Cloning and sequence analysis of the coat protein gene of barley mild mosaic virus

l4ru.s Research, 27 (1993) 79-89 0 1993 Elsevier Science Publishers

VIRUS

79 B.V. All rights reserved

0168-1702/93/%06.00

00852

Cloning and sequence analysis of the coat protein gene of barley mild mosaic virus I.J. Foulds a, V.J. Lea a, C. Sidebottom b, C.M. James a, R.E. Boulton a, T. Brears ‘, A.R. Slabas b,l, P.L. Jack a and R. Stratford a aPlant Breeding International, Trumpington, Cambridge, UK b U&ever Research Laboratory, Colworth House, Shambrook, Bedford, UK and ’ Laboratory of Plant Molecular Biology The Rockefeller University, New York, NY 100214399, USA (Received

2 September

1992; revision

received

9 October

1992; accepted

13 October

1992)

Summary The sequence of the 3’ 1462nts of RNA-l of a UK isolate of the fungal-transmitted virus barley mild mosaic (BaMMV) has been determined. An open reading frame encoding the coat protein gene was identified within this region using amino acid sequence information obtained by cyanogen bromide cleavage of virus particles. The amino acid sequence of the full-length coat protein was deduced from the nucleotide sequence. Amino acid sequence comparisons revealed highest homology to the coat protein of barley yellow mosaic virus. In addition, a significant, but limited, number of the amino acid residues that are conserved between aphid-transmitted potyviruses were also conserved between BaMMV and potyviruses. BaMMV; BaYMV; Bymovirus; Potyvirus

Introduction Barley yellow mosaic disease is one of the most important diseases of winter barley in parts of Europe and Asia (Inouye and Saito, 1975). It has recently been Correspondence too: R. Stratford, Plant Breeding International, Maris Lane, Trumpington, CB2 2LQ, UK. t Current address: University of Durham, Department of Biological Sciences, Science South Road, Durham, DHl 3LE, UK.

Cambridge, Laboratory,

80

recognised that the disease is caused by two viruses: barley yellow mosaic virus (BaYMV) and barley mild mosaic virus (Kashiwazaki et al., 1989a; Huth and Adams, 1990; Prols et al., 19901, although it was previously thought that BaMMV was a mechanically transmissible isolate of BaYMV. The two viruses have identical particle morphology comprised of flexuous rods with two modal lengths of approximately 275 nm and 550 nm and produce similar symptoms (Huth et al., 1984). The particles encapsidate bipartite, polyadenylated single-stranded RNA genomes of similar size (approximately 8 kb and 4 kb; Huth et al., 1984; Usugi et al., 1989; Prols et al., 1990). However, cross-hybridisation studies and restriction enzyme analysis have suggested that the two viruses are substantially different at the nucleotide sequence level (Batista et al., 1989; Prols et al., 1990). Furthermore, there is no cross-reaction in serological tests (Huth et al., 1984; Kashiwazaki et al., 1989a; Chen and Adams, 1991). BaYMV and BaMMV have been considered to be possible members of the potyvirus group based on a number of similar features including the formation of cytoplasmic inclusions and their flexuous rod particles (Huth et al., 1984; Shukla et al., 1991). However, this classification has been questioned and it has been proposed that the barley mosaic viruses, wheat yellow mosaic, wheat spindle streak mosaic, oat mosaic and rice necrosis mosaic viruses should form a separate bymovirus group Wsugi et al., 1989). This grouping is on the basis of particle morphology with two modal lengths, transmissibility by the fungus Polymyxa graminis and the absence of a serological relationship with potyviruses. In this report, we present the nucleotide sequence of the coat protein gene of the UK isolate of BaMMV and assess further the taxonomic relationship between BaMMV, BaYMV and the potyvirus group.

