Journal of General Virology (1992), 73, 2473-2477.
2473
Printed in Great Britain
The nucleotide sequence of red clover mottle virus bottom component RNA
M. Shanks and G. P. Lomonossoff*
Department of Virus Research, John Innes Institute, John Innes Centre, Colney Lane, Norwich NR4 7UH, U. K.
The complete nucleotide sequence of the bottom
component RNA (B RNA) of red clover mottle virus
strain S has been determined. The sequence consists of
6033 nucleotides and contains a single long open
reading frame sufficient to encode a protein of Mr
210258. The proteolytic processing sites within this
protein have been deduced by comparison of its
sequence with that of the B RNA-encoded protein of
cowpea mosaic virus. Comparison of the amino acid
sequences of the individual proteins confirms that the
two viruses have a similar genome organization.
Red clover mottle virus (RCMV) is a member of the
comovirus group of plant viruses. Its genome consists of
two molecules of positive-strand (messenger-sense)
RNA which are encapsidated separately in isometric
particles termed middle (M) and bottom (B) components.
Three distinct strains of RCMV, S, N and O, have been
described, and these differ in host range and symptomatology (Oxelfelt, 1976). In the case of strain S, both
RNAs have been shown to be polyadenylated (Oxelfelt,
1976; Shanks et al., 1986), and the M RNA has been
shown to have a small protein (VPg) linked to its 5' end.
We have previously determined the complete nucleotide
sequence of the M RNA of RCMV strain S (Shanks et
al., 1986) and the sequence of a 771 nucleotide portion of
the B RNA sequence which allowed us to deduce the
amino acid sequence of the virus-encoded protease (the
24K protease) and the VPg (Shanks & Lomonossoff,
1990). In this paper we report the complete nucleotide
sequence of B RNA, thereby completing the sequence of
the entire genome of RCMV strain S.
The construction of cDNA clones specific for the 3'terminal 4-5 kb of the B RNA of RCMV strain S has
been described previously (Shanks & Lomonossoff,
1990). The clones, including Sma/Pst-2 and Taq-7, the
partial sequences of which have already been reported
(Shanks & Lomonossoff, 1990), were sequenced using
the dideoxynucleotide method (Biggin et al., 1983) either
directly or after subcloning restriction enzyme fragments
derived from them into bacteriophage M13 vectors. In
this way, the sequence of 4373 nucleotides extending
from the PstI site at the left end of clone Sma/Pst-2
(position 1660 of the final sequence) to the 3' end of the B
RNA was determined. To obtain further sequence data,
double-stranded cDNA was synthesized as described
previously for M RNA (Shanks et al., 1986) using the
oligonucleotide d(GGTATTTTCCTAACACC), complementary to nucleotides 1680 to 1696 of the B RNA
sequence, as a primer for first-strand synthesis. The
resultant cDNA was digested with a variety of restriction
enzymes, and the fragments were cloned into appropriately linearized bacteriophage M 13 vectors. Analysis of
the resulting clones enabled the sequence of all but the 5'terminal 33 nucleotides to be determined.
To complete the sequence of B RNA, the oligonucleotide d ( G T C T C A A G C A G A A A A G A G A G ) ,
complementary to nucleotides 67 to 86 of the final sequence, was
kinase-labelled and used to prime cDNA synthesis in the
presence of dideoxynucleoside triphosphates (Meshi et
al., 1983). The sequence obtained enabled all but the
extreme Y-terminal base to be identified. This was
identified by labelling the VPg presumed to be attached
to the 5' terminus of B RNA with 125I as described
previously (Lomonossoff et al., 1985), and digesting the
labelled RNA with a variety of specific nucleases. The
digests were analysed by electrophoresis on SDSpolyacrylamide gels and the identity of the Y-terminal
base was deduced as described previously for RCMV M
RNA (Shanks et al., 1986). The results obtained
confirmed the presence of the VPg at the 5' end of B
RNA and showed that the 5'-terminal residue of B RNA
is a U (data not shown).
