G T A C T A T A ~ C A ~ C G A C
ISK
G A K H I K N R E H L E E F R T S L C
V A G S L N N C A Y Y T H L N D
~3OK
4801 GCTGTCGGTGAGG~A~GACCGCACCTC~GG~CG~T~TATAGAGCA~AGTT~GTAC~GTGTGATA~
AGG~A~CAAACA~Gr~2"~GGAGT~TGGCG~AGTA
AVG
V I K T A P L G S F V Y R A L V K Y L
D K R L F Q T L F L E * M A L V
4921 G T C ~ G G A C G A C G ~ G A ~ C T G A G ~ C A T C ~ G T C T G C C G C T G A G ~ C ~ C T G C T G ~ A T G A C ~ C G G T C ~ G A c G G T A C G ~ T T T C G A A A G ~ G A C A A A G T G A ~ G C A
VKD
V K I S E F I N L S A A E K F L P A V M T S V K T V R I S K V D K V I A
5041 ATGGAAAACGA~CG~ATCCGATGTG~GC~AAAGGTGTAAAGCTTGTT~GGATGG~ATGTGTG~AGcAGGG~AG~GTGTCCGGGGAGTGG~CCTAcCCGAC~CTGC
M E N D S L S D V N L L K G V K L V K D G Y V C L A G L V V S G E W N L P D N C
5161 AGAGGTGGAGT~GCG~GTTTGG~GAT~GAG~TGCAAAGAGATGACG~GC~CAC~GGATC~ATAG~CCAGTGCAGCT~GAAACGATTTGC~CAAA~GATCCCG~T
R G G V S V C L V D K R M Q R D D E A T L G S Y R T S A A K K R F A F K L I P N
5281
TATAGCA~ACTACCGCCGATGCTGAGAGAAAAGTTTGGC~GTTTTAG~TA~A~GGTG~GCCATGGAAAAGGGTTTCTGTCCT~ATCTTTGGAGTTTGTCTCAG~GTA~
Y S I T T A D A E R K V W Q V L V N I R G V A M E K G F C P L S L E F V S V C I
5401 GTACACAAATCC~TATAAAA~AGGC~GAGAGAGAAAATTACTAGTGTGTCAG~G~GGACCCG~G~C~ACAG~GCAGTCG~GATGAG~CATCG~TCAG~CC~TGGCT
V H K S N I K L G L R E K
T S V S E G G P V E L T E A V V D E F I E S V P M A
5521
5641
GACA~ACGTAAATTTCGC~TC~TCT~GAAAGG~GT~T~GTATGTAGGT~GAGAAATGAT~T~GGG~G~T~GG~GGG~GCTGTTTGAT~GG~AG~GGG
D R L R K F R N Q S K K G S N K Y V G K R N D N K G L N K E G K L F D K V R I G
~CPCAGAACTCGGAGTCATCGGACGCC~GTc~C~CGTT~F~CTATGGC~ACACAG~CCAGTGCC~TC~AGTGTA~AGG~CTGTATGGGCTGATCCA~A~G~Ac~
Q N S E S S D A E S S S F *
M A Y T V
S A N Q L V Y L G
V W A D P L E L Q A
5761
ATCTGTGTAC~CGGCG~AGGC~TCAG~CAAACAC~CAGGCTAG~CTACGG~C~CAG~G~CTCTGATGTGTGG~GACTA~CCGACCGCTACAG~AGA~CCTGcTA
L C T S A L G N Q F Q T Q Q A R T T V G Q Q F S D V W K T
P T A T V R F P A
5881
CTGGTTTCAAAGTTTTCCGATAT~TGC~GTGCTAGA~CTCTAGTGTCGGCAC~CTCGGAGCC~GATACTAG~CAGGAT~TAG~G~GAAAATCCGCAAAATCCTAC~CTG
G F K V F R Y N A V L D S L V S A L L G A F D T R N R I I E V E N P Q N P T T
6001 CC~GACGC~TGCGAC~GGCGGGTAGACGATGCGACGGTGGCC~AGGGCCAGTAT~GT~CCTCATG~TGAG~AG~CGTGGCACGGG~TGTAC~TC~GCTCTG~CG
E T L D A T R R V D D A T V A I R A S I S N L M N E L V R G T G M Y N Q A L F
6121
AGAGCGC~GTGGACTCACCTGGGCTAC~TC~AAACATGATGGCATAAAT~G~G~CG~A~AAACGTCCGTGG~GAGTACGAT~CTCGTA
S A S G L T W A T T P *
GTQ'I'I~I'I'I'CCCTCCAC~
6241
ATCGAAGGG~GTCG~GGGATGG~CGC~A~ATACATGTGTGACGTGTA~GCG~CGACGT~A~i.i.f~£CAGGGG~CG~TCC~C~CCG~CGCGGGTAGCGG~CCA~H
Fig. 2. The entire nucleotide sequence of the PMMV-S genome, shown as DNA, and deduced amino acid sequences of the ORFs.