Materials

and Methods

Virus strains, purification and RNA extraction

The Streatley strain of BaMMV was originally obtained from Dr M.J. Adams (Rothamsted, UK) and was propagated in Hordeum vulgare cultivar Maris Otter by mechanical inoculation (Adams et al., 1986). Virus was purified as described by Huth et al. (1984) but with the addition of 1 mM phenyl methyl sulphonyl fluoride as a protease inhibitor. BaMMV RNA was isolated from the purified virus by incubation in 0.1 mg/ml proteinase K, 0.1% SDS for 20 min at room temperature followed by phenol-chloroform extraction and ethanol precipitation. SDS PAGE and amino acid sequencing

A virus preparation was dried on a centrifugal evaporator (Un’ivap) and resuspended in 70% formic acid containing 2.5 mg/ml cyanogen bromide. This was then incubated at room temperature for 16 h in the dark. The cyanogen bromide cleaved peptides were separated either by SDS PAGE followed by blotting onto a

81

PVDF membrane (Matsudaira, 1987) or by HPLC using a Brownlee Aquapore C8 RP300 cartridge column (2.1 X 30 mm). Samples were loaded onto the HPLC column previously equilibrated in 0.1% trifluoroacetic acid (TFA) and eluted with a O-70% linear gradient of 90% acetonitrile/0.085% TFA over 70 min. Selected peaks were further chromatographed over the same column and eluted with a linear gradient of 90% acetonitrile/O.l% TFA as before but the gradient was adjusted to be one-third shallower. N-terminal protein sequencing was performed on an Applied Biosystems model 470 protein sequencer. SDS PAGE was performed according to Laemmli (1970). Northern blotting and oligonucleotide probing

RNA was separated on formaldehyde/ agarose gels and transferred to HybondN nylon filters (Amersham International) according to the manufacturer’s instructions. The low redundancy 14mer oligonucleotides CP-1 and CP-2 (5’-GGRTCXGGYTCYTC-3’ and 5’-GTRAAYTGRTCXGG-3’, respectively, where R = A or G, X = A, C, G or T and Y = C or T) were synthesised to be complementary to BaMMV RNA in regions of the coat protein where the amino acid sequence had been determined. CP-1 and CP-2 were radioactively labelled with 32P using T4 polynucleotide kinase and blots were probed at 5°C below the minimum predicted Tm as described by Sambrook et al. (1990). cDNA synthesis and cloning

Oligo dT primed cDNA was synthesised from total BaMMV RNA using the cDNA Synthesis System Plus (Amersham International) following the manufacturer’s instructions except that first strand synthesis was performed using Moloney murine leukemia virus RNase H- reverse transcriptase and buffer from GIBCOBRL. cDNA was fractionated on a sucrose gradient (Davis and Pearson, 1978) and centrifugation was at 35,000 rpm for 16 h at 20°C in a Sorvall AH650 rotor. Fractions containing fragments of greater than 500 bp were ligated into pUC13 and transformed into E. coli MAX Efficiency DHScz cells (GIBCO BRL). Plasmid DNA was screened by restriction mapping and Southern blotting using CP-1 and CP-2 as probes. DNA sequence analysis

Recombinant clones containing the coat protein gene were sequenced using the dideoxy method of Sanger et al. (1977) with the Sequenase kit from USB. A combination of exonuclease III deletions (Sambrook et al., 19901, subcloning and custom-made oligonucleotide primers were used to obtain full-length sequence from both strands of clones pBM-217, -55, -249, -322. DNA sequence was compiled and analysed using the DNASTAR Inc. computer package. Protein sequences were compared using version 1.68 of the Amino Acid Align (AANW) program with a gap penalty of 2 and a deletion penalty of 12.

82

Results BaMMV coat protein gene is encoded on RNA-1

SDS polyacrylamide gel electrophoresis of purified BaMMV revealed three protein bands at 32.5 kilodaltons &Da), 26 kDa and 25 kDa (Fig. 1). The proportion of these bands differed between virus preparations. In some experiments all three bands were present in roughly equal proportions but in others the high molecular weight band was predominant. All three bands cross-reacted with antibodies affinity purified to the 32.5 kDa band (data not shown). This suggests that the 32.5 kDa band is the major coat protein polypeptide and that the two minor bands are likely to be degradation products. Initial attempts to sequence the N-terminus of the three coat protein bands were unsuccessful and, therefore, we chose to use cyanogen bromide cleavage. The peptide digestion products were separated by gel electrophoresis and PVDF blotting or by HPLC. The sequence of the two most abundant peptides were AGHEEPDPIVPPA-DT-L-N and IPDQFTSRTALETLKQT-LAAIGV were the gap (-1 refers to positions where the amino acid could not be unambiguously identified. Two areas of limited redundancy were selected from these sequences

Mr

69-

Fig.