The complete nucleotide sequence of B RNA from
RCMV strain S is shown in Fig. 1, and is 6033
nucleotides long, excluding the poly(A) tail. Apart from
the 5'-terminal 33 bases, the entire sequence was
determined in both directions using cDNA clones and
each base was sequenced 3.5 times on average. In
The nucleotidesequencedata reported here have beensubmittedto
the EMBLdatabase and assignedthe accessionnumber X64886.
0001-1010 © 1992 SGM
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~ L K J ~ C C G C A A A C G ~ C ,
i0
30
A A A H C C A A A A ~ C U ~ C A A A ~ C U U U U C U C ~ C ~ ~ C U ~
50
70
90
ii0
~CAAAUUL~Ut~CUGGAACAUCGAGCGAUCC~CCAAHACUACCJt~~CL~~CCUA~CUCA~~
13o
15o 3 2 K
17o
19o
21o
M Y M L T F E P C L C V A G I
250
270
290
230
I ~ Q V R S N P F M H V V Q A Y
310
330
350
A R T T E T Y R E D I E M T K S M L K L K A D E P L L V M S I V A A A M D F Q T
AUC~CGC.ACAA~GAAACL"./ACCG~GAUALK3C~CAAAAUC~U
G C U C ~ C U U U A C U ~ ~ ~
370
390
410
430
450
470
M V M A P I E M E A S E F L Y G F Y A E R M S Y I V T N R G M S E L H E Y I Q L
C A A ~ C C A A ~ . G C U U C U ~ ~ C U A U C - C A C ,
A A A ~ U U G U G A C A A A U A G ~ C ~ C ~ ~
490
510
530
550
570
590
Q C Q R H L L V K V E I D G Q Y L V Q E H E Y E A Q G F N I K R V K E L I T D V
UGCA~CAAAGA~UCUACU~CAAGGUGGA~UUC~CAGUACt~CC~
_UCAA~GAAUUAAU~
610
630
650
670
690
710
A T W V P K K V K G M I G W S V D A V L D S F Q E Y F Y K V I T E R I P M A M K
UUG~CUUC~CCCAAAOAAA~CAAC~UGA~G~CUGUA~CGCA(VJUCUAGA~CUUUU~GC~CU~UAA~~C~
730
750
770
790
810
830
V C S W V A T V W D Q I K T W I E D A M T A M S S F L Q G C N E L L T W G L A T
AC<~J~J~7JUCALv'.C~r'~C~CCAAAUCAAGA~UGGAL~.C*~UGACUGC~U(~JcUAGUUUU~GC~cGAAUUACUAA~~
850
870
890
910
930
950
L A A C C A L N V L E R I L I F M E F L D E S I D I A G I F L R T G V V A A A C
~CUGGCAGCAUGCUGUGCUcUAAAUGU~~CUAAUA~UGGAAU~3UAUUGA~GCAGGCA~CCUUCGGACUGGAGUGG~GCAGC~U
970
990
1010
1030
1050
1070
Y H F S S T A K G F T E M M S V L S V A T T A V A A V V C A N Y F G G S K T K K
GUUACCAU~f~JUC~CC~GGAUU~CAGA~CUGUCCUUU~CGCAACAACAGCCGUAGCUC~CU~UUACL~CCAAAA
lO9O
W 58K
nlo
1130
1150
i170
119o
!