Arrows indicate initiation codons. Terminationcodons and the amber readthroughcodon are indicated by an asterisk. The predicted
position ~r the origin of assembly is underlined.
protein has been mapped to amino acids 833 to 1086, and
is known as the helicase domain (Hodgman, 1988;
Gorbalenya & Koonin, 1989; Habili & Symons, 1989). It
is implicated in nucleic acid unwinding, and possibly in
other processes such as recombination and transcription.
Of the six conserved motifs common to Sindbis-like
viruses in which the tobamovirus group has been
classified (Goldbach & Wellink, 1988), the NTP-binding
activity has been ascribed to domains I and II (amino
acids 833 to 850 and 902 to 913, respectively). The
domain I motif is strictly conserved in the 126K protein
of PMMV-S with respect to those of ToMV and TMV,
but differs from that of T M G M V in which the consensus
Thr at position 842 has been substituted by Tyr, and the
following Glu-Ile-Leu-Ser sequence has evolved to GlyAsp-Phe-Glu, the first substitution being of the semi-
conservative type and the other three of the nonconservative type. It is therefore possible that the amino
acid exchanges occurring in this domain in T M G M V
could result in its lower replicability, in comparison with
TMV or ToMV (Wetter, 1986). In the domain II to V
motifs (amino acid positions 902 to 913,930 to 940, 966 to
974, 1038 to 1055 and 1070 to 1085, respectively), all of
the amino acid substitutions that occur in the PMMV-S
protein with respect to the ToMV and TMV ones are of
the conservative type: Ile to Leu (position 906), Tyr to
Phe (position 930), ¥al to lle (position 974), Tyr to Phe
(position 1048), Ala to Glu (position 1049), Val to Leu
(position 1070), Lys to Arg (position 1079) and Leu to Ile
(position 1081). The same occurs with the amino acid
substitutions in T M G M V (Fig. 3).
The carboxyl end of the 183K protein results from
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2880
E. Alonso and others
•
PMMV-S
ToMV
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.
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A V V S N E S G L T L T F E R P T E A N V A L A L Q P T IT S K E E G S L K IVS SDVGE S S IREVVRKSE ISML GLTGNTVSDEFQRSTE IE SLQQFHMVSTET I I R K Q M H A M
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V Y T G P L K V Q Q C K N Y L D S L V A S L SAAVSNLKKI IKDTAAI DLETKEKF GVYDVCLKKWLVKPLSKGHAWGVVMD SDYKCFVALLTYD GEN IVCGETWRRVA
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V S S E S L V Y S D M G K I R A I R S V L K 6 G E P H I S SAKVT~ VDGVPGCGKTKE IL ~ V N F D E D L V L V P GKQAAEM IRRRANSSGL I V A T K E N V R T V D S F L M N Y G R G
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L K P V I R T A A E K P R K P G L L E N L V A M I K R N F N S P E L V G V V D IEDTASLVVDKFFDAYL IKEKKKP -KNIP-LLSRASLERWIEKQEKSTI GQLADFDF IDLP 1294
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Fig. 3. Alignment of the deduced amino acid sequences of the 126/183K proteins from different tobamoviruses. Source of amino acid
sequences as in Table 1. Only amino acid exchanges are indicated. Gaps are indicated by ( - ) . Numbering corresponds to that from
PMMV-S. Numbers in parentheses indicate the total length of each protein. The sequence motifs defined by Gorbalenya & Koonin
(1989), Poch et al. (1989), and Rozanov et al. (1990) are boxed.
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PMMV-S
readthrough of the UAG stop codon at nt 3421 to 3423.
The suppression of a termination codon is a widespread
phenomenon among animal and plant RNA viruses, and
is related to the regulation of the expression of the
different components of the viral RNA polymerase
(Ishikawa et al., 1986; Strauss et al., 1988). The fact that
the surrounding nucleotide sequences (ATAGCAATTACAG) at positions 3420 to 3432 (Fig. 2) are strictly
conserved in all the tobamoviruses reveals the functional
importance of this particular region in their genomes, as
it also occurs in alphaviruses (Strauss et al., 1988). The
so-called polymerase module is found in the carboxy
portion of the protein, in which four domains (A to D)
have been defined (Poch et al., 1989). This module is
common to all of the DNA- and RNA-dependent RNA
polymerases, and it is known to be involved in the
elongation of pre-existing chains (Quadt & Jaspars,
1989). The Gly-Asp-Asp motif first identified by Kamer
& Argos (1984) is found in the C domain surrounded by
hydrophobic residues. The alignment of the PMMV-S
183K protein with those from other tobamoviruses (Fig.
3) also shows that all the amino acid substitutions in
these domains are of the conservative type.
The only non-conservative differences between
TMGMV and PMMV-S occur in the D domain, Cys to
Gly (position 1496) and Asn to Leu (position 1500). The
higher sequence variability in this readthrough part of
the 183K protein is found in the region located at amino
acids 1250 to 1291. There is also lower amino acid
sequence identity in both the N- and C-terminal
segments (Fig. 3), a feature common to other RNA
viruses (Haseloff et al., 1984; Allison et al., 1989).
It remains to be determined whether the attenuated
biological behaviour of PMMV-S in tobacco, in comparison with TMV and ToMV, could be ascribed to
regions of maximum sequence heterogeneity or to
segments of the non-highly conserved sequence of the
126K/183K protein, as previously described for the
attenuated L11A strain ofToMV (Nishiguchi et al., 1985)
in which the amino acid substitutions responsible for this
characteristic have been mapped in regions, not highly
conserved, of the 126K protein (amino acid residues 348,
759 and 894). It is also unknown whether the ability of
PMMV-S to break the resistance against tobamoviruses
conferred by the E 1 and L 2 genes in pepper (Boukema et
al., 1980; Garcia-Luque et al., 1990) is due to any of the
amino acid changes which take place in this protein, as
described for the Ltal strain of ToMV, in which two
amino acid substitutions (Glu to Gin and Tyr to His) at
positions 979 and 984, respectively (980 and 985 in the
PMMV-S protein) have been identified as those responsible for the ability of this strain to break the Tm-l
resistance gene in tomato (Meshi et al., 1988). However,
since none of the resistance conferred by the L 1 and L z
genes in pepper is expressed in protoplasts (unpublished
R N A sequence
2881
results), it is plausible to consider that other regions of
the PMMV-S genome may be implicated also.
Organization o f the 3 0 K protein gene
The third ORF of PMMV-S encodes the 30K protein
(Fig. 2). Translation initiates at nt 4909 and terminates at
nt 5682; thus the coding region for this protein overlaps
with neither the coding region for the 183K protein nor
with that for the coat protein. Its putative translation
product is 257 amino acids long with a calculated Mr of
28 347. Therefore, this is the first tobamovirus in which
none of the reading frames overlap, since in ToMV,
TMV, cucumber green mottle mosaic virus (CGMMV)
and TMGMV the 5' end of the genes encoding the 30K
protein overlap with the 3' end of those encoding the
183K proteins (Goelet et al., 1982; Ohno et al., 1984;
Saito et al., 1988; Solis & Garcia-Arenal, 1990), and in
sunn-hemp mosaic virus (SHMV) as well as in CGMMV
(Meshi et al., 1982; Saito et al., 1988) their 30K-coding
regions overlap at the 3' end with their coat protein
genes.
The 30K proteins of tobamoviruses are responsible for
cell-to-cell spread of the viral infection (Deom et al.,
1987; Meshi et al., 1987), by modifying the plasmodesmata (Wolf et al., 1989). Although the exact mechanism
of action is unknown (Hull, 1989), a domain responsible
for binding to nucleic acids which maps between amino
acid positions 65 and 87 has been defined (Citovsky et
al., 1990). As with the consensus sequences in the
126K/183K proteins, the amino acid substitutions that
take place in this region of the PMMV-S 30K protein are
of the conservative type with respect to TMV, ToMV
and TMGMV, except for the semi-conservative exchange Ala to Val (position 70) in the ToMV 30K protein
(Fig. 4).
As in other tobamoviruses (Ohno et al., 1984; Solis &
Garcia-Arenal, 1990) the PMMV-S 30K protein is
encoded in the least conserved part of the entire genome,
both at the nucleotide and amino acid levels (Table 1). Its
alignment with those from the most closely related
tobamoviruses (Fig. 4) shows that the PMMV-S 30K
protein shares a higher degree of amino acid sequence
identity with that of TMV than with those of ToMV or
TMGMV, in contrast to other proteins encoded by
PMMV-S. It contains two well conserved regions located
at amino acid positions 46 to 125 and 151 to 204. In the
first one, only one non-conservative amino acid substitution takes place in the PMMV-S 30K protein with
respect to ToMV and TMV (Arg to Tyr, both at position
109) and TMGMV (Ala to Cys at position 113). In the
second well-conserved region, all of the amino acid
changes among the TMV, ToMV and PMMV-S 30K
proteins are of the conservative type, but it is less
conserved compared to TMGMV, with three non-
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2882
E. Alonso and others
i00
PMMV-S
ToMV
TMV
TMGMV a
TMGMV b
PMMV-S
ToMV
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E . . . . . . . . . . V P E N - K E M - - - V G N N NN ..... K K I - - - N S g K GF I - ~ E I E D N V S D D E I S T I
E R F R K T K K G K K R K K E K - K R V - - - V G N S NN ..... K K I - - - N S G ~ GL V - E ~ I E D N V S D D E I S T J
S E
E
K
E
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~
M NI
M D[
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(264)
(268)
(256)
(266)
Fig. 4. Alignment of the amino acid sequence of the 30K protein of PMMV-S with those from the most related tobamoviruses.
Numbering, symbolsand sourceof the amino acid sequencedata as in Fig. 3 and Table 1. The amino acid sequencedomainsdefinedby
Saito et al. (1988) are boxed.
conservative substitutions (Leu to Lys, Thr to Leu and
Ala to Lys at positions 172, 179 and 192, respectively)
(Fig. 4). Several amino acid changes in the central
segment of the tobamoviral 30K proteins have been
identified in temperature-sensitive mutants defective in
cell-to-cell movement (Ohno et al., 1983; Zimmern &
Hunter, 1983) as well as in the Ltbl strain of ToMV,
known to overcome the resistance conferred by the Tm-2
gene in tomato plants (Meshi et al., 1989). This capability
resides in two amino acid substitutions, at positions 68
and 133 (Cys to Phe and Glu to Lys, respectively). In this
region of the PMMV-S 30K protein (Fig. 4), there are
three non-conservative amino acid changes with respect
to the proteins of TMV (Glu to Met, Ala to Lys and Lys
to Ala at positions 133, 147 and 150, respectively) and of
ToMV (Ala to Lys, Ala to Lys and Lys to Ala at positions
130, 147 and 150) but only one (Val to Pro at position 136)
with respect to T M G M V . Although some of these
substitutions may be of a compensatory type, they could
be responsible for the ability of PMMV-S to overcome
the pepper L 1 and L 2 resistance genes. The carboxy
region of the tobamoviral 30K proteins are the most
variable in terms of length or amino acid sequence.
However, Saito et al. (1988) found that all of these
proteins have a particular charge distribution with a
basic domain flanked by two acidic domains. In this
sense, the content of acidic amino acids (Glu, Asp) in the
extreme C terminus of the 30K protein of PMMV-S is
lower (four) than that of T M V and ToMV (six and seven,
respectively). These changes could be involved in the
adaptability of PMMV-S to its pepper host, although
they may only represent, as stated above, the high degree
of variability in this area of the tobamoviral 30K
proteins.
Coat protein gene
The fourth ORF of PMMV-S encodes the coat protein. It
ranges from nt 5685 to 6158, with an intergenic region of
two nucleotides between the 30K and coat protein ORFs
(Fig. 2). The resulting protein consists of 156 amino
acids, with a calculated Mr of 17110. This value differs
from the previous report of 158 amino acids for the coat
protein of an Italian isolate of P M M V (Wetter et al.,
1984), determined by amino acid analysis. Although
PMMV-S and P M M V are different isolates, which can
be distinguished by their responses in Capsicum spp. with
different resistance genes and therefore have been
identified as different pathotypes (Garcia-Luque et al.,
1990), sequencing of the Italian P M M V coat protein
gene has shown that it also consists of 156 amino acids
(M. L. Ferrero, I. Garcia-Luque, E. Alonso, A. de la
Cruz, J. F. Rodriguez, M. T. Serra & J. R. Diaz-Ruiz,
unpublished results).