1. SDS PAGE

of BaMMV

coat protein weight markers

polypeptides from purified (Mr) are shown (kDa).

virus

particles.

Molecular

83 Mr

1

949f-46-

2

+-RNA

1

+-RNA

2

2*37-

Fig. 2. Northern blot of RNA extracted from purified BaMMV probed with the radioactively labelled oligonucleotides CP-1 (1) and CP-2 (2) derived from amino acid sequence data from BaMMV coat protein. The position of RNA-1 and RNA-2 is indicated by arrows. RNA markers (Mr) are shown (kb).

RNA-1

_____?~EcoR’

EcoRi

1462 'IAl,

_____

_____

I

*

pBM-217

pBM-55,-249

VBM~322

IAI,

Fig. 3. Nucleotide sequencing strategy. The position of cDNA clones, relative to the 3’ end of RNA-l, is shown. Clones pBM-217, -322, -55 and -249 were sequenced in full on both strands. The region containing open reading frame is indicated by the shaded box and the putative cleavage site of the coat protein is shown ( A ).

84

for the synthesis of oIigonucIeot~de probes complemental to regions of the coat protein gene (CP-1 and CP-2, see Methods). Since BaMMV consists of two RNA components of approximately 8kb (RNA-11 and 4 kb (RNA-2; Batista et al., 19891, the location of the coat protein gene was investigated by Northern blotting and probing with radioactively-labelled coat protein oligonucleotides CP-1 and CP-2 (Fig. 2). Both oligonucleotides hybridised predominantly with RNA-l (Fig. 2) which suggests that the coat protein is encoded on the longer of the two RNA components. On long exposures there was slight cross-reaction of CP-2 to RNA-2; it is likely that this is due to conditions of low stringency since weak hybridisation to the RNA markers was also observed (data not shown). 140 -20 ” v&3 GAAUFCGACGAAGiCACUGAAGdCAUUUGCG~ddC CC~JICA:.L'GUCCUUCdCGAUGGUCCGCACCUCEULTd OICEHPY~SLTYVRTSFGIGFSLS~~~lVAlLO EFOEAli

vsa

~,tca

“ldcl “LSC 660 “zac r227 UGCAGC:GCGiUGi:;iiGUCCUGCdUGCCUdCCUCUCLGGCA~UGC:GCUCUUUUCGAAUCCUUCAAC~CACCCdAGCUCUUUA~UCUCGUGCAC~C~~ACCUCCU~~GG~~C~~C~C~ Y 5 I A G G VCH A Y L 5 Gl A A I FE S F N T P K I_ F N L"" T Y L L

;i L

I

T

V250 "290 V300 "329 "3dO GAGiACGdGGI\GGAACUCUUCUCUCUAUGdUGGAACUCAAAGAtAUG~UCAtiGCCCCUGCCCACUAAGGAACAGAUAGCCUUGUUGCACUACGUCG~G~CLGAGCCCA~C~~GGAGG~C~CU EHEiELFSHHELKO~F~?LPTK~~~ALtHYVGT~PlV~Di

vsao

VW0

V&O 443 "32'3 dUCUUCCGCUGUUAUUCCAUCGAAdCGdAACUGAUGUUCGUAAAGCUGAGGCGCGUGCCGCAUUGGGCCAUAAAGCACGGAUG:CUUGA~GAAAUC~UC~UUGAU~UCA~Gd~CCCC~~C IFRCYS I ElKLHFVKLRRVPHVA [KHGCCOE iV[mF]H

_______

vtooo

--_-__ IPO

vtozo

v980 VI040 VI060 CAGUI;CACCUCCAGGACUGCACUGGAAACdCUGAAGCAGdCCAAGCUUGCCGCCAUUGGUGUUGGCACAAGCAAUUCUCUUCUCdCCUCUGAGCAGdC~AACAUGAGG~CCAC~GAAACC 0 FTS R r ALE 1L K 9 T K L A A IG" G T S N 5 L L T 5 E 0 T N H RTT

El

V1100 VI120 V1140 vi160 V1160 CGAAGGiGCAAUWCWCGAUGGUCdCGAGGCGCUUCUCCGGUAGAUCUCUCAUGCAGUUUCdUUUCCUUUCdAGCdGUGCAUUUAAAUACGCUUUUAAUUACAUCdAGCGUCAAGAAUU RRRHOYOGHEALLR VI3GO V1220 VI240 vtao c vl230 CCUCCCGCUUGCdGCGAAUCCUUAAAGGGUGGCdCUUUGUGUUAUGCAA~~~dUUCAGU~CGCAdCCdACCAUCAUUG~~UGGUGdCddCGCAGUdCUUUCUU~CUUUCdCAUCAd vt330 "1X.0 GCGACCdGCdCAUCGiiCGCUUACdGCUddUCKUdCCUGAGGGUGGC~CUCUGUG~~~

r!423 Vi‘IOO " 380 !a AUGGAAUGUGCUAOCUCGCAACCAdCCGUCAUUGGUUG~GG~G~G~UA~CACCCGC

VIP60 GCUCUUGUdUdCCGGA~GGUUdddAdAAddAAAdd

Fig. 4. The sequence of the 3’ 1462 nucleotides of BaMMV RNA-l. The predicted amino acid sequence of the longest open reading frame is shown under the nucleotide sequence. The putative cleavage site of the coat protein is shown (A ). Positions where the nucleotide and predicted amino acid sequence varied between different cDNA clones are also shown. The peptide sequences obtained by direct sequencing of purified virions are underlined and the position of the oligonucleotides CP-1 and CP-2 is indicated by a dashed line above the complementary nucleotide sequence. Two amino acid sequence motifs that are conserved between potyviruses are boxed (solid line). The potential polyadenylation motif is also boxed (dashed line) and the direct repeat sequence in the 3’ untranslated region is indicated by arrows.

85

Nucleotide sequence of BaMMV coat protein gene

To clone the gene for the BaMMV coat protein cDNA clones were synthesised and identified using CP-1 and CP-2 as probes. Four overlapping cDNA clones (Fig. 3; pBM-217, -55, -249 and -322) containing sequences homologous to the coat protein gene were obtained and were fully sequenced in both strands. Regions of partial single-stranded sequence data were also obtained from a further 8 cDNA clones (not shown) such that all regions of the 3’ 1462 nts of RNA-l were covered by at least two independent clones. The nucleotide sequence is presented in Fig. 4. Computer analysis revealed one large ORF consisting of 1122 nts in the ( + ) strand virion polarity terminated by a stop codon located 340 nts upstream of a poly (A) tail (Fig. 4). The two peptide sequences from which the oligonucleotide coat protein probes had been derived were identified within the large ORF as indicated BdMV BaYMV

BdMMV BaYMV

Bi%MtU B&XV

B&&-V BaYHV

BaMMV BaYMV

BaHKV BaYHV

Bd4MV BaYMV

EFDEATEDICENPmSLTMVRTSFGI[GFSLSIERIVAILQWSRAGGVLHAYLSGIAALFESFNTPK 66 RFD T DXCENP~SLT~ T FG GFSL ERI AI QWS GGVLH YX GI A ESFNTPK EFDDITSDICENP~SLTMVKTPFGVGFSLPVERIIAIMQWSKKGGVIl1SYLACISAIYESFNTPK 66 v LFNLVH~LWLITEHEEELFSMME~MFMPLPTKEQIAL~GTEPIVEDTFLqACHEEPDPI YLLWL EHE E M PP LF LHYE LQA D LFKSIYAnL~TEEHEAEILSSTALPIPSM~~~CDDEIW-----LQAADPLTDAQ A