V N A Q G N P V D L L E R I A A G L S S I S Q D S L V S L G K S C S A I N S I A
AGG~GCAAG~UC~GUA~UL~UU~F~<L~GGUCUGUCUA(~/AUAUCCCAAGAUU~UUC~UCCCUC~CCUGUAGUGCUA~G
1210
1230
1250
1270
1290
1310
T S Y G H L R N F A G R V L T M L R D F A W K I L G L E T R F L A D A A L V F G
CUACAAGCUAUGGA~U~GUAACUUCGCAGGUAGAGUUUUAACUAUGCUUAGGGAV~UGCCt~UL~GAAA~CGU~UCCU~~~CG
1330
1350
1370
1390
1410
1430
E D V D G W L Q R I S A L R E A Y V S K A Y S S Q D E V F E M N V L L E R G Y K
G A G A A G A C ~ ~ I ~ U U A ~ J C < T J U U G ~ F J A G A G G C C U A U G L ~ C A ~ ~ ~ C C U ~ ~
1450
1470
1490
1510
1530
1550
M R H L M A T G S R V S P A I G N M L M Q G L A D L E R L H R N A A V Q G V K G
AAAUGAGACAUCUA~CAGGCUCUAGA~CCCCUGCA~CAUGCUCA~Ct~~JACAGGGCCKrJAAAG
1570
1590
1610
1630
1650
1670
V R K I P F T V F A H G N S R C G K S L L I G K L I S D F Q E H K G L G E D T V
GU(~CCU~CAGUCUUUGCUCA~GGUAA~C~GAU~CUACUUA~CUCAUAAGCGAUUUCCAAGAACA~CUUC~GA~
1690
1710
1730
1750
1770
1790
Y S R N T T E T H W S G Y R R Q P I V V I D D F A A V E S D I S A E A Q L I N L
UGUA~CUCC~C~CUGAAACGCACUC~G~AA~CAACCUA~(~JGGUGAt~
_UCUGAUAU~CUC~GGCGCAAC~UC
1810
1830
1850
1870
1890
1910
V S S T P Y S V V M A A I E E K G M T F D S Q F I F A S T N F L E V S P N G K I
UUG~CCCUAUU~
~ C U ~ C U C ~ d U C A ~ C U A C C A A ~ C U G G A A ~ C C U A A U ~
1930
1950
1870
1990
2010
2030
R C D D A F R N R R H V L I D V K L K P E V E Y Q S D D F T A N Q S Y N I L E H
UAAGAL~UGAUGCUUU~GAAAUAC~CAU~UUGAUGUGAAGC~CCUC~CCAGAGUGAUC~~~~
2050
2070
2090
2110
2130
2150
S H G R Y N V V A T F D N Y E E L L A Y C L T K H E Q H E A E Q E A N L A K L R
A~UGGAAGAUACAAUGL~CU~UAACUAUGAGC~,C~CUGU~UGACCAAACAUGAACAA~UGAAGCCGAGCAGGAAGCCAA~C~
2170
2190
2210
2230
2250
2270
R T N K F E S H F K K F E Q V L Q L S T Y F S S S I E R I K R E A L A T T D G A
GU~D_C~
_ ~ C C ~ V O U ~ C A A G U G C U A ~ C ~ L r 0 U C A G C U C U U C ~ U A G A G A G A A U ~ ~ C ~
2290
2310
2330
2350
2370
2390
D D Y H L L Y V V P R N G S Y L H V A A N K D F Q I Q Q W Y G P V E E V A E E D
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2410
2430
2450
2470
2490
2510
I L R A S E R M L L G A Y E F L L L S T E L N V V V K N H L P E L I C T D N Y D
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C~(Xru-JtrdACUACUUUC~AACt~C-AAAAAUCAUCUAC
_ C
2530
2550
2570
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2610
2630
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H N L E F C G V V G D P V Y H Q Q L L K N I R A L K P W H R A V L F G I G T L M
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2650
2670
2690
2710
2730
2750
G A K N P T P W Y K R M W E G I K D V L Y K A Y S T E I S Q W P V P L K I T C G
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2770
2790
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2810
2830
2850
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3030
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3350
Short communication
S
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3410
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3850
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3810
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4550
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4650
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4390
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4630
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5790
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5930
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D
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F N E I L V R
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4790
L
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4910
N
A
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K T S A T L E F R R L E
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5010
5030
D
C
4890
M
Q
V
T
T
D
L
P D H N D S L D M L T S V D L L G P A
CCOJC~CCACAAUGACA6~CUUGA~~CtL~CUAGGACCAGCAAUCCUGG
5350
5370
5390
I
L
G
Q
L
H
Y
V
N
V
N
N
C
5130
G
R
C
L R Y H T V
~ C A C U G U U C
5470
P
Q
P
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R
G
G
L
L
A
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L
R
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L A L K S I P W
C C A C A G A U C
5250
N
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T Q H W L
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5610
N
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S
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U
5950
L
N
5270
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E
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L F N T M
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5510
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N
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5630
E
C
Q
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5850
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S
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A R N K V
C.AC~~ C A A G G U G A
5750
M
~
K D N I L P I N V I A V L L
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JC~ CUAU ~ C U C - C H A C ,
5710
5730
5830
U
5150
5490
5590
5810
C
T
5570
I
L
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5690
L
I
G F
C
4750
5110
5450
~
5910
(
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H D P T E M V E F
C A U U C C U U C ~
5210
5230
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4990
5330
S N C H S F T
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5550
A
I
~
N
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4670
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V
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Q
4430
L
F
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4410
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I
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4070
P
L
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4950
P C L S G
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3950
4050
L K R S S V L W D A
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5070
5090
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4730
H
L
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3930
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4610
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5770
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F
5670
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C
3690
3710
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5890
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4250
5430
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4970
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4490
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3590
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A C A ~ C U G A ~
4850
4870
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5410
S
L
S
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3470
S
P T L Y Q V K
U A C C A A C A ~ C ~
5290
K L I
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~
5650
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N L V N V L R E L
C C U A C U G A ~ C
5190
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W
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CCUUUC~CAOJCAU~
4770
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5050
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G C ~
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4830
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4130
4150
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4190
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4810
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3550
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6030
Fig. 1. The complete nucleotide sequence of RCMV strain S B RNA. The amino acid sequence encoded by the long ORF is shown
above the nucleotide sequence using the standard one-letter code. The proteolytic cleavage sites proposed to be used to release the
RCMV equivalents of the CPMV 32K, 58K, VPg, 24K and 87K proteins are indicated by arrowheads. The CPMV 32K, 58K, 24K and
87K proteins are believed to be a protease cofactor, a membrane-binding protein with a nucleotide-binding site, the viral protease and
the viral RNA-dependent RNA polymerase, respectively.
addition, most o f the s e q u e n c e was deduced from more
than one i n d e p e n d e n t l y isolated clone. T h e overall base
c o m p o s i t i o n o f B R N A is 2 9 . 2 ~ U , 1 8 . 2 ~ C, 2 9 . 5 ~ A
and 23.1 ~o G, figures w h i c h are similar to those reported
for M R N A (Shanks et al., 1986). T h e g e n o m i c R N A s o f
R C M V strain S have previously been s h o w n to have
similar sequences at their 3' ends (Shanks et al., 1986), a
situation also found in c o w p e a m o s a i c virus ( C P M V )
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2476
Short communication
(Davies et al., 1979). Comparison of the sequence
reported here with that of M R N A shows that the 5'terminal sequences of the R N A s are also very similar,
with 50 of the first 55 residues being identical. R C M V B
RNA
also possesses the 5' structure V P g U A U U A A A A U which is common to all comovirus
RNAs sequenced to date (Stanley & van Kammen, 1979;
Shanks et al., 1986; MacFarlane et al., 1991).
Inspection of the B R N A sequence reveals the
presence of a single long open reading frame (ORF)
beginning with an A U G codon at position 270 and
terminating with a U G A codon at position 5862. This
O R F is sufficient to encode a protein of 1864 amino acids
with a calculated Mr of 210258. Unlike the situation
found for CPMV B RNA, the A U G at the start of the
long ORF is not the 5' proximal one, an upstream out-ofphase A U G occurring at position 14. This A U G is in
phase with a U A A termination codon at position 92 and
thus could direct the synthesis of an oligopeptide of only
26 amino acids. Also, in contrast to the situation found
with CPMV B RNA, the A U G that commences the long
O R F is not in an optimal context despite having an A
residue at the - 3 position (Liitcke et al., 1986) because it
has a U rather than the optimal G residue at the + 4
position. Both these features might be expected to reduce
the efficiency of translation of R C M V B R N A in
comparison with that of the B R N A from CPMV.
Comparison of the amino acid sequences encoded by the
long ORFs of the B R N A s of C P M V and R C M V reveals
that they are clearly homologous. As calculated by the
Gap program (Devereux et al., 1984), the proteins are
56.6% identical, a figure which rises to 74.7% when
similarities in amino acids are taken into account. The
similarities between these B RNA-encoded polyproteins
strongly suggest that they will undergo a similar pattern
of proteolytic processing, and it was on this basis that the
potential cleavage sites between the R C M V equivalents
of the CPMV B RNA-encoded 58K protein, VPg, 24K
protease and 87K protein were deduced (Shanks &
Lomonossoff, 1990). The data presented here now allow
the potential cleavage site between the R C M V equivalents of the CPMV B RNA-encoded 32K and 58K
proteins to be assigned (Fig. 1). All the cleavage sites
identified in the RCMV B RNA-encoded polyprotein
have glutamine (Q) at the - 1 position and alanine at the
- 2 position, and either glycine (G), serine (S) or
methionine (M) at the + 1 position. In this respect they
closely resemble the cleavage sites found in the CPMV B
RNA-encoded polyprotein, except that the latter have
either alanine or proline at the - 2 position (Lomonossoft & Shanks, 1983; Wellink et aL, 1986).
Assuming the cleavage sites within the R C M V B
RNA-encoded polyprotein have been correctly identified, the sizes of the individual B RNA-encoded proteins
Table 1. Properties of the R C M V B RNA-encoded proteins
Similarity with
CPMV proteins (%)t
Protein
No. of
aminoacids
Mr*
Direct
Familial~
32K
58K
VPg
24K
87K
315 (326)§
600 (593)
28 (28)
208 (208)
713 (712)
35580(36449)§
67130 (66292)
3480 (3538)
23583 (23319)
80557 (79970)
41.4
50-3
75.0
54.8
61.6
63.2
71.2
89.2
74-0
75-9
* Calculated from the amino acid sequence.
Calculated using the Gap program (Devereuxet al., 1984)with a
gap weight of 3-00 and a gap length weight of 1-00.
Using the substitution of related amino acids as defined by Kamer
& Argos (1984).
§ Figures for the equivalent CPMV proteins are shown in
parentheses.
can be calculated and their homology to their CPMV
counterparts assessed (Table 1). The motifs associated
with nucleotide binding, the proteolytic activity and the
RNA-dependent R N A polymerase activity found in the
CPMV 58K, 24K and 87K proteins, respectively, are
strictly conserved in the equivalent R C M V proteins. The
protein with the least similarity to its C P M V counterpart
is the 32K protein, a protein which, in the case of
CPMV, has been shown to act as a cofactor enabling the
24K protease to cleave the M RNA-encoded polyproteins (Vos et al., 1988). Though it had been demonstrated
that the proteolytic activity associated with CPMV is
unable to cleave the polyproteins encoded by the M
R N A of RCMV (Goldbach & Krijt, 1982), comparison
of the sequences of the respective 24K proteases did not
provide any explanation for this specificity (Shanks &
Lomonossoff, 1990). The comparatively low similarity
between the 32K proteins of R C M V and C P M V makes
it plausible that it may be this protein, rather than the
24K protease itself, which determines substrate specificity in regard to cleavage of the M RNA-encoded
polyproteins.
We thank Dr J. Stanley for critically reading the manuscript.
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