The alignment of the deduced amino sequence for the
coat protein gene of PMMV-S with those of other
tobamoviruses (Fig. 5) shows that there is a strict
conservation of those amino acid sequence motifs (36 to
41, 88 to 94, 113 to 120) which correpond to the RNAbinding site in the coat protein (Altschuh et al., 1987).
The lower amino acid sequence identities are located at
the N, C and central regions of the proteins (Fig. 5). It
remains an open question as to whether any of these
changes also affect the ability of PMMV-S to overcome
the resistance conferred by the L 1 and L 2 genes in pepper
plants, as described for several mutant strains of TMV
and ToMV in which changes in their coat proteins
make these mutants either able to be localized by the N'
gene in N. syh,estris or to escape its action (Knorr &
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2883
P M M V - S RNA sequence
PMMV-8
ToMV
TMV
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AYTVssANQLX/YLGSVWADPLELQNLCTSALGNQ
S SIT PS F F S
I L V NS
S SITTPS F F S A
I I
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VQI
.
PMMV-S
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~o~
~
TMV
TMGMV
A N T IVN ~
QQQF SDVWKT IPTATVRFPATGFKVFRYNAVLD
E
PF QS
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IV R
E
PS QV
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Y
[
AA p v s M
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I
L
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sAN
v
SAN
IV
A
N
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S
~
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F
G
I
I
P
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T
~IF
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VENPQ
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QA
DQP
(156)
M
V~SAAS
11581
S
V TSG AT
V T
AT
(158)
(158)
T
SLVSALLG
P IT
Fig. 5. Alignment of the coat protein sequence of PMMV-S with those from the more closely related tobamoviruses. Numbering and
source of amino acid sequences as in Fig. 3 and Table 1. The RNA-binding domains are boxed.
Dawson, 1988; Saito et al., 1988, 1989; Culver &
Dawson, 1989).
Based upon nucleotide sequence homology with the
TMV origin of assembly (Zimmern, 1977), the predicted
position of this region in PMMV-S is located between nt
5458 and 5517, in accordance with the absence of
encapsidation of its coat protein m R N A as shown by
electron microscopy observation (Wetter et al., 1984) and
electrophoretic analysis of the virion particles (GarciaLuque et al., 1990).
Functional and evolutionary considerations
The determination of the nucleotide sequence of
PMMV-S R N A has allowed us to confirm that the entire
genome of this virus has diverged from other related
tobamoviruses at a similar rate. The grouping of the
tobamoviruses based on the amino acid composition of
their coat proteins (Gibbs, 1986) and on the basis of the
peptide pattern of the 126K proteins (Fraile & GarciaArenal, 1990) corresponds well to what is deduced from
the entire genome, i.e. PMMV is located in the same
cluster as TMV, ToMV and TMGMV, being more
closely related to ToMV and TMV. These data also
confirm the relationship between T o M ¥ and PMMV-S
previously found by serological analysis (Wetter et al.,
1984; Alonso et al., 1989).
The possession by PMMV-S R N A of all the conserved
sequence motifs necessary for replication and virion
stability, its biololgical properties such as the diminished
capability to replicate and/or accumulate in tobacco
plants, its ability to overcome the tobamoviral resistance
genes in pepper and its inability to infect tomato plants,
should be ascribed to the requirements for the establishment of a functional interaction(s) with the host factor(s)
known to be necessary for the efficient multiplication of
the viruses in their host plants. Whether these requirements are related to changes at the amino acid level
(polymerase, 30K and/or coat proteins) or at the
nucleotide level (3' non-coding region) is under current
study.
The authors thank M. V. Lafita for typing the manuscript. B.W. and
E.A. were supported by fellowships from MEC and Fundacibn Ram6n
Areces, respectively. The work was supported by grants from
PLANICYT (AGR88-0082) and Fundaci6n Ram6n Areces.
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