132 127

_____~--~~~~-~---------.-~~~~~~-----------.--VPPASDTDLTN~PpD~

153 V T KEDARIAAADGARFEL4DADRRRKVEADRVEAARVKKAADAALKPVNLTATRTPTEDDGKLKTPSG 193

SRAVIPRGTSDWSMPEPTMRTLGFKSKIKIETLADVPEG~TFASVATESQRRKWEEATRGDFGI 219 G L VP M R ws P KI SVA ES WAR GI ARIPSSAADGMJSVPATKQVNAGLTLKIPLNKLKSVPKSVMEHNNSVALESELKAWTDAVRTSLGI 259

TDDE~LLIAAGIYFADNGTSPNFDEELTMEVNSGLNSIKEYP~PFWRAKK ISTLRRIFRCY NGTS E M SG TDEW LX E PF V A LRRIRY ~D~WIDALIPFIGWCC~~SD~RNQVMQIDSGKGA~~SLSPFI ~GG~I~

285 325

SIETKZMFVKLRRVPHWAIKHG--CLDEIVFDFMIPDQl%RTALETl.KQTKLAAIGVGTSNSLLT 349 S ET L VHW KHG FDF P E KQ IAA G GT N LT SDE~LI~LV~S~GAS~~~DF~PRS~PQDI~VSKQ~LGTG~TMLT 391

SEQTNMRTTETRRRNDYDGHEALLR R DDGH L S TNRT SDTTNLRKTTNHRVLDSDGHPELT

Fig. 5. Alignment of the predicted amino acid sequences at the 3’ end of RNA-1 of BaMMV and BaYMV. Gaps (- ) have been introduced to maximise homology. The arrow indicates the putative site of cleavage for the release of the coat protein.

Fig. 6. Dot plot comparison of the predicted amino acid sequence of the coat protein of (A) BaMMV (BMCP.PRO) and BaYMV (BYCP.PRO), (Bl BaMMV and PVY (PVYCP.PRO) and (Cl BaMMV and PPV (PPVCP.PRO).

(Fig. 4). The amino acid sequence (LGFTVPID) of a third peptide that was detected only at a low level could not be located within any ORFs and might be derived from host components that co-purified with the virus preparation. The peptide beginning with the amino acid sequence AGHEEPDP is likely to be at the N-terminus of the coat protein since it does not follow a methionine residue which usually precedes a cyanogen bromide cleavage site (Allen, 1981). In addition, the A residue is preceded by LQ to form a putative LQ/A cleavage site characteristic of several potyvirus cleavage sites as well as BaYMV polyprotein cleavage (Kashiwazaki et al. 1989b; Shukla et al., 1991). Sequence comparisons

between

~a~~,

BaYMV and potyuiruses

To assess the relationship of the BaMMV coat protein sequence to other viral coat proteins, computer-aided searches were made using the predicted amino acid sequence. The highest level of homology (36% identity overall) was to the coat protein of BaYMV and the homology was lowest at the N-terminus (Figs. 5 and 6A). Lower levels of homology (20-22% identity) were found to potyviruses such as potato virus Y (PVY) and plum pox virus (PPV) and, again, the homoiogy was weakest at the N-terminus (Fig. 6B,C). The lack of conservation of the N-terminus of the coat protein of potyviruses has been noted previously (Shukla and Ward, 1988; Shukla et al., 19881. The sequence motif’s NGTS and FDF, known to be conserved within the potyvirus group and between potyviruses and BaYMV (Kashiwazaki et al., 1989b; Timmerman et al., 1990; Niblett et al., 19911, were also conserved between BaMMV and potyviruses (Fig. 4). We detected only 13.5% identity between the amino acid sequence of the coat proteins of BaMMV and the mite-transmitted potyvirus wheat streak mosaic (WSMV; Niblett et al., 1991). We observed approximately 55% identity between the predicted amino acid sequence of the BaMMV ORF upstream of the coat protein (from BaMMV amino acids 1-123; Fig. 5) and the putative Nib product of BaYMV which is also located immediately upstream of the coat protein gene (Fig. 5; Kashiwazaki et al., 1990).

However, we could detect no significant homology between BaMMV and BaYMV in the 3’ untranslated region. The 3’ untranslated region of BaMMV (340nts) is longer than that of BaYMV (231nts) and contains an imperfect direct repeat of approximately 120nts within which short palindromic sequences exist (Fig. 4). Repeats within the 3’ untranslated region have been observed in other potyviruses (Dougherty et al., 1985; Hay et al., 1989). In addition, there is a potential polyadenylation motif (UAUGU) 85nts upstream from the poly(A) tail (Fig. 4) which has also been noted in the sequence of potyviruses (Maiss et al., 1989).

Discussion We have determined the nucleotide sequence of the coat protein gene of a UK isolate of BaMMV and report that it is located at the 3’ terminus of RNA-l. The sequence of the 3’ 1462nts of BaMMV reported here contains a continuous ORF of 374 amino acids (Fig. 4) and alignment with BaYMV and potyviruses revealed homology to both the putative replicase product Nib and the coat protein. The nucleotide sequence of the 3’ end of RNA-l of two Japanese isolates of BaMMV was published recently (Kashiwazaki et al., 1992). There is 91% homology between the nucleotide sequence of BaMMV RNA-l reported here and the Japanese Kal isolate (data not shown). We detected only 36% amino acid sequence identity between the coat proteins of BaMMV and BaYMV (Fig. 5) which is consistent with their designation as separate viruses (Kashiwasaki et al., 1989a; Huth and Adams, 1990; Prols et al., 1990). The coat protein gene of BaMMV appears to have no methionine initiation codon and, therefore, is likely to be expressed by processing of a polyprotein at a LQ/A cleavage site located between amino acids 123 and 124 in a similar manner to BaYMV and potyviruses (Fig. 4; Kashiwazaki et al., 1989b; Shukla et al., 1991). We do not know why our initial attempts to sequence the N-terminus directly were unsuccessful. However, since one of the sequences we obtained from the cyanogen bromide cleavage products contained a sequence corresponding to the N-terminus, it seems likely that the N-terminus was not blocked in vivo but became blocked during sample preparation. The molecular weight of the coat protein of this isolate of BaMMV was estimated by SDS PAGE to be approximately 32.5 kDa (Fig. 1) which is similar to that reported for a BaMMV German isolate (31 kDa-36 kDa; Kashiwazaki et al., 1989a; Ehlers and Paul, 1986; Huth et al., 1984). This figure is significantly more than that predicted from the nucleotide sequence (28.5 kDa, Fig. 4). It is likely that this difference is due to aberrant mobility on SDS PAGE gels rather than post-translational modification since in vitro transcription and translation of the coat protein ORE; produces a protein of 32.5 kDa (data not shown). We observed significant homology between sequences upstream of the BaMMV coat protein to the putative Nib product of BaYMV (Fig. 5; Kashiwazaki et al., 1990) indicating that gene order is conserved between BaYMV and BaMMV, at least at the 3’ end of RNA-l. Sequence data from a number of other clones from

88

BaMMV RNA-l revealed 22% and 30% identity to the CI and NIa proteins of tobacco vein mottling virus respectively (data not shown). This suggests that the organisation of genes upstream of Nib and the coat protein is also conserved between BaMMV, BaYMV and potyviruses, In a recent review, Ward and Shukla (1991) suggested that the potyvirus group should be elevated to family status and that vector transmission (which correlates with the major sequence diversity) should define genera. It was concluded that transmission by aphids should not be considered an essential taxonomic parameter for potyvirus classification. This concept is simiiar to the proposal of Barnett (1991) to establish the family of plant viruses called Potyviridae to include at least three genera based largely on vector type (potyvirus, baymovirus and ryemovirus). Some of the key features of the Potyviridae include gene organisation and the 3’ location of the coat protein gene; production of the coat protein by proteolytic processing; significant sequence identity with potyvirus coat proteins and the formation of pinwheei cytoplasmic inclusions (Barnett, 1991; Ward and Shukla, 1991). The conse~ation of these features provides strong evidence that BaYMV and BaMMV should both be regarded as fungal-transmitted potyviruses, possibly within the genus baymovirus (Barnett, 1991). This classification would fulfill one of the main functions of taxonomy which is to reflect evolutionary relationships.

We wish to thank Dr Robