cDNA libraries representing the P-PAV and NY-RPV. BYDV isolates were similarly prepared in 2gtl 1, pUC18 and pGEM-. 3Z. The 2gtl I libraries were screened ...
Journal of General Virology(1990), 71, 2791-2799. Printedin Great Britain
2791
Nucleotide sequences of coat protein genes for three isolates of barley yellow dwarf virus and their relationships to other luteovirus coat protein sequences J. R. Vincent,* P. P. Ueng,t R. M. Lister and B. A. Larkins~ Department of Botany and Plant Pathology, Lilly Hall of Life Sciences, Purdue University, West Lafayette, Indiana 47907, U.S.A.
Barley yellow dwarf virus (BYDV) can be separated into two groups based on, among other criteria, serological relationships that are presumably governed by the viral capsid structure. Nucleotide sequences for the coding regions of coat proteins of approximately 22 K were identified for the MAV-PS 1, P-PAV (group 1) and NY-RPV (group 2) isolates of BYDV, The MAV-PS1 and P-PAV coat protein sequences shared 71% deduced amino acid similarity whereas that of the NY-RPV isolate shared no more than 51% similarity with either the MAV-PSI or the P-PAV sequence. Other comparisons showed that these and other BYDV coat protein sequences examined to date share a high
degree of identity with those identified from other luteoviruses. Among luteovirus coat protein sequences in general, several highly conserved domains were identified whereas other domains differentiate MAVP S I and PAV isolates from NY-RPV and other luteoviruses. Sequence similarities and differences among BYDV coat proteins (approx, 22K) are consistent with the serological relationships exhibited by these viruses. Amino acid sequence comparisons between BYDV isolates that share common aphid vectors indicate that it is unlikely that these coat proteins are involved in aphid specificity.
Introduction
logical ultrastructure of infected cells (Gill & Chung, 1979), and the profiles of dsRNAs obtained from infected tissue (Gildow et al., 1983). Group I includes the representative isolates: MAV, transmitted by Macros# phum (= Sitobion) avenae Fabr. ; PAV, transmitted by M. avenae and Rhopalosiphum padi L. ; and SGV, transmitted by Schizaphis graminum Rond. (Rochow, 1970a). Group 2 includes the representative isolates: RMV, transmitted by R. maidis Fitch. ; and RPV, transmitted by R. padi (Rochow, 1970a). The differences in both serological and aphid vector relationships among BYDV isolates are presumed to reflect differences in viral coat proteins (Rochow, 1970b). The characterization of coat protein genes from different isolates should therefore provide a more detailed understanding of the serological and vectorial relationships exhibited by these viruses. Coat protein nucleotide sequences have been obtained from several luteoviruses, including potato leafroll virus (PLRV) (Kawchuk et al., 1989; Prill et al., 1989), beet western yellows virus (BWYV) (Veidt et al., 1988) and a PAVlike BYDV isolate from Victoria, Australia (Miller et al., 1988a), herein referred to as Vic-PAV. Furthermore, complete genome nucleotide sequences have been reported for BWYV (Veidt et al., 1988), PLRV (Mayo et
Luteoviruses cause yellowing diseases on a wide range of host plants (Matthews, 1982). They are transmitted obligately by aphid vectors in a persistent, circulative manner and are limited to the phloem tissue of the host plant. Many luteoviruses exhibit some degree of serologically inter-relatedness (Waterhouse et al., 1988; Rochow & Duffus, 1981), and yet they can be grouped into clusters based on the closeness of the serological relationships. Barley yellow dwarf virus (BYDV), the type member of the luteoviruses (Matthews, 1982), comprises a group of serologically related viruses that infect barley, oats, wheat, rice and other graminaceous hosts (Rochow, 1970a). Isolates of BYDV were originally distinguished by, and named according to, their predominant aphid vectors (Rochow, 1970a). They can also be separated into two major groups based on serological relationships (Aapola & Rochow, 1971; Rochow, 1970a; Rochow & Carmichael, 1979; Rochow & Duffus, 1981), cytopathot Present address: USDA-ARS,BARC-West,Building 006, Beltsville, Maryland 20705, U.S.A. :~Present address: Department of Plant Science, University of Arizona, Tucson, Arizona 85721, U.S.A. 0000-9689 © 1990SGM
2792
J. R . V i n c e n t a n d others
al., 1989; K e e s e et al., 1990) a n d the V i c - P A V isolate of B Y D V (Miller et al., 1988b). W e previously reported the c o n s t r u c t i o n of a c D N A library r e p r e s e n t i n g a n M A V isolate of B Y D V a n d a d e s c r i p t i o n o f clones believed to c o n t a i n the coat p r o t e i n coding region ( B a r b a r a et al., 1987). I n this work we present the coat p r o t e i n nucleotide sequences for three different isolates o f B Y D V r e p r e s e n t i n g a range of serotypes, a n d e x a m i n e the relationships a m o n g these isolates a n d other luteoviruses based o n the d e d u c e d a m i n o acid sequences of the coat proteins.
3
4
IIIK-,~••:Li:~•i~i-
58"5K-:•';•7"."••• ~••
•••
35.4K-"
25.7K........... ~""~
Methods Viruses, cDNA cloning, and library screening. The BYDV isolates used were as follows: (i) a subculture of the New York MAV (NY-MAV) isolate of Rochow, maintained at Purdue University and designated MAV-PSI (Barbara et al., 1987; Lister & Sward, 1988), (ii) the P-PAV isolate of Hammond (Hammond et al., 1983) and (iii) the New York RPV isolate of Rochow (NY-RPV) (Rochow, 1970a) as maintained at Purdue University. All virus isolates were maintained in oats (Arena sativa L. cv. Clintland-64) and checked by ELISA for crosscontamination as previously described (Barbara et al., 1987). Virus was purified and the RNA extracted as described for the MAV-PS1 isolate (Barbara et aL, 1987). The construction of the MAV-PS1 cDNA library, the cloning of the cDNA into 2gtl 1, and the subcloning from 2gtl 1 into pUC 18 (Vieira & Messing, 1982) were previously described by Barbara et al. (1987). Subsequently, the MAV-PS1 cDNA was cloned into pGEM-3Z (Promega). cDNA libraries representing the P-PAV and NY-RPV BYDV isolates were similarly prepared in 2gtl 1, pUC18 and pGEM3Z. The 2gtl I libraries were screened for immunologically recognizable lacZ fusion proteins (Young & Davis, 1983) with serotypespecific polyclonal antisera prepared against purified virus, and therefore presumed to identify the viral coat protein-coding region. Immunoreactive 2 clones were then used as hybridization probes in colony hybridizations to identify similar sequences in clones from the plasmid libraries. Hybridization probes were labelled with [~32p]dATP (Amersham) by either nick translation (Maniatis et al., 1982) (nick translation kit, Amersham) or random multiprimer labelling (Feinberg & Vogelstein, 1983) (multiprime labelling system, Amersham). Restriction maps representing each viral genome were generated by single and double enzyme restriction digests and by Southern blot hybridization between different restriction fragments. cDNA sequencing and verification of the N Y - R P V coat protein gene. Plasmid DNA isolated by alkaline lysis techniques (Birnboim & Doly, 1979) was sequenced by the dideoxynucleotide chain termination method (Sanger et al., 1977) with a modified T7 DNA polymerase (Sequenase). Both DNA strands from clones representing the antigenic regions of the viral genomes and neighbouring regions were sequenced by one or more of the following strategies: (i) restriction fragments representing overlapping regions of the genome were subcloned; (ii) Bal 31-generated deletions in existing cDNA clones were identified (Guo et aL, 1983); (iii) exonuclease III/mung bean nuclease-generated nested deletions were identified in existing cDNA clones (Putney et al., 1981); (iv) to sequence regions inaccessible by other methods, synthetic oligonucleotide sequencing primers were prepared (Laboratory for Macromolecular Structure, Department of Biochemistry, Purdue University). Sequence analyses were performed with Microgenie (Beckman) and the Genetics Computer Group sequence analysis software, version 6.1 (Devereux et al., 1984).
20-1K--
16.2 -
- :--7••••
••
•••
i,
•/:
Fig. 1. SDS-PAGE of coat proteins from three isolates of BYDV. Virions were disrupted with SDS and the coat proteins were separated through 15% polyacrylamide. Lane 1, Mr standards; lane 2, P-PAV; lane 3, MAV-PSl; lane 4, NY-RPV.
A 654 bp AccI fragment containing the entire putative NY-RPV coat protein gene was cloned into pGEM-3Z (to make pRPVCP5) in the sense orientation relative to the T7 RNA polymerase promoter. A restriction fragment containing the putative translation initiation AUG codon of the NY-RPV coat protein gene was removed from pRPVCP5. This construction (pRPVCP5d46) deleted the 5' 46 bp from pRPVCP5. After linearization with HindIII, T7 RNA polymerase (Promega) was used to generate in vitro transcripts (Krieg & Melton, 1987) from pRPVCP5 and pRPVCP5d46. These transcripts were translated in vitro with rabbit reticulocyte lysates (Pelham & Jackson, 1976) (Promega) containing [3SS]methionine(Amersham). The translation products were separated by discontinuous SDS-PAGE (Laemmli, 1970) through 15% polyacrylamide and compared with purified viral proteins. The gels were stained with Coomassie blue R250 prior to autoradiography.
Results Identification a n d verification o f the coat p r o t e o l - c o d i n g regions
Based o n s e p a r a t i o n by S D S - P A G E , m i n o r differences in the size of the capsid proteins of the M A V - P S 1 , P - P A V a n d N Y - R P V B Y D V isolates were detected (Fig. 1). W i t h reference to Mr markers, the m i g r a t i o n of M A V - P S 1 , P - P A V a n d N Y - R P V coat proteins corres p o n d e d to 21.8K. 21.9K a n d 22-9K respectively. I n the course of a n a l y s i n g the nucleotide sequence of the M A V PS1, P - P A V a n d N Y - R P V g e n o m e s (P. P. U e n g et al. a n d J. R. V i n c e n t et al., u n p u b l i s h e d results), single o p e n
B YD V coat protein sequences
2793
Table 1. Comparison of luteovirus coat proteins and 17K internal O RF-encoded proteins Internal Coat
protein
ORF-encoded protein
Virus
Mr
Bases
Mr
Bases
Reference
MAV-PS1 P-PAV NY-RPV Vic-PAV BWYV (FL1) BWYV (GB1) PLRV-1 PLRV-2
21.9K 22.0K 22.2K 22.0K 22.5K 22-5K 23"2K 23-2K
597 600 612 600 606 606 624 624
17'2K 17.1K 17-2K 17.1K 19.6K 19.7K 17.3K 17.4K
459 456 459 456 525 525 465 465
This work This work This work Miller et al. (1988a) Veidt et al. (1988) Veidt et al. (1988) Prill et al. (1989) Kawchuk e t al. (1989)
reading frames (ORFs) were identified that corresponded to the apparent size of these capsid proteins. The coding region for the putative MAV-PS1 coat protein is 597 bp and would encode a 21934 protein (Fig. 2a). The coding region for the putative P-PAV coat protein is 600 bp and would encode a 21981 Mr protein (Fig. 2b), whereas the NY-RPV putative coat proteincoding region is 612 bp and corresponds to a 22190 Mr protein (Fig. 2c). All three regions of these cDNA sequences also contained a second ORF within the coat protein-coding region in a different frame (Fig. 2). These internal ORFs, which would produce proteins of Mr 17181, 17124 and 17211 for MAV-PS1, P-PAV and NYRPV, respectively, are thought to correspond to the VPg for each of these BYDV isolates (Miller et al., 1988a; Murphy et al., 1989). Sequencing of the MAV-PS1 and P-PAV coat proteincoding regions also identified occasional nucleotide heterogeneities. These are located at position 300 for MAV-PS1 and positions 225 and 370 for P-PAV (Fig. 2). Although these variations generally did not alter the amino acid sequence (except P-PAV position 225), it is not known whether these differences represent authentic variability within the viral genome or whether these substitutions represent cloning artefacts. The lengths of the identified coding regions, and the predicted molecular sizes of the capsid proteins and the VPgs correspond to similar proteins that have been characterized from other luteoviruses (Table l). In vitro translation of the m R N A transcript from pRPVCP5 yielded three major proteins, the largest of which comigrated with the authentic NY-RPV coat protein (Fig. 3, lanes 2, 3 and 4); this provided additional evidence that these ORFs corresponded to the coat protein sequences. The 22K protein was also immunoprecipitated with NY-RPV antisera. Removal of the first A U G from pRPVCP5 yielded only smaller Mr products (Fig. 3, lane 5). Based on the Mr markers, both
of these proteins migrated corresponding to Mr values larger than expected for the VPg. Presumably one of these proteins corresponds to the internal ORF for the 17K protein; the other may represent a truncated in vitro translation product.
Sequence compar&ons between coat protein-coding regions Jbr B YD V and other luteoviruses
The amino acid sequence identity of the NY-RPV coat protein ORF with those of MAV-PS1 and P-PAV, as well as the similarity of these proteins with coat proteins of other luteoviruses (Kawchuk et al., 1989; Miller et al., 1988a; Prill et al., 1989; Veidt et al., 1988), provide convincing evidence for their identification. When the nucleotide and amino acid sequences of these luteovirus coat proteins were gapped and aligned to maximize their similarities, obvious relationships between them were apparent. At the nucleotide level, identities extend from near 55%, in comparisons between NY-RPV (or BWYV or PLRV) and the MAV-PS1 or Vic-PAV isolates, to 95% when the different PAV isolates are compared to each other. Specifically, comparisons of MAV-PSI and NY-RPV with P-PAV show 76% and 56~ identity, respectively (Table 2). Most noticeable in these comparisons is that the NY-RPV sequence has greater identity to the sequences from BWYV and PLRV than to those from the other BYDV isolates. Coat proteins of the various luteoviruses can be separated into two domains: an amino-terminal domain of variable length which ranges from 60 to 69 amino acids (Fig. 4), and a carboxy-terminal (C-terminal) domain which essentially contains an identical number of amino acids for all luteovirus coat protein sequences (Fig. 5). A distinctive feature of the amino-terminal domain of the luteovirus coat protein is the occurrence of two, highly basic regions separated by a region of non-
2794
J. R. Vincent and others (a) BYDV coat protein gene: MAV-PS1 isolate M
N
S
V
G
R
R
N
N
R
R
R
N
G
M
P
A
R
g
R
G
A
E
R
g
R
G
V
S
A
L
A
A
V
Q
R F
R
G
M
E
V
V
~g L
V
Q
g
S
P
N
N
R
P
l
A
E
P
AUGAAUU•AGUAGG•CGUAGAAA•AAC•GCAGGAGGAAUGGC••AAGGAGAG•AAGG•G•GUUAG•GCAGUUCGGAGAAUGGUUGUGGU•CAACC•AAU•GAG•C 20
10
G P D
K
Q
R
N
R A
D
E
30
R L
R
V
R
D
40
T
A
50
R G G G A
O
E
E
E
G
N
Q
60
L
]
]
L
S Y
70
G P
L
D
A
O
80
G R
Q
A
T
G
E
L
90
V
R
F
Y
V
S
100
F
Y
S
S
V
Q
N D
S
T
L
T
L
GGACCAAAA•GA•GAGCUCGUCGACGCA•AAGAGGAGGAGGGGCAAAUCUUAUAUCUGGACCAGCAGGCAGGACUGAGGUAUUCGUAUU•U•AGUCAA•GACCUU 110 120 130 140 150 160 170 180 190 200 210 (c) I(
A
R
N
P
S
T
S
P
G
Q
T
G
I
Q
K S
F
S
G
S
P
V
D P
L
S
T
F
Q
R
C
N
P
A
A
Q
L
S
R
F
G
Q
G
V
I
E
L Y
K S
S
S
Y
P
H T
L
Y
T
F
K T
I
R
T S
M
Q
T
AAGGCCAACUCCUCAGGGACAAUCAAGUUCGGUCCCGACCUUUCGCAAUGCCCAGCGCUUUCAGGUGGAAUACUCAAGUCCUACCACCUUUACAAGAUCACAAAC (G) 220 V
K
S
V
R
230 E
L
F
S
K
L
240
S
S
H A
H
T
250
S
R
A
P
S
P
T
L
260 V
Q
G A
$
A
270
M F
O
C
I
L
280
E L
L
N
D S
T
T
290 W C
L
G
S
A
300
O
H
S
N
T
O
L
P
310 G S
W V
Y
A
l
T
N L
T
GUCAAGGUUGAGUUUAAGUCACACGCGUCCGCCUCUACAGUCGGCGCAAUGUUUALAJGA;~CUCGACACLAJGGUGCUCACAAUCAACCUUGGGUAGCUACAUUAAC
320 S
330
F
T
H S
l
340
S
P
S
K
S
A
350 T
O N Q Q
K
P
T
K
360
F
P
T
S
A
P
370
O O
P
N
l
D
R
L
380 G K
T
E
G R
390
F
N
R E
S
n
400
S
T
R V
V
N
410 O
F
Y
420
14 L
Y
K
R U
UCAUUCACCAUcUcAAAAUCAGCAAC~AAAAC~UUCACCGCCCAACAGAUUGACGGGAAGGAAUUCAG~`GAGAGUACGGUGAAC~AAUUUUACAUGCUAUACAAG
430 A
N
G
440 S
T
S
450
D
T
A
460
G
O
F
I
470 I
T
I
480
R
V
A
490
N
M
T
P
500
K
5 I0
520
U
GCGAAUGGUAGUACAUCUGAUACCGCCGGGCAAUUCAUCAUUACAAUACGCGUUGCCAACAUGACUCCCAAAUAG 530
540
550
560
570
580
590
600
(b) BYDV coat protein gene: P-PAV isolate M
N
S
V
G
R
R
G
P
R
R
A
N
Q
N
G
T
14 A
R
Q
R
E
R
G
R
G
R
A
R
V
T
E
V
O
R F
P
G
V
Q
V
Id
V L
V
Id
0 S
P N
N
P
R I
E
AUGAAUUCAGUAGG•CGUAGAGGAC•UAGACGCGCAAAUCAAAAUGGCACAAGAAGGAGGCGCCGUAGAACAGUUCGGCCAGUGGUUGUGGUCCAACCCAAUCGA 10 A
G P
Q
D
20
R
P
R
D
R
D
30
N
E
40
G R R
M
V
D
K
A
G R
R
E
50 G G A
E
E
G
60
N
Q
P
l
V
L
70
F
Y
R
L
P
D
T
O
80 G G T
Q
A
G
90
E
L
V
R
F
Y
V
S
1O0 F
Y
S
S
V
O
D
S
N
T
T
GCAGGACCCAGACGACGAAAUGGUCGACGCAAGGGAAGAGGAGGGG•AAAUCCUGUA•AJUAGACCAACAGGCGGGACUGAGGUAUUCGUAUUCUCAGUCGACAAC 110
120
130
140
150
160
170
180
190
200
210
(L) L
K L
A
K
N
P
S
T
S
P
G A
P
I
G Q
K S
F
N
G P
S
A
S
P
L
V
S
Y
Q C
R
P
N A
A
L
Q R
S
D
F Q
G
T
I
E
L Y
K L
S
S
Y
P
H R Y
T
T
V
K
T
I
R
T S
Q
CUUAAAGCCAACUCCUCCGGGGCAAUCAAALAJCGG•CCCAGUCUAUCGCAAUGCCCAGCG•UUUCAGACGGAAUACUUAAGUCCUACCACCGUUACAAGAU•ACA (U) 220
230
240
250
260
270
280
290
300
310
(L) S
[
V
R
S
V
V
E
L
S
F
L
K
S
S
H
H
T
A
R
S
A
P
Q
T
T
L
A
R P
G
k
A
]
L
F
S
L
i
E
L
L
N
O
S
T
T
A
P
R
C
A
K
N
O
S
N e
k
L
P
G
M V
S
A
Y
l
T
L
AGUAUCCGUGUUGAGLAJUAAGUCACACG•GUC•GCAACUACG•cCGGCGCUAUCUUUAUUGAACUCGACACCGCGUGCAAACAAUCAGCCCUGGGUAGCUACAUU (C) 320
330
340
350
360
370
380
390
400
410
420
B YD V coat protein sequences N
S I
F
P
T
S
I
Q
S S
R
A
T
G
A P
A
P
K
O
V
R
F
S
R
S
k
E
E P
A
K
l
R
N L
G
T
K
G
E
R
F
N
Q
S
E
R
S
N
T
Q
I R
D
Q
F
W 14 L
2795
Y
U
AAUUCCUUCACAAUCAGCAGGACCGCC~CAAAGGUCUUCAGAGCCGAAGCGAUUAACGGGAAGGAAUUCCAGGAAUCAACGAUAGACCA~GGAUGCUCUAC
430 K
A
440
N G T
T
450
T
D
T
A
460 G Q
470
F
l
I
T
480
14 S
V
S
490 L
N
T
500
A
K
510
520
U
AAGGCCAAUGGAAC•ACCACUGACACGGCAGGACAA•AJCALAJAUCACGAUGAGUGUCAGLAJUGAUGACGGCCAAAUAG 530
540
550
560
570
580
590
600
(c) BYDV coat protein gene: NY-RPV isolate H
S
T
V
V
L
R
S
N
G
N
A
N
G
N
V
S
R
R
R
A
R
O
R
A
Q
D
R
R
V
E
A
S
R L
R
G
R
E
P
G
A
L
A
L
R
Q
T
E
O R
P
S
V
Q
V
V
14 L
V
W S
AUGAGUAC~GUCGUCCUUAGAUCCAAUGGCAAUGGUUCGCGCAGACGCAGACAGAGAGUCGCUCGGCGAAGGCCUGCUGCAAGAACGCAGCCAGUGGUUGUCGUC
I0 A
S L
N
P
20 G
T
P
A
A
Q
30
R
P
R
G R
G A
E
40 R
R
D A
R
D
50 P
D
V
G
O L
60
P
V
R L
R
G
G E
70 R
E
T
E
P L
80 R
S
Q D
G L
90
G
E
E
G
S
E
R
A
IO0 G
V
E
A
T R
F
V
H S
F
G•UU•CAA•GGCC•AG••AGGC•CGGAAGACGC•GA•GACCAGUUGGU••UCGGCGAGGAAGAACU•CAAGAUCUGGAGGAGGAAGCCGUGG•GAGACAUU•GUU
110 F
120
S
S
K
Q
D
R
S I
130 L
H
A S
R
140
G N A
S
T
P
S
150
G S
L
E
V
I
T S
160 F
P
G P
S
G
170 S
R
L
L
S Y
180
E
Q
Y
S
P I
A
R
190 F
H
Q
S
R
N
200 G V
H
E
L
Y
210
K S
R
A P
Y
H
1" Iq
UUCUCAAAGGAUUCACUCGCGGGCAACUCCUCUGGAAGUAUCACCUUCGGGCCGUCUCUAUCAGAGUAUCCGGCAUUCCAGAAUGGAGUACUCAAGGCCUA•CAU 220 E
Y
N
K l
230 l
T
R
S
N
Q
240
C V
I
V
L
S
Q
Y
S
250 F
V
S
S
S A
260 E
A
R
P
S
270
S
L
P
T
A
Q
A
Q
P
280 G S A
l
P
290 S
S
Y
L
E
T
300
L
S
I,/
D
P
T
H
P
I
310 C
K
A
K
A H
S
S
L
H
GAAUAUAAGAUCACAAAUUGUGUCUUACAGUUCGUCAGCc`AGGCcUCUUCCACAGCAGCCGGCUCCAUCUCUUACGAGUUGr`ACCCCCAUUGCAAAGCAUCUUCA
320 L
330
A S
S
H
q
T
!
Q
340 N
S
I(
I
S
F
350 T
S
I
Q
T S
360
K
P
K
T L
G A V
R
370 R
S
G A
F
380 P
S
A
Q
K
R
R
390
14 I
N
G
400 L
E
4 I0
W H P
S
D
420 E
D
Q
U
CUCGCAUCAACAAUCAAUAAGUUCACAAUCACCAAAACUGGUGCGCGGAGCUUCCCAGCGAAGAUGAUCAACGGGUUAGAGUGGCACCCCUCAGAUGAGGAUCAG 430 F
R
I
440 L
Y
g
450
G N
G A
460 S
S
V
470 A
G S
F
480 K
I
T
490 L
R
V
500 Q
L
g
N
510 P
I(
520
U
uU••GAA•U••UUA•AAAGGGAA•GGUG•UUCUU•UG•GG••GGGUC••U•AAGAUCACUCUUAGAGU••AGC•A•AAAAC•CAAAAUAG
530
540
550
560
570
580
590
600
610
Fig. 2. Nucleotidesequences for the coat protein genes of MAV-PS1, P-PAV andNY-RPVisolatesofBYDV. The deduced amino acid sequence for the coat protein is shown on the top line for each isolate. The internal ORF-encoded sequence corresponding to a 17K protein is shown on the second line. Alternative nucleotides determined from the sequencing of multiple clones representing the coat protein region are indicated in parentheses below the nucleotide sequence. The corresponding alternative amino acids are indicated in parentheses above the amino acid sequence. (a) MAV-PS1; (b) P-PAV; (c) NY-RPV.
polar amino acids (Fig. 4). The amino acids found within the two basic regions are nearly all arginine residues and, as a result, arginine constitutes 60 to 70~ of the amino acids found within the amino-terminal domain. The C-terminal domains from luteovirus coat proteins show a high degree of amino acid conservation. Thirtyfive percent of the amino acids in the C-terminal domains are positioned identically (Fig. 5). Many of
these amino acids are clustered together forming highly conserved regions. Multiple sequence analyses (Devereux et al., 1984) of the C-terminal amino acid sequences from all of these luteoviruses identified a consensus sequence, in addition to separate consensus sequences for the MAV-PS1/PAV group and the NYRPV/PLRV/BWYV group. These analyses showed that whereas some regions are conserved among all luteo-
2796
J. R. Vincent and others
1
2
3
4
5
6
viruses, other regions divide t h e m into a group containing B Y D V isolates M A V - P S 1, V i c - P A V and P - P A V and a group containing B Y D V N Y - R P V , B W Y V and PLRV.
Discussion
Fig. 3. SDS-PAGE separation of NY-RPV coat protein and in vitro translation of putative NY-RPV coat protein mRNA. The coat protein from SDS-disrupted NY-RPV virions and radioactive proteins from in vitro translation of transcripts from pRPVCP5 or pRPVCP5d46 were analysed separately or in combination. Lane 1, Coomassie blue R250stained proteins from a rabbit reticulocyte lysate. Lane 2, Coomassie blue R250-stained proteins from a rabbit reticulocyte lysate containing 20 ~tg of purified NY-RPV. Lane 3, Coomassie blue R250-stained proteins from 20 p.g of purified NY-RPV. Lane 4, autoradiogram of [3sS]methionine-labelled translation products from a rabbit reticulocyte lysate containing in vitro transcripts from pRPVCP5. Lane 5, autoradiogram of [35S]methionine-labelled translation products from a rabbit reticulocyte lysate containing in vitro transcripts from pRPVCP5d46. Lane 6, autoradiogram of [35S]methionine-labelled translation products from a rabbit reticulocyte lysate to which no exogenous RNA was added. The arrows indicate the two major in vitro translation products produced by pRPVCP5 in vitro transcripts. Mr values are indicated on the right.
The viral coat protein sequences for the B Y D V isolates M A V - P S 1 , P - P A V and N Y - R P V share a high degree o f homology. However, nucleotide and a m i n o acid sequences o f N Y - R P V are more similar to those o f B W Y V and P L R V than to other B Y D V isolates. The degree o f sequence similarity a m o n g these coat protein sequences is consistent with the serological relationships by w h i c h B Y D V isolates have been grouped. Thus the group 1 isolates M A V - P S 1 and P - P A V share 7 6 ~ nucleotide and 71 ~ a m i n o acid identity and, depending on the antisera and serological test procedures used, can show varying degrees of serological cross-reactivity. T h e P - P A V and V i c - P A V (Miller et al., 1988a) isolates share 9 5 ~ nucleotide and 9 7 ~ a m i n o acid identity and are o f the same serotype. The distantly related group 2 isolate N Y R P V shares only 56~o nucleotide and 5 0 ~ a m i n o acid identity with the P - P A V isolate and has long been regarded as serologically distinct. Consistent with these observations is the high degree o f sequence identity between N Y - R P V and B W Y V , w h i c h have been shown to be serologically related ( R o c h o w & Duffus, 1978, 1981). The amino-terminal sequences (roughly 60 a m i n o acids) o f B Y D V , B W Y V and P L R V contain a high proportion o f basic a m i n o acids clustered into two regions. T h e y are c o m p o s e d p r e d o m i n a n t l y of arginine residues and are separated by relatively non-polar a m i n o acids. The conservation of this organization a m o n g luteoviruses and the presence of two highly basic regions suggest that these features m a y be involved in structural p r o t e i n - R N A interactions within the viral capsid (Harrison, 1983).
Table 2. Nucleotide and deduced amino acid sequence similarity between the M A V-PSI, P-PA V, N Y-RP V isolates o f B YD V and other luteovirus 22K coat protein-coding regions* Nucleotide similarity (%)
MAV-PS1 P-PAV NY-RPV
P-PAV 76-6 -
NY-RPV 54.8 56.4
Vic-PAV 76.1 95.0 55.2
BWYV (FL1) 57.0 57-4 65.4
BWYV (GBI) 57-8 56-3 65.3
PLRV-1 53.0 57-6 69.4
PLRV-2 53-1 57.6 69.1
Vic-PAV 71.8 97.0 48.8
BWYV (FL1) 48.1 48-1 66.2
BWYV (GBI) 49.0 47-4 65.2
PLRV-1 PLRV-2 46.9 47.4 47-4 47-8 65.6 66.2
Deduced amino acid similarity (%)
MAV-PS1 P-PAV NY-RPV
P-PAV 71.3 -
NY-RPV 47.8 50.7 -
* Reciprocal comparisons between isolates and self-comparisons are not presented and are indicated by (-). References for other luteovirus coat protein sequences are: Vic-PAV, Miller et al. (1988a); BWYV (FL1) and BWYV (GB1), Veidt et al. (1988); PLRV-1, Prill et al. (1989); PLRV-2, Kawchuk et al. (1989).
2797
B Y D V coat protein sequences
MAV-PS1
1 MNSVGRR
N
N R
P-PAV Vic-PAV
1 MNSVGRR i .......
G
P RRANQNGTRR
RRNGPRRAR
NY-RPV
1 MSTVLLI/_SNGNG
PLRV- 1 PLRV-2
1 MSTVVVKGNVNGGVQQ 1 ................
BWYV (FL1) BWYV (GB1)
1 MNTVVGRRI ING RRRPRRQTRR i ........ T ..................
RVS
A VR~QPN
R RRR
• J . . . . . . . . . . . . . .
S RRRRQRVARR
' . . . . . . . . . . .
RPAAR
RAGPKRRARRR
TE
TVI~_PVVVVQPN R A G P R R R N G R R
QPAR~GRRRRPVGPRRGR
_RATQRRPRRRRR GNNRTGR ................. RG
RT
PVFRPTGG F ........ T
TVPTR GAGS ..........
60
T 61 61
PRSGGGSRG
GQPRRRRRRR GGNRRSRR TGVPR GRGS ............ • " .... ' . . . . . . "'"
PRRRRRQSLR RRANR VQpVVMVTAS '............... ' ......... P PQPVVVVQTS S .......
LISGPAG
' . . . . . . . . . . . . . . . . . .
T QpvvvvAsN
AQ_R
GGGAN
KG RGGAN
66 S 69 • 69 S 64 64
Fig. 4. Comparison of the amino acid sequences from amino-terminal regions of luteovirus coat proteins. The amino acid sequences of the luteovirus coat proteins were aligned to maximize their relatedness. When similar isolates are presented, only the amino acid differences between those isolates are shown; identical amino acids are represented by dots. Regions high in basic amino acid content are represented as boxed areas, and the basic amino acid residues are underlined. References: PLRV-1, Prill et al. (1989); PLRV-2, Kawchuk et al. (1989); BWYV, Veidt et al. (1988).
MAV-PS1
61
EVFVFS
V NDL
P-PAV Vic-PAV
62 62
EVFVFS
y
NY-RPV
67
ETFVFS
K DSL A
PLRV-1 PLRV-2
70 70
ETFVFT
BWYV (FL1) 65 BWYV (GB1) 65
ETFVFS
KANSSG
T
IKFGPDLSQCPA
LSG
9 ~
S
GILKSYHLYKIT
NVK
VEFKSHASA
STV
GAMFIELDTWC
S
132 132
......................................... ITFGPSLSEYPA
FQN
GVLKAYHEYKIT
NCV
LQFVSEASS
131
TA AGSISYELDPHCK
FKD
140 140 135 135
MAV-PS1
132 QST
LGSYINSFTISK
SATKTFTAQQ
IDGKE I FRESTVNQFYM
LYKANG
STSDT
AGQFIIT
I R V A N ~
199
P-PAV Vic-PAV
133 133
QSA "'"
LGSYINSFTISR TAAKVFRAEA ........... K "'S'T''S'"
INGKE I FQESTIDQFWM
LYKANG
TTTDT
AGQFIIT
MSVSLOI~,AKI
200
.....
200
NY-RPV
138
ASS
LASTINKFTITK
INGLE
WHPSDEDQFRI
LYKGNG
ASS
I.~VQI~-~
204
PLRV-1
141 VSS LQSYVNKFQITK GGAKTYQARM INGVEI WHDSSEDQCRI 141 ............................... '
LWKGNG
KSSDT P
AGSFRVT IRVALQINPK ] .............
208 208
LYKGNG
SSS
AGSFRIT
ILCQFHINPN I
202 202
PLRV-2
BWYV (FL1) 1 3 6 L N S BWYV (GB1) 136 • • •
TGARSFPAKM
I
~STINKFGITK PGKRAFTASY INGTE[ ............... A . . . . . . . . . v-,
WHDVAEDQFRI
V
I
AGSFKIT
Fig. 5. Comparison of the amino acid sequences from carboxy-terminal regions of luteovirus coat proteins. BWYV and the NY-RPV isolate of BYDV contain 138 amino acids in this region whereas PLRV and the PAV and MAV-PS1 isolates of BYDV contain 139 amino acids. When similar isolates are presented, only the amino acid differences between those isolates are shown; identical amino acids are represented by dots. Conserved regions representing consensus sequences identified from multiple sequence analyses (Devereux et al., 1984) are indicated by boxed areas. Asterisks above the amino acid sequence indicate that an amino acid is identically located in the carboxy-terminal region of all the luteoviruses. References: PLRV-1, Prill et al. (1989); PLRV-2, Kawchuk et al. (1989); BWYV, Veidt et al. (1988).
Although similarity comparisons among BYDV and other luteovirus coat protein sequences may provide an explanation for serological differences, the similarity in the C-terminal sequences of these proteins suggests that they all might show some degree of serological interrelatedness. In this regard, polyclonal and monoclonal antisera to MAV isolates, and to P-PAV and NY-RPV have been shown to recognize both homologous and
heterologous BYDV isolates in indirect ELISA (Diaco et al., 1986a) and in serologically specific electron microscopy (Diaco et al., 1986b). Indirect ELISA techniques also indicated serological cross-reactivity among a BYDV isolate ('BYDV-PAV-IL'), a BWYV isolate ('BWYV-CA') and an isolate of soybean dwarf virus, another luteovirus (Hewings & D'Arcy, 1986). Because BYDV dissociates in the alkaline buffers used for
2798
J. R. Vincent and others
antigen coating (Diaco et al., 1986a), these indirect ELISA procedures probably analysed the serological relationships of dissociated virus particles. Because of the high degree of amino acid sequence similarity between luteovirus coat proteins, even serotype-specific antisera may thus also identify common epitopes and could, in fact, be used as general probes for luteoviruses, providing virus preparations are appropriately treated (e.g. dissociated). Immunological detection procedures which tend to preserve the structural integrity of luteoviruses, such as double antibody sandwich ELISA, emphasize isolate-specific detection (Rochow & Carmichael, 1979), but procedures involving disrupted virus, including some indirect ELISAs or Western blot analyses, may permit immunological cross-reactions detecting many luteoviruses, and indicating a broader spectrum of serological relationships. Often, BYDV isolates are most efficiently transmitted by specific aphid vectors. Such specificity is a property of specific interactions of the viral capsid with receptor sites on the accessory salivary gland membranes of the vector (Gildow & Rochow, 1980; Gildow, 1987). Of the isolates used in the present study, the P-PAV isolate and other PAV isolates are non-specifically transmitted by both R. padi and M. avenae (Rochow, 1970a). Thus R. padi can transmit both P-PAV and NY-RPV, and M. avenae can transmit both P-PAV and MAV-PS1. NYRPV shares close sequence similarity with both BWYV and PLRV, but is efficiently transmitted by R. padi whereas BWYV and PLRV are both efficiently transmitted by Myzus persicae. These vector relationships allow evaluation of the coat protein sequences for regions likely to be involved in aphid specificity. Thus amino acids which are similarly located in NY-RPV and BWYV or PLRV are unlikely to be specifically involved in the aphid transmissibility of BYDV. Similarly, those amino acids similarly located in P-PAV and MAV-PS1 are unlikely to be specifically involved in the transmissibility of NY-RPV and P-PAV by R. padi. Elimination from consideration of the above amino acid pairs then allows us to identify amino acid pairs which are unique to PAVs and NY-RPV, and thus sequences potentially involved in R. padi transmission of PAVs and NY-RPV. Analyses of deduced amino acid sequences of the Cterminal regions of luteovirus coat proteins reveal that there is only one pairing of amino acids which is unique to both P-PAV and NY-RPV, alanine at P-PAV position 120 and NY-RPV position 125 (Fig. 5). The other luteoviruses have different amino acids at this location, but these differences result from single base changes from the GCC codon found in both P-PAV and NYRPV. Furthermore, both BWYV and PLRV have the related amino acid serine at this position. At all other positions within the P-PAV or NY-RPV C-terminal
domains, either P-PAV and NY-RPV have different amino acids, or an identical amino acid can be found in one of the other luteovirus sequences. In the light of these observations, it seems unlikely that the C-terminal region of luteovirus coat proteins is responsible for vector specificity. In icosahedral R N A viruses, the N-terminal regions of the coat proteins are typically located within the interior of the virus particle (Rossmann & Johnson, 1989), and thus this region is also unlikely to be involved in aphid specificity. Therefore, it is likely that another protein on the viral capsid is involved. In this regard, it has been proposed that a second structural protein which has been identified in Vic-PAV virions is involved in aphid specificity (Waterhouse et al., 1989). In recent work in our laboratory (P. P. Ueng et al. and J. R. Vincent et al., unpublished results), similar proteins have been identified by Western blot analyses, the sizes of which correspond to ORFs identified by sequencing of cDNA produced from the three BYDV isolates discussed herein. We thank C. Logan and H. Laffoon for their photographic services. This work was funded by Grant 88-37263-3855 from the Competitive Research Grants Office of the U.S. Department of Agriculture and Grant 1484670 from The Quaker Oats Company. The Genetics Computer Group Sequence Analysis Software Package for the VAX was supported by NIH grant No. AI27713. The nucleotide sequence data reported in this paper will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession numbers X17260 for BYDV MAV-PSI, X17261 for BYDV P-PAV and X17259 for BYDV NY-RPV. This is Journal Series Article 12326 of the Purdue University Agricultural Experiment Station.
References AAPOLA, A. I. & ROCHOW, W. F. (1971). Relationships among three isolates of barley yellow dwarf virus. Virology 46, 127-141. BARBARA, O. J., KAWATA, E. E., UENG, P. P., LISTER, R. M. & LARKINS, B. A. (1987). Production of cDNA clones from the MAV isolate of barley yellow dwarf virus. Journal of General Virology 68, 2419-2427. BIRNBOIM, H. C. & DOLY, J. (1979). A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Research 7, 1513 1523. DEVEREUX, J., HAEBERLI, P. & SMITHIES, O. (1984). A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Research 12, 387 395. DIACO, R., LISTER, R. M., HILL, J. H. & DURAND, D. P. (1986a). Detection of homologous and heterologous barley yellow dwarf virus isolates with monoclonal antibodies in serologically specific electron microscopy. Phytopathology 76, 225-230. DIACO, R., LISTER, R. M., HILL, J. H. & DURAND, O. P. (1986b). Demonstration of serological relationships among isolates of barley yellow dwarf virus using polyclonal and monoclonal antibodies. Journal of General Virology 67, 353-362. FEINBERG, A. P. & VOGELSTEIN, B. (1983). A technique for radlolabeling DNA restriction endonuclease fragments to high specific activity. Analytical Biochemistry 132, 6-13. GILDOW, F. E. (1987). Virus-membrane interactions involved in circulative transmission of luteoviruses by aphids. Current Topic's in Vector Research 4, 166-209.
B Y D V coat protein sequences
GILDOW, F. E. & Rocnow, W. F. (1980). Role of accessory salivary glands in aphid transmission of barley yellow dwarf virus. Virology 104, 97-108. GILDOW, F. E., BALLINGER, M. E. & ROCHOW, W. F. (1983). Identification of double-stranded RNAs associated with barley yellow dwarf virus infection of oats. Phytopathology 73, 1570-1572. GILL, C. C. & CHUNG, J. (1979). Cytopathological evidence for the division of barley yellow dwarf virus isolates into two subgroups. Virology 93, 59~9. Guo, L-H., YANG,R. C. A. & Wu, R. (1983). An improved strategy for rapid direct sequencing of both strands of long DNA molecules cloned into a plasmid. Nucleic Acids Research 11, 5521-5540. HAMMOND, J., LISTER, R. M. & FOSTER, J. E. (1983). Purification, identity and some properties of an isolate of barley yellow dwarf virus from Indiana. Journal of General Virology 64, 667~i76. HARRISON,S. C. (1983). Virus structure: high resolution perspectives. Advances in Virus Research 28, 175-240. HEWINGS, A. D. & D'ARcY, C. (1986). Comparative characterization of two luteoviruses: beet western yellows virus and barley yellow dwarf virus. Phytopathology 76, 1270-1274. KAWCHUK,L. M., MARTIN,R. R., ROCHON,D. M. & MCPHERSON,J. (1989). Identification and characterization of the potato leafroll virus putative coat protein gene. Journal of General Virology 70, 783-788. KEESE, P., MARTIN, R. R., KAWCHUK,L. M., WATERHOUSE,P. M. & GERLACH,W. L. (1990). Nucleotide sequences of an Australian and a Canadian isolate of potato leafroll luteovirus and their relationships with two European isolates. Journal of General Virology 71, 719-724. KRIEG, P. A. & MELTON,D. A. (1987). In vitro RNA synthesis with SP6 polymerase. Methods in Enzymology 155, 397-415. LAEMMLI, U. K. (1970). Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature, London 227, 680685. LISTER, R. M. & SWARD, R. L. (1988). Anomalies in serological and vector relationships of MAV-like isolates of barley yellow dwarf virus from Australia and the U.S.A. Phytopathology 78, 766-770. MANIATIS, T., FRITSClt, E. F. & SAMBROOK,J. (1982). Molecular Cloning: A Laboratory Manual. New York: Cold Spring Harbor Laboratory. MATTHEWS, R. E. F. (1982). Classification and nomenclature of viruses. Intervirology 17, 1-199. MAYO, M. A., ROBINSON,D. J., JOLLY, C. A. & HYMAN, L. (1989). Nucleotide sequence of potato leafroll luteovirus RNA. Journal of General Virology 70, 1037-1051. MILLER, W. A., WATERHOUSE,P. M. & GERLACH, W. L. (1988a). Sequence and organization of barley yellow dwarf virus genomic RNA. Nucleic Acids Research 16, 6096-6111. MILLER, W. A., WATERHOUSE,P. M., KORTr, A. A. & GERLACH,W. L. (1988 b). Sequence and identification of the barley yellow dwarf virus coat protein gene. Virology 165, 306-309.
2799
MURPHY,J. F., D'ARCY, C. J. & CLARK,J. M., JR (1989). Barley yellow dwarf virus RNA has a 5"-terminal genome-linked protein. Journalof General Virology 70, 2253-2256. PELHAM, H. R. B. & JACKSON, R. J. (1976). An efficient mRNAdependent translation system from reticulocyte lysates. European Journal of Biochemistry 67, 247-256. PRILL, B., MAISS, E., TIMPE, U. & CASPER, R. (1989). Nucleotide sequence of the potato leafroll virus coat protein gene. NucleicAcids Research 17, 1768. PUTNEY, S. D., BENKOVIC, S. J. & SCHIMMEL, P. (1981). A DNA fragment with an ~-phosphothiolate nucleotide at one end is asymmetrically blocked from digestion by exonuclease III and can be replicated in vivo. Proceedings of the National Academy of Sciences, U.S.A. 78, 7350-7354. ROCHOW, W. F. (1970a). Barley yellow dwarf virus. CMI/AAB Descriptions of Plant Viruses, no. 32. ROCHOW,W. F. (1970b). Barley yellow dwarf virus: phenotypic mixing and vector specificity. Science 167, 875-878. ROCHOW,W. F. & CARMICHAEL,L. E. (1979). Specificity among barley yellow dwarf viruses in enzyme immunosorbent assays. Virology95, 415-420. Rocnow, W. F. & DUFFUS,J. E. (1978). Relationships between barley yellow dwarf and beet western yellows viruses. Phytopathology 68, 51-58. ROCHOW, W. F. & DUFFUS, J. E. (1981). Luteoviruses and yellows diseases. In Handbook of Plant Virus Infections and Comparative Diagnosis, pp. 147-170. Edited by E. Kurstak. Amsterdam: Elsevier/North-Holland. ROSSMANN,M. G. & JOHNSON, J. E. (1989). Icosahedral RNA virus structure. Annual Review of Biochemistry 58, 533-573. SANGER,F., NICKLEN,S. & COULSON,A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proceedings of the National Academy of Sciences, U.S.A. 74, 5463-5467. VELDT, I., LOT, H., LEISER, M., SCHEIDECKER, D., GUILLEY, H., RICHARDS, K. & JONARD, G. (1988). Nucleotide sequence of beet western yellows virus RNA. Nucleic Acids Research 16, 9917-9932. VIEIRA, J. & MESSING, J. (1982). The pUC plasmids, an ml3mp7derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19, 259-268. WATERHOUSE, P. M., GILDOW, F. E. & JOHNSTONE, G. R. (1988). Luteovirus group. AAB Descriptions of Plant Viruses, no. 339. WATERHOUSE,P. M., MARTIN,R. R. & GERLACH,W. L. (1989). BYDVPAV virions contain readthrough protein. Phytopathology 79, 1215 (abstract). YOUNG, R. A. & DAVIS,R. W. (1983). Efficient isolation of genes using antibody probes. Proceedings of the National Academy of Sciences, U.S.A. 80, 1194-1198.
(Received 21 May 1990; Accepted 23 August 1990)
