Journal of General Virology (1991), 72, 1505-1514. Printedin Great Britain
1505
Nucleotide sequence of tomato ringspot virus RNA-2
M . E. Rott, 1 J. H . Tremaine 2 and D. M . Rochon 2.
1Department of Plant Science, University of British Columbia, Vancouver, British Columbia V6H 2A2 and 2Agriculture
Canada, Research Station, 6660 N.W. Marine Drive, Vancouver, British Columbia V6T 1X2, Canada
The sequence of tomato ringspot virus (TomRSV)
RNA-2 has been determined. It is 7273 nucleotides in
length excluding the 3' poly(A) tail and contains a
single long open reading frame (ORF) of 5646
nucleotides in the positive sense beginning at position
78 and terminating at position 5723. A second in-frame
A U G at position 441 is in a more favourable context
for initiation of translation and may act as a site for
initiation of translation. The TomRSV RNA-2 3' noncoding region is 1550 nucleotides in length. The coat
protein is located in the C-terminal region of the
large polypeptide and shows significant but limited
amino acid sequence similarity to the putative coat
proteins of the nepoviruses tomato black ring (TBRV),
Hungarian grapevine chrome mosaic (GCMV) and
grapevine fanleaf (GFLV). Comparisons of the coding
and non-coding regions of TomRSV RNA-2 and the
R N A components of TBRV, GCMV, GFLV and the
comovirus cowpea mosaic virus revealed significant
similarity for over 300 amino acids between the coding
region immediately to the N-terminal side of the
putative coat proteins of T o m R S V and GFLV; very
little similarity could be detected among the noncoding regions of TomRSV and any of these viruses.
Introduction
part of the picornavirus-like supergroup which includes
the comoviruses, potyviruses and piconorvaruses (Goldbach, 1987). Some common features within this group
include genomic structure and organization, as well as
regions of nucleotide and amino acid sequence similarity. Martelli (1975) suggested that members of the
nepovirus group could be divided into three subgroups
based on the Mr of their RNA-2 components. Subgroup I
would include grapevine fanleaf (GFLV), raspberry
ringspot, tobacco ringspot and arabis mosaic virus with
RNA-2 components of Mr 1"4 × 106 to 1"5 × 106,
subgroup II would include TBRV, Hungarian grapevine
chrome mosaic (GCMV) and artichoke Italian latent
virus with RNA-2 components of Mr 1"5 × 106 to
1"6 × 106; and subgroup III would include TomRSV,
peach rosette mosaic, cherry leaf roll and myrobalan
latent ringspot virus with RNA-2 components of Mr
> 1"6 x 106.
We report here the nucleotide sequence of TomRSV
RNA-2. This is the first sequence of a nepovirus from the
subgroup having a large RNA-2 component. Comparisons between this sequence and other nepovirus sequences are presented also.
Tomato ringspot virus (TomRSV), a member of the
nepovirus group (Harrison & Murant, 1977), is found
mainly in North America around the Great Lakes and
along the Pacific coast where populations of its nematode vectors Xiphinema sp. occur. The most serious
disease problems caused by TomRSV occur in Prunus sp.
such as peach and cherry, but TomRSV also causes
diseases in apple, raspberry and grapevine. Chronically
infected plants do not show obvious symptoms but are
characterized by a general decline in productivity (StaceSmith, 1984).
Like other nepoviruses, TomRSV consists of 28 nm
isometric particles composed of 60 copies of a single coat
protein species encapsidating each of the two components of the bipartite, single-stranded, positive-sense
R N A genome (Harrison & Murant, 1977; Schneider et
al., 1974). The 5' terminus of each viral R N A component
is covalently attached to a small protein (VPg) (Mayo et
al., 1982) and their 3' termini are polyadenylated (Mayo
et al., 1979). Mature TomRSV proteins are probably
released from two large polyprotein precursors corresponding to RNA-I and -2 by proteolytic processing as
shown for the nepovirus tomato black ring (TBRV)
RNA-1 (Demangeat et al., 1990).
It has been suggested that nepoviruses be included as
Methods
cDNA clones. Viral RNA used for preparation of cDNA was
obtained from a raspberry isolate of TomRSV. Two overlapping
0001-0085 © 1991 SGM
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1506
M . E. Rott, J. H. Tremaine and D. M . Rochon
eDNA clones derived from TomRSV RNA-2 were used to determine
most of the nucleotide sequence of RNA-2; clone K6 which has been
described previously (Rott et al., 1988), and clone 035. Generation of
035 was essentially as described by D'Alessio et al. (1987), except that
first-strand eDNA synthesis was primed from virion RNA using
the phosphorylated synthetic oligonucleotide, oligonucleotide no. 2
(5" TTCTGGTTCCTCTTCC 3'), which was complementary to
TomRSV RNA-2at nucleotide positions 2201 to 2216. The amount of
first-strand product was estimated using agarose gel electrophoresis.
Solution containing first-strand eDNA was adjusted to 0.2 M-sodium
acetate pH 5-8, and eDNA was precipitated with 2.5 volumes ethanol,
and then washed with 70~ ethanol before continuing with secondstrand synthesis (D'Alessio et aL, 1987). Synthesized double-stranded
eDNA was ligated into the EcoRV site of Biuescript (Stratagene) and
used to transform competent Escherichia coli DH5ct cells (BRL).
Nortbern-blotted TomRSV RNA (Vrati et al., 1987) was used to
confirm that clone 035 originated from TomRSV RNA-2. Clone 035
(approximately 2.2 kb) was analysed further by restriction enzyme
mapping and dideoxynueleotide sequencing (see below).
Sequencing and sequence analysis. Subclones used to sequence clones
K6 and 035 were generated by restriction enzyme digestion and/or
exonuclease III-generated nested deletions (Henikoff, 1984). The
dideoxynucleotide chain termination method of Sanger et al. (1977) was
used to sequence double-stranded plasmid DNA templates using
modified T7 DNA polymerase (Sequenase) as described by Toneguzzo
et al. (1988). All portions of RNA-2 were sequenced completely in both
directions except for the 5' extreme 28 nucleotides which were not
present in any cDNA clone analysed. These 28 nucleotides were
sequenced in one direction only using viral RNA as a template (see
below). On average each nucleotide was sequenced over three times.
Two regions of TomRSV RNA-2 were sequenced using viral RNA
as template with synthetic oligonucleotide primers no. 1 (5'
GCCTTCGATGGAACC 3', complementary to positions 115 to 130)
or no. 2 and Moloney murine leukaemia virus reverse transcriptase in
the presence of dideoxynucleotides (Ahlquist et al., 1981).
Sequence data were assembled and analysed using the Gene-Master
(Bio-Rad) sequence analysis software package on a Compaq 386
computer. Further analysis was performed using programs and
databases available through the Canadian National Research Council's Scientific Numeric Database Service on a DEC VAX. We would
like to thank Bill Ronald and Jim Purves of Agriculture Canada for
writing a program used to test the goodness of fit between the amino
acid composition of the TomRSV coat protein (Tremainc & StaceSmith, 1968) and the calculated amino acid composition of segments of
the deduced polyprotein sequence encoded by RNA-2 (see Meyer et aL,
1986). The raspberry isolate of TomRSV used by Tremaine & StaceSmith for the coat protein amino acid composition analysis and the
raspberry isolate we have sequenced were both obtained from the
British Columbia Frazer Valley growing area and are therefore
probably very similar. The coat protein multiple sequence alignment
was 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 predicted amino acid sequences were compared using the
TFASTA program (Pearson & Lipman, 1988) with sequences in the
protein sequence librariesof the NBRF, Swiss-Prot and Pseqlp, and the
nucleotide sequence libraries of the NBRF, GenBank and EMBL. The
5' region between two potential AUG initiation codons was analysed
for a possible coding function using the program TESTSCORE
(Fickett, 1982) and a window size of 67. Scores within the top 77 to
100~ predict coding regions. This was followed with the absolute
positional base preference method (Staden, 1984), using a window size
of 50, to determine the coding frame.
Results and Discussion
Sequencing and primary structure o f R N A - 2
T w o o v e r l a p p i n g c D N A clones, d e s i g n a t e d K 6 a n d 0 3 5 ,
were used to d e t e r m i n e m o s t o f the n u c l e o t i d e s e q u e n c e
o f T o m R S V R N A - 2 . C l o n e K 6 w h i c h has b e e n
p r e v i o u s l y d e s c r i b e d ( R o t t et al., 1988), c o r r e s p o n d s to
5498 n u c l e o t i d e s at the 3' e n d o f R N A - 2 (Fig. 5 a). C l o n e
0 3 5 , w h i c h c o r r e s p o n d s to the 5 ' - t e r m i n a l r e g i o n o f
T o m R S V R N A - 2 , was d e r i v e d using the s y n t h e t i c
o l i g o n u c l e o t i d e p r i m e r o l i g o n u c l e o t i d e no. 2, w h i c h was
c o m p l e m e n t a r y to R N A - 2 at a r e g i o n a p p r o x i m a t e l y 400
n u c l e o t i d e s f r o m the 5' e n d o f clone K 6 (Fig. 5a). T w o
o l i g o n u c l e o t i d e s (no. 1, c o m p l e m e n t a r y to T o m R S V
R N A - 2 n u c l e o t i d e s 115 to 130 a n d no. 2, c o m p l e m e n t a r y
to n u c l e o t i d e s 2201 to 2216) were used to c o n f i r m a n d / o r
extend the sequence obtained by sequencing cDNA
clones. T h e s e q u e n c i n g r e a c t i o n s using o l i g o n u c l e o t i d e
no. 1 y i e l d e d 28 n u c l e o t i d e s at the 5" t e r m i n u s o f R N A - 2
n o t p r e s e n t in 0 3 5 a n d resulted in t w o s t r o n g stop points.
T h e s e stop p o i n t s c o r r e s p o n d to n u c l e o t i d e s 1 a n d 2 o f
T o m R S V R N A - 2 a n d a r e e a c h d e n o t e d N in F i g . 1.
O l i g o n u c l e o t i d e no. 2 was used to s e q u e n c e p a r t o f the
v i r a l R N A to c o n f i r m the p r e s e n c e o f t h r e e t a n d e m
r e p e a t s d e d u c e d f r o m the n u c l e o t i d e s e q u e n c e w i t h i n the
o v e r l a p p i n g p o r t i o n s o f clones K 6 a n d 0 3 5 (see below).
T o m R S V R N A - 2 d e t e r m i n e d as d e s c r i b e d a b o v e is
7273 n u c l e o t i d e s in l e n g t h e x c l u d i n g the 3' p o l y ( A ) tail
(Fig. 1). T h e c a l c u l a t e d Mr o f R N A - 2 is 2.35 x 106
[excluding the 3' p o l y ( A ) tail a n d 5' VPg], w h i c h is
s i m i l a r to the 2.4 x 106 M r value d e t e r m i n e d b y
d e n a t u r i n g gel e l e c t r o p h o r e s i s ( M u r a n t et al., 1981). T h e
base c o m p o s i t i o n o f R N A - 2 is 22.5~o A , 23"3~o C, 24-9~o
G a n d 29.3Y/o U.
Open reading f r a m e s
A single long o p e n r e a d i n g f r a m e ( O R F ) c o n s i s t i n g o f
5646 n u c l e o t i d e s is p r e s e n t in t h e v i r i o n sense o r i e n t a t i o n
o f R N A - 2 . T h i s O R F , w h i c h a c c o u n t s for 7 8 ~ o f t h e
R N A - 2 sequence, b e g i n s at the first A U G at p o s i t i o n 78
( A U G 78) a n d t e r m i n a t e s at a U A A stop c o d o n at
p o s i t i o n 5723. T h e p r e d i c t e d t r a n s l a t i o n p r o d u c t w o u l d
h a v e a n Mr o f 207K. A U G 78 ( U U U G A U G U C ) d o e s
n o t h a v e e i t h e r the o p t i m a l K o z a k ( C G / A C C A U G G ) o r
Liitcke ( A A C A A U G G C ) c o n t e x t for t r a n s l a t i o n initiat i o n in a n i m a l s o r plants, r e s p e c t i v e l y ( K o z a k , 1986;
Liitcke et al., 1987). T h e n e x t i n - f r a m e A U G , w h i c h is
the fourth A U G f r o m t h e 5' t e r m i n u s ( U G C A A U G G A )
occurs at p o s i t i o n 441 a n d is in a f a v o u r a b l e K o z a k
c o n t e x t for the i n i t i a t i o n o f t r a n s l a t i o n w i t h a G in the
- 3 a n d + 4 positions. T h i s raises t h e p o s s i b i l i t y t h a t
A U G 441 m a y be a n i n i t i a t i o n site for t r a n s l a t i o n . T h e
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T o m R S V R N A - 2 sequence
predicted translation product beginning at A U G 441
would have an Mr of 194K. However, there is further
evidence that A U G 78 may act as an initiation site for
translation. The 5' 800 nucleotides of TomRSV RNA-2
were analysed using the program TESTSCORE (Fickett,
1982). The region beginning from A U G 78 gave a score
of between 50 and 77~o for the first 100 nucleotides, and
then rose to 100~o through to the end of the sequence
analysed. Scores between 77 and 100~ indicate coding
regions (Fickett, 1982). Further analysis using the
absolute positional base preference method (Staden,
1984) indicates that there is only one coding frame in this
region beginning at approximately nucleotide 180 to the
end of the sequence analysed (the first 800 nucleotides).
A U G 78 is the only potential in-frame initiation site
upstream of the coding region indicated by TEST
SCORE and the absolute positional base preference
method. Translation initiation of TomRSV RNA-2
therefore, may be similar to that described for the
comovirus cowpea mosaic virus (CPMV) M RNA. The
latter has at least two in-frame sites for translation
initiation, one at position 161 and the other at position
512 and/or 524 (van Wezenbeek et al., 1983). Both sites
appear to be functional (Holness et al., 1989) and
preliminary results suggest both leaky scanning and
internal initiation as mechanisms for initiation at the 512
site (Wellink et al., 1990). Initiation of translation at
internal A U G codons has been established to occur in
both poliovirus (PeUetier & Sonenberg, 1988) and
encephalomyocarditis virus (Kaminski et al., 1990) by a
mechanism of internal ribosome binding. For TomRSV
RNA-2, it is not known whether A U G 78 and/or A U G
441 act as the site for initiation of translation and further
work is required to address this question.
All other potential ORFs in the positive-strand were
less than 355 nucleotides in length. Two ORFs in the
negative-strand orientation are present which are 603
and 582 nucleotides in length (positive-strand nucleotide
positions 956 to 353 and 3103 to 2521, respectively). A
search of the NBRF, Swiss-Prot, Pseqlp, GenBank and
EMBL databases failed to detect sequences with
significant amino acid sequence similarity to either of
these two ORFs.
Non-coding regions
The 5' non-coding region of TomRSV RNA-2 was
analysed and compared to the corresponding regions of
the two R N A components of TBRV (Meyer et al., 1986;
Greif et al., 1988) and GCMV (Brault et al., 1989; Le
Gall et al., 1989), RNA-2 of GFLV (Serghini et al., 1990)
and the B and M components of the comovirus CPMV
(Lomonossoff & Shanks, 1983; van Wezenbeek et al.,
1983). It has been reported previously that TBRV,
1507
GCMV, GFLV and CPMV share a conserved
U G A A A A A U sequence downstream from the 5' terminus (Serghini et al., 1990). TomRSV RNA-2 has a similar
sequence (CGAAAAAU). This short octanucleotide
was the longest region of sequence identity that could be
detected between the 77 nucleotide 5' non-coding region
of TomRSV RNA-2 and the 114 to 287 nucleotide 5' noncoding regions of any of these other viruses. The base
composition of the 5' TomRSV RNA-2 non-coding
region is similar to those of TBRV, GCMV, GFLV and
CPMV in having a high U content (44.2%) and a low
G + C content (35.1%).
The 3' non-coding region of TomRSV RNA-2 is 1550
nucleotides excluding the poly(A) tail. This is much
longer than the sizes of the 3' non-coding regions of
TBRV, GCMV, GFLV and CPMV, as well as those of
most other positive polarity R N A viruses. The 3' noncoding regions for TBRV, GCMV, GFLV and CPMV
are less than 305 nucleotides. Comparison of the 3' noncoding region of TomRSV RNA-2 with those of the
RNA-2 components of GFLV, TBRV and GCMV, the
M R N A from CPMV, and poliovirus R N A did not
reveal any significant sequence similarity except for the
sequence AAAAGC found immediately preceding the
poly(A) tail in RNA-2 of TomRSV, TBRV and GCMV.
The conserved blocks of sequence among the 3' noncoding regions of TBRV, GCMV, GFLV and CPMV
previously reported by Serghini et al. (1990) could not be
detected in the 3' non-coding region of TomRSV RNA-2.
The 3' non-coding regions of TBRV, GCMV, GFLV
and CPMV have a high U content (40.5 to 48.0~o) which
is similar to that of their 5' non-coding regions. The
entire 3' non-coding region of TomRSV RNA-2 has a
lower U content of 31.2%. However, its extreme 3' end
(approximately 110 nucleotides) has a U content of
44-2~o which is similar to the high U content of the 3'
non-coding regions of the other viruses.
The possible significance of the very long 3' noncoding region on TomRSV RNA-2 is unknown. In a
previous paper (Rott et al., 1988) we reported that the 3'terminal regions of TomRSV RNA-I and RNA-2 share
an extended region of nucleotide sequence homology.
Nucleotide sequence analysis of part of RNA-1 has
confirmed that RNAs 1 and 2 share 3'-terminal sequence
identity over a 1533 nucleotide region (unpublished
results). The possible significance of the 3'-terminal
identity of RNAs 1 and 2 in viral R N A replication will
be discussed in that communication.
Analysis o f the RNA-2-coding region
(i) Location of the putative coat protein-coding region
Tremaine & Stace-Smith (1968) reported the coat protein
amino acid composition for a raspberry isolate of
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1508
M. E. Rott, J. H. Tremaine and D. M. Rochon
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Downloaded from www.microbiologyresearch.org by
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E
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TomRSV RNA-2 sequence
1509
R G G N T M L S L D A V E V V I D P V G M P G D D T D L T V M V L W C Q N S D D 1 1 3 5
3361 AGCGUGGU~U~UACUA~CUG~GAUGC~GR~UCGUUAUCGACC~AG~GG~ADG~cUGGUGAUGACACUGAU~GACUG~AUGGU~GUGGUGUC~CAGAUG
O R A L I G A M S T F V G N G L A R A V F Y P G L K L L Y A N C R V R D G R V L I I 7 5
~AUCAGCGCGCU~UGAU~GGGGCCAUGUCUAC~GUGGGC~AUGG~CU~CCAGAGC~G~UAUC~CGGGC~A~A~AUAUG~C~GUAGAGUGCGAGAUGGCCGAGU~
K V I V S S T N S T L T H G L P Q A Q V S I G T L R O H L G P G H D R T I S G A I 2 1 5
3~1 U~AGGUCAUUGUGAGCA~A~A~CAACGCUCACGCAUGGU~GCCCCAGGCUC~GUCUCCA~GGGAC~UG~GCCAGCAUUUGGGGCCAGGUCAUGAUCGCACUAUCUCUGGUG
L Y A S Q Q Q G F N I R A T E Q G G A V T F A P O G G H V E G I P S A N V Q M G I 2 5 5
3721 CCCUGUAUGC~CCC~C~CAGGG~UC~UAUACGCGCCACGGAACAAGGUGGUGCUGUAACAU~GCCCCCC~GGGGGCCAUGUUGAGGGUAUCCCCAGCGCC~UGUACAGAUGG
A G E H L I Q A G P M Q W R L Q R S Q S S R F V V S G H S R T R G S S L F T G S I ~ 5
3841 GCGCUGGGGAGCAU~GCGGGUCCCAUGCAGUGGCGC~GCAGAGGUCGC~UCUUCUCGA~UGUGGUCU~UGGUCA~CGCGAACGCGUGGAAGC~CU~GU~ACUGG~
V D R T O Q G T G A F E D P G F L P P R N S S V Q G G S W Q E G T E A A Y L G K 1 3 3 5
3961 GUGUCGAUAGGACGCAGCAGGGAACGGGGGC~G~GACCCGGGUUU~UACCACCCAGG~CUUCUG~CAGGGCGGGUCCUGGC~GAAGGUACUGAAGCCGC~A~UAGGCA
V T C A K D A K G G T L L H T L D I I K E C K S Q N L L R Y K E W Q R Q G F L H I ~ 5
~8~AAG~ACCUGUGCG~GGACGCC~GGGUGG~C~UA~GCACACUUUGGAUAUUAUAAAAGAGUGC~AUCCC~UA~GGUAU~AG~UGGC~CGUCAAGGCU~CUUC
G K L R L R C F I P T N I F C G H S M M C S L D A F G R Y D S N V L G A S F P V 1 4 1 5
4~I AUGGA~GCUUAGA~GCGCUGC~UAUACCCACUAACAU~UGUGGGCAUUCCAUGAUGUG~CC~GGACGCG~UGGUCG~AUGAUUCGAACGUGCUAGGUGCUAG~CCAG
K L A S L L P T E V I S L A D G P V V T W T F D I G R L C G H G L Y Y S E G A Y I 4 5 5
4321UGAAG~GGC~GU~AUUGCCAACGGAGGUGA~AGUCUGGCUGAUGGGCCCGUGGUCACGUGGACG~UGAUA~GGGCGUCUGUGUGGUCAUGGUCUCUA~AUUCCGAGGGCGC~
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A R P K I Y F L V L S D N D V P A E A D W Q F T Y Q L L F E D H T F S N S F G A I 4 9 5
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V P F I T L P H I F N R L D I G Y W R G P T E I D L T S T P A P N A Y R L L F G I 5 3 5
4561CGG~CCU~UA~ACCUUACCCCAUA~UAAUAGA~AGAUAUAGG~AUUGGCGCGGGCC~CAGAGAUAGAU~CAUC~CUCCCGCACCA~CGCCUAUCG~UAC~CG
L S T V I S G N M S T L N A N Q A L L R F F Q G S N G T L H G R I K K I G T A L I 5 7 5
~81GCUUGUCCACUGUCAUUAGUGGU~CAUGUCGAC~UG~UGCC~UCAAGCC~AUUGCGU~UCCAGGG~CG~UGGCACUUUACAUGGGCGCA~AGAUAGGGACAGCAC
T T C S L L L S L R H K D A S L T L E T A Y Q R P H Y I L A D G Q G A F S L P I I 6 1 5
4801UUA~AACCUG~CCCU~UA~AUCGUUGCGCCACA~GAUGCGAGUCUCACA~GGAGACCGCAUAUCA~GGCCCCA~ACAUUUUGGCUGACGGAC~GGGGCU~CAUUACC~
S T P H A A T S F L E D M L R L E I F A I A G P F S P K D N K A K Y Q F M C Y F 1 6 5 5
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P D I L D D K S E V L L R Q H P L S L L I S S T G F F T G R A I F V F Q W G L N I 7 3 5
5161UUCCAGAUAUAC~GAUGAU~GUCAG~GUGCUCUUAAGGC~CAUCC~UAUCUCUGC~AUCUCAUCUACCGGU~ACGGGUAGAGCCA~UUUGU~UCCAGUGGGGU~GA
T T A G N M K G S F S A R L A F G K G V E E I E Q T S T V Q P L V G A C E A R I 1 7 7 5
5281AUACUACUGCUGGG~UAUGAAAGG~CA~CUGCGCGCCUGGCC~UGGC~GGGCG~GAGG~AUUGAGC~ACGUCAACAGUGC~CCAC~G~GGCGCUUGUG~GCCCGCA
P V E F K T Y T G Y T T S G P P G S M E P Y I Y V R L T Q A K L V D R L S V N V I S I 5
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I L Q E G F S F Y G P S V K H F K K E V G T P S A T L G T N N P V G R P P E N V I 8 5 5
5521UUA~ACAGGAGGGAU~UCU~CUAUGGACCUAGUGUCA~CAUUUUAAG~AG~GUCGGCACGCCUAGUGCCACCCUAGGGAC~AU~UCC~G~GGGCGCC~ACCUGAG~CG
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5761CAGCUUCC~GGUGGC~UC~AGCU~U~UAGGGG~AUCCAGCCUUAAGCAAGCUGGCACCGGUCCUGAUGGACUA~CAGGA~GCACCUGGU~GG~GAAUUCGAGUA~
5881A~CUU~AUCUUG~UACUCGUGACUUAUAGUA~A~CAAGAGGAAUGAcUCAUGU~UGU~AU~ACAUGAUGGCAUA~GAG~CGGCUCAUAUGGUGCUCAUUACGUUC~GU
6~1G~GAAGGAUCC~UAG~CUUGAACUGUGGUG~CAUGUGAGG~AUCCACG~AUCUCUGAUUGUC~UAGACUAGUCUAGGAGACGAUA~UCCUAUGUGGGUGAGUCcCACUCUGG
6121 CGAGACACGCAGUGCC~A~UG~UGAGGUUAUCA~CAUCAUAUC~GAGUCUGCA~UA~CG~U~UGUAGUUGUCAUAGCCUACCGAUGAGUCUGCGAG~AGG~CCAU
~41GAGGAC~GGG~GGCUAACCCCCACUUAAUCUC~CAUAGAUCAUUCGACAGUGUGUCGAG~ACUAUGGGUUUUGACACCUUAAGGGAAGCGAGAG~CUCGUAUGGAUAUCACUCUA
636! AU~AUGGUAC~ACCAUG~CAUG~UAGAGU~AUCAUCG~CUCGACGGUGUGAUAC~UCC~GU~UAGUC~A~GAUA~CUCGUUGAUCGUAUGUGAAGCGUGGAGCGAG~CG
~8[ A~CG~CUGAUUA~CAGAGGUAGGACGCUA~G~CCAGGCGU~C~AUGGGCAU~GCUGU~AC~GGt~3CGCAAGCCAUGCAGCACCUCCCG~ACUUGUGUACU~CUAGGGG
6601CUCCCGGCCUU~UU~CGGUACAAUACCUAGUG~GCAAGUAAUUGCGUUGAGGGAU~GAGUAGCAUGUUCCUACUUAAGGAAGG~UAUGUCGUGUUUUCCACACGUUAGUG~GC~
6721UG~UGU~UGGCACUGCAGUG~AGG~UGGUUCCCAGCCACU~CUGGGAUUCUAAUCGUACGUCACAAUUGUGUGUGUAU~G~ACGGAGGAGUAGCGAUCCUCUACCACGCGAGU
6841CUGGAAGUGA~ACCAGGGCCUAAGAUGGCCAGCACACGGUACGAUU~AU~AGCUGU~UGUAGUGGUAUG~GUUGAGACU~CUUACCCGUACGAGUUA~CUCU~GAUGGAU
~ 6 1 GUGUG~CUGCCAUCUUAGAGGAAGUAGAUGUGU-~U~ACCAAUCUGAGACGAGCCG~UUCGGUGCU~UACGUCAAUGAU~UACUCGUGCAG~GCAGCUGCACGAGUAUG~
7081 ~UA~A~A~AGU~CUCGGAUACG~U~G~ACCCU~C~UA~G~A~CUCUCUC~UCUUA~CU~CUGCAAG~ACG~GUUUUCGCA~GGU~UGUU~UCCGU~UG~UC~
7201 AAC GC U G C ~ U G C A A U U U U C U ~ U G ~ A U U G C
~ U C GUAGUGUCG~CU~GUCC ~ G ~ C A U A ~ A G C
Fig. 1. Nucl~tide sequence of TomRSV RNA-2 and deduced amino acid ~quence of the long pol~rotein. The first two nuci~tides
which could not be read ~om the sequencing gel are each repre~nted by an N. The second in-~ame A U G proposed to be the initiation
site ~ r translation is indicated by three asterisks. The underlined regions are complementa~ to oligonucleotides no. 1 and no. 2.
Nucl~tides are numbered on the le~ beginning at the first N. Amino acids are numbered on the right and begin at the ~ s t in-~ame M.
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1510
M. E. Rott, J. H. Tremaine and D. M. Rochon
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V
C
S
S
I
F
H
H
R
I
L
L
L
P
A
A
P
T
P
P
A
N
N
S
N
I
A
Q
A
*
***
.
TomRSVI421
TBRV
929
GCMV
~5
GFLV
771
L
S
S
L
L
F
L
S
P
D
C
V
T
P
P
P
E
H
H
H
V
V
V
W
I
Q
H
L
S
V
V
I
L
L
L
H
TomRSVI47!
TBRV
9~
GCMV
955
GFLV
821
V
L
L
K
P
A
P
E
A
R
W
L
E
S
S
A
A
T
A
A
D
A
Q
D
W
T
V
W
Q
K
T
Q
F
F
Y
A
*
A
R
R
H
D
D
D
K
T
R
R
V
*
G
V
S
L
Y
L
L
V
P
S
K
G
Q
E
E
E
L
F
A
L
V
T
T
T
L
Y
L
Y
V
S
S
F
*
T
T
V
C
F
T
A
A
W
W
W
S
E
R
Q
E
T
V
T
E
D
G
G
L
. J
.
F
I
I
I
DIGRLCGHG
DFHKICGQT
DFHKICGQS
DYGELCGHA
H
E
D
E
T
K
E
E
F
L
I
A
S
V
A
T
N
R
H
S
S
G
G
F
F
L
L
L
G
A
A
G
A
E
T
K
V
Q
R
P
P
P
S
T
F
I
L
I
L
V
V
T
S
T
F
L
Y
Y
D
P
P
P
P
H
I
I
G
t
TomRSV[5~
TBRV
1028
GCMV
1~5
GFLV
870
I
I
M
F
D
A
A
D
L
V
I
L
T
G
G
T
S
T
N
A
T
Y
A
V
P
A
G
T
A
M
S
A
P
I
I
L
N
T
N
R
A
S
Y
F
F
A
R
P
P
G
L
V
L
L
L
S
S
T
F
L
F
L
G
A
A
G
L
A
V
Q
S
K
Q
V
T
L
Q
P
V
Q
K
M
TomRSVI570
TBRV
1077
GCMV
1054
GFLV
919
K
R
Q
H
K
T
C
I
I
T
T
C
G
S
S
A
T
S
S
P
S
K
R
V
L
L
L
L
L
R
R
W
L
L
L
S
R
W
W
E
H
W
K
N
D
G
G
G
A
T
D
T
S
V
T
T
L
P
I
M
T
E
T
D
L
T
L
W
E
D
E
N
T
Q
Q
E
A
L
L
L
T o m R S V 1620
TBRV
1124
GCMV
1101
GFLV
968
F
F
Y
H
g
Y
H
A
S
A
R
A ......
T ......
T ......
T P A R L L A G
T
A
A
Q
S F L E D
N F G N S
N F G D S
S
Q R D
M
G
G
M
L
S
A
S
R
A
R
S
L
F
F
L
E
Y
W
N
I
V
V
F
F
S
T
Y
A
T
P
A
I
L
M
I
A
C
S
A
G
A
S
G
P
P
P
P
F
M
M
I
S
A
A
A
TomRSVI~3
TBRV
1168
GCMV
[145
GFLV
1017
E
P
P
P
G
G
P
D
V
L
M
L
P
C
C
S
R
R
R
L
T
E
Q
P
I
I
I
S
A
N
N
F
G
Y
Y
E
E
K
D
D
Q
Q
Q
D
Q
R
R
Y
F
F
F
F
N
A
A
V
W
W
W
W
F
-
C
C
T
V
S
L
L
D
F
L
L
F
R
E
R
S
C
P
-
L
P
-
D
D
-
N
N
P
E
F
S
K
F
K
K
L
T
I
A
S
L
D
S
K
D
D
P
I
K
W
I
L
E
TomRSVI709
TBRV
1217
GCMV
1195
GFLV
1~3
R
F
Y
G
Q
V
V
E
H
N
N
N
P
A
A
P
L
L
F
F
S
A
A
A
L
I
I
A
L
L
M
M
I
C
C
I
S
A
A
A
S
T
T
C
T
T
T
H
G
G
G
G
F
M
M
L
F
H
H
H
T
H
A
S
G
G
G
G
R
N
K
V
A
C
C
L
TomRSV1759
TBRV
12~
GCMV
1243
GFLV
II~
I
N
S
K
T
A
I
-
N
G
M
E
E
E
G
Q
H
H
L
T
I
H
D
S
G
G
G
T
D
E
P
V
T
F
S
Q
M
H
H
P
C
L
V
L
Y
G
F
V
N
G
A
G
S
P
I
A
L
L
Q
C
S
S
K
E
N
S
L
A
T
S
E
TomRSVI803
TBRV
1312
GCMV
1293
GFLV
1157
T
P
A
D
Q
D
H
Q
A
F
W
A
K
S
D
K
L
W
W
S
V
V
L
I
D
A
T
K
R
S
S
V
L
L
L
L
S
H
T
R
V
V
V
V
N
S
D
L
V
I
I
C
I
E
Q
K
L
V
V
P
Q
H
L
R
E
E
P
P
G
G
G
G
F
F
F
F
*
A
A
A
I
L
F
F
F
T
V
V
Y
T
T
T
S
C
S
A
I
L
I
V
V
S
A
A
V
I
S
S
V
S
T
S
G
G
S
S
T
N
G
G
T
M
R
~
K
S
T
I
V
T
A
A
-
L
Y
Y
Y
.
N
S
S
N
A
Y
Y
L
*
Q
A
A
S
*
Y
A
S
F
Q
Q
Q
K
A
G
G
T
L
L
L
L
L
L
L
V
* *
F
H
H
C
F
F
F
V
Q
L
L
L
G
G
G
G
S
V
I
M
N
G
G
G
*
* *
**
Y
-
I
V
V
V
L
D
D
Y
A
V
C
V
D
E
D
E
G
V
V
E
Q
N
D
D
G
V
V
G
A
D
V
S
F
A
S
F
S
S
S
E
P
P
P
P
K
E
E
S
D
T
T
G
N
V
M
E
K
E
E
T
A
T
S
A
K
G
K
Q
Y
S
L
L
Q
E
E
P
F
Y
Y
I
M
M
Y
V
C
I
I
V
Y
Q
Q
Q
F
I
I
I
K
K
K
E
F
V
L
I
E
K
T
E
W
I
L
I
P
P
P
G
A
S
S
S
R
R
R
R
L
I
V
F
P
G
C
F
D
N
N
D
I
L
I
F
L
S
A
T
D
S
Y
S
I
T
I
D
F V F Q W G L N T T A G N M K G
I H F S W L W H - - P A E L G K
L H F S W T L N - - K G T S F K
L K L Q W S L N
....
T E F
S
Q
D
G
F
L
L
K
S
Q
S
A
G
G
S
K
R
H
G
L
L
I
S
A
K
S
V
H
L
G
R
S
A
T
I
V
V
T
P
P
P
E
V
F
F
L
S
K
R
S
F
F
F
F
Y
Y
Y
Y
G
G
G
G
P S V K H F K K E V G T P S A T L G T N N P V G R P P
R S A G P L T I P A T V A D V S A V S G S
RSAGPLTIPS
RTSFPV
F
F
F
V
K
G
G
G
P G G Q Y A A A L Q A A Q Q A G K N P F G
T
S
S
N
P
Q
K
K
I
I
I
I
S
Q
Q
R
D
K
L
D
H
G
G
E
I
I
I
I
E
E
D
-
L
A
A
V
V
N
D
R
D
K
K
N
K
H
E
T
S
V
A
C
E
K
T
R
V
V
V
V
L
T
T
S
L
N
N
M
F
F
F
T
G
V
Y
I
K G V E E
Q G M G I
S G M G D
T K L V G D
**
Y
F
F
F
T
A
A
A
G
G
G
G
L
L
L
V
*
H
H
H
G
E
Q
E
L
R
M
M
Y
R
S
S
S
P
P
P
P
*
TomRSV 1853 E N V D T G G
N
A
A
N
*
**
P
P
-
Y
I
V
A
T
T
T
N
T
S
S
P
S
G
G
N
G
G
G
T
P
K
T
R
P
A
P
F
G
D
F
S
S
E
T
L
M
A
S
Y
E
E
E
S
P
N
N
R
T
S
S
S
P
P
P
P
*
Y
W
W
W
I
I
L
M
Y
E
R
A
V
I
V
I
R
Q
E
K
T
S
T
L
RG
Fig. 2. Alignment of the amino acid sequences of the TomRSV, TBRV, GCMV and GFLV putative coat proteins. Alignment was
generated using the multiple sequence alignment program of Feng & Doolittle (1987). A double asterisk indicates amino acids common
to all four sequences. A single asterisk indicates three of four amino acids at that position are identical. Underlined dipeptides are
known or potential cleavage sites. Numbers to the left of each line of sequence refer to amino acid position on each nepovirus RNA-2
polyprotein.
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TomRSI/ RNA-2 sequence
*
*
*
*
*
*
**
**
*
**
*
**
1511
*
TomRSV 984 A R K R W L S - R K Q E D S Q E D N I K R Y - - - A D K H G I S F E E A R A V Y K A P K E G V P T Q R S I L P D V R D A Y S A R S A G A R V R S L F G
GFLV
360 GRAQWI S E R R Q A L R R R E Q A N S F E G L A A Q T D M T F E Q A R N A Y L G A A D M I E Q G L P L L P P L R S A Y ....... A P R G L W R
*
*
***
*
**
*
*
*****
**
*
*
**
*
*
*
****
*****
TomRSV 1059' G S P T T R A Q R T E D F V L T S P S A G D A S S F S F Y F N P V S E Q E M A E Q E R G G N T M L S L D A V E V V I D P V G M P G D D T D L T V M V L
GFLV
435 G-P S T R A N Y T L D F R L N G I P TG -TNTLE I L Y N P V S E E E M E E Y R D R G M S A W I D A L E IAI N P F G M P G N P T D L T V V A T
*
**
**
***
*******
*
***
*
*
*
***
*
*
*
******
TomRSV 1134 W C Q N S D D Q R A L I G A M S T F V G N G L A R A V F Y P G L K L L Y A N C R V I % D G R V L K V I V S S T N S T L T H G L P Q A Q V S I G T L R Q H
GFLV
510 Y G H E R D M T R A F IGSASTFLGNGLARAI F F P G L Q - -YSQE EPRRES I I R L Y V A S T N A T V D T D S V L A A I S V G T L R Q H
*
*
**
*
**
***
*
*
*
*
*
*
*
*
*
*
*
**
**
*
TomRSV 1209 L G P G H D R T I S G A L Y A S Q Q Q G F N I R A T E Q G G A V T F A P Q G G H V E G I P S A N V Q M G A G E H L I Q A G P M Q W R L Q R S Q S S R F
GFLV
585 V G S M H Y R T V A S T V H Q A Q V Q G T T L R A T M M G N T V V V S PEGS L V T G TPEARVE I GGGS S I RMVGP LQWE SVE E PGQ TF
TornRSV
GFLV
1284
WSGHSRT
660 S I R S R S R S
Fig. 3. Amino acid sequence alignment of the regions N-terminal to the TomRSV and GFLV coat proteins. Dashes indicate gaps
introduced to maximize the alignment. Asterisks above the sequences indicate positions of amino acid identity. The numbers to the left
of the sequences indicate amino acid positions on the long polyproteins of TomRSV RNA-2 and GFLV RNA-2.
Table 1. Amino acid composition analysis of the TomRSV
coat protein
Amino
acid
Relative
mole ratio*
Scaledt
Determined
from sequence:~
A
C
D+N
E+ Q
F
G
H
I
K
L
14-9
5.1
16-0
18-1
14-5
18.1
5-2
12-7
9-7
24-1
36
12
38
43
35
43
12
30
23
58
43
10
45
49/47
37
53/51
13
28
26
60
M
P
2-7
11-5
10.5
16.2
14-9
9-9
5-2
7.3
7
28
25
39
36
24
12
18
7
32
26
38/37
40
26
9/8
20
R
S
T
V
W
Y
* From Tremaine & Stace-Smith (1968).
t Relative mole ratio values were rescaled for a coat protein with an
Mr of approximately 58000 as determined by Allen & Dias (1977).
:~ Value to the left of the slash is determined from the potential Q - G
cleavage site, the value to the right of the slash is determined for the
potential Q-E cleavage site.
TomRSV assuming an Mr of 24000. Later, Allen & Dias
(1977) reported that the TomRSV coat protein had an Mr
of 58 000. We have rescaled the coat protein amino acid
composition determined by Tremaine & Stace-Smith for
a protein with an Mr of 58K (Table 1) and compared this
with the predicted amino acid composition encoded by
the RNA-2 long ORF by chi-squared analysis. Values of
chi-squared were greater than 27.6 for sequences
C-terminal to residue 1037 and were less than 5-4 for
sequences between residues 1235 and 1882. This would
suggest that the TomRSV coat protein is encoded at the
C-terminal region of the RNA-2-encoded polyprotein. A
comparison of the amino acid composition of the
TomRSV coat protein and the C-terminal region of the
RNA-2-encoded polyprotein is shown in Table 1. The
coat protein-coding regions for the nepoviruses TBRV,
GCMV and GFLV have also been localized to the
C-terminal region of the RNA-2-encoded polyproteins
(Meyer et al., 1986; Brault et al., 1989; Serghini et al.,
1990). An alignment of the amino acid sequence at the
C-terminal region of the TomRSV RNA-2 polyprotein
and the putative coat protein sequences of TBRV,
GCMV and GFLV is shown in Fig. 2. The putative
TomRSV coat protein sequence shared sequence identity
of 21.4~, 22.9~ and 23.4~o with the putative coat
protein regions of TBRV, GCMV and GFLV, respectively. The N-terminal region of the putative coat protein
was analysed for the following potential protease
cleavage sites: Q-S, Q-G, Q-M, E-S, E-G, R-A, R - G
and K - A (Palmenberg, 1990; WeUink et al., 1986;
Serghini et aL, 1990; Brault et aL, 1989; Demangeat et
al., 1990). The sites Q - G and E-G, at amino acid
positions 1320-1321 and 1325-1326, are tentatively
identified as potential cleavage sites for the TomRSV
putative coat protein based on their location and the
previously determined size of the TomRSV coat protein
(Fig. 2). The Q - G and E - G sites proposed for TomRSV
are different from the R-A, R - G and K - A cleavage sites
proposed for GCMV, GFLV and TBRV, respectively,
but are similar to those of the como-, poty- and
picornaviruses (Hellen et al., 1989). A third potential
cleavage site may occur at the K - G site at position 13431344 which would conform to a nepovirus consensus (K
or R - G or A). The putative TomRSV coat protein would
have a calculated size of 60K to 62K which is similar to
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1512
M. E. Rott, J. H. Tremaine and D. M. Rochon
the 58K determined by S D S - P A G E (Allen & Dias,
1977). The TomRSV coat protein is larger than the coat
proteins o f T B R V , G C M V and GFLV. The larger size of
the TomRSV coat protein is at least partially due to an
additional 36 to 48 amino acids at the C terminus (Fig. 2).
(ii) Coding region N-terminal to the putative coat protein
The region to the N-terminal side of the putative coat
protein has the capacity to encode a 132K to 145K
polypeptide depending on which A U G acts as the
translation initiation site. It is not known how many
different protein products are encoded by this region or
what their functions are. However this region can be
divided into several domains based on similarities in
amino acid sequence and genomic organization with
other nepo- and comoviruses as well as some unique
features of the TomRSV RNA-2 polyprotein itself.
The TBRV and G C M V RNA-2 polyproteins share
amino acid sequence similarity throughout their entire
length. However, the most highly conserved region is
immediately N-terminal of the putative coat protein
sequences (Brault et al., 1989). We were unable to align
this conserved region with the corresponding region in
TomRSV. Interestingly however, we did detect sequence
identity of 36.7% for over 300 amino acids between the
regions immediately N-terminal of the TomRSV and
G F L V putative coat proteins (Fig. 3). This was the
highest match obtained in comparisons between the
TomRSV RNA-2 polyprotein and those of other
nepoviruses. Only a short region of identity could be
detected in this region between TomRSV RNA-2 and
(a)
TomRSV
(7273 nt)
Repeat 1 554
Repeat 2
SWSSPLPLFANFKVNRGACFLQVLPQRWLPDECMDLLSLFEDOLPEGPLPSF
*****************************************************
607 S W S S P L P L F A N F K V N R G A C F L Q V L P Q R V V L P D E C M D L L S L F E D Q ~ E G P L P S F
**********
**************
* *
** ** *
Repeat 3 660
SWSSPLPLFASFRVNRGACFLQV/a~ARKVVSDEFMDVLP
Fig. 4. Alignment of the amino acid sequence of the three tandem
repeats near the N-terminal region of the TomRSV RNA-2 polyprotein. Asterisks between repeats indicate positions of amino acid
identity. Proline residues and the L-P dipeptides are underlined. The
numbers at the beginning of each line refer to amino acid position on
the long TomRSV RNA-2 polyprotein.
AUGAUG
1781 441
VpG
Coat protein
[
~11
(b)
RNA-2 of TBRV or GCMV. It has been suggested, by
analogy with the M RNA-encoded proteins of comovirus
CPMV, that this region may encode a cell-to-cell
transport function (Meyer et al., 1986; Wellink & van
Kammen, 1989). If so, the difference between the
T o m R S V / G F L V and T B R V / G C M V sequences may
reflect slightly different mechanisms to potentiate virus
movement throughout infected plants.
An internal region of TomRSV RNA-2 from nucleotides 1812 to 2244 consists of three tandem repeats. Two
of the repeats were near perfect and 159 nucleotides in
length whereas the third was degenerate and 114
nucleotides in length. This region fell almost completely
within the overlap portion of clones K6 and 035, and the
presence of the repeats was therefore confirmed by two
independent clones. In addition, part of the repeated
sequence was confirmed by dideoxynucleotide sequence
analysis using oligonucleotide no. 2 as a primer and viral
R N A as template. Sequence analysis indicated that this
region encodes two identical amino acid repeats 53
amino acids in length, and one partial and degenerate
UAA
[ 5724
I
po|y(A)
035
,~.
2
AUG AUG
I 8] 233
K6
Coat protein
UAG
[ 3560
GFLV
(3774 nt)
AUG
I 288
Coat protein
AUG
[ 218
Coat protein
U~G4361
TBRV
(4662 nt)
U/~G4190
GCMV
(4441 nt)
Fig. 5. (a) Locationof the cDNA clones 035 and K6 relative to TomRSVRNA-2. Arrowslabelled 1 and 2 refer to oligonucleotidesno. 1
and no. 2, respectively.(b) Comparison of the TomRSV, TBRV, GCMV and GFLV RNA-2 components. Thick lines represent
nucleotidesequenceand the largeboxedregionsindicate the longORFs. Amino acid sequencesimilarityis indicatedby similar shading
within boxed regions.The diagonal shading within the TomRSV sequenceindicates the three tandem repeats. Boxedregions without
shading do not share amino acid sequence similarity. Numbers refers to nucleotide (nt) positions on the respective RNAs.
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T o m R S V R N A - 2 sequence
repeat of 38 amino acids (Fig. 4). The sequence has a
high proline content (12.1%) and the dipeptide L-P is
repeated 13 times. The significance of this repeated
region, the high frequency of P or the presence of the L-P
dipeptide is unknown. A search of the NBRF, GenBank,
EMBL, Swiss-Prot and Pseqlp databases failed to detect
significant amino acid sequence similarity with any
other sequence.
In conclusion TomRSV RNA-2 has many similarities
with the RNA-2 components of the nepoviruses TBRV,
GCMV and GFLV, as well as with the M R N A of
CPMV. These include a 5' VPg, 3' poly(A) tail,
expression by cleavage of a polyprotein, location of the
coat protein-coding region at the C terminus of the large
polyprotein, and some sequence similarities within the
N-terminal region of the long polyprotein. TomRSV
RNA-2 also has some unique features which can account
for the much larger size of its R N A in comparison to
TBRV, GCMV and GFLV. These features include a
very long 3' non-coding region of 1550 nucleotides, a
larger putative coat protein-coding region and repeated
elements within the N-terminal coding region. A
comparison of the genomic organization of the RNA-2
components of TomRSV, TBRV, GCMV and GFLV is
shown in Fig. 5. We believe that the unique features of
TomRSV as compared to TBRV, GCMV and GFLV
confirm the inclusion of TomRSV in a separate subgroup
of nepoviruses (Martelli, 1975; Harrison & Murant,
1977).
A grant from NSERC of Canada to J. H. T. providing financial
support for M. E. R. is gratefully acknowledged.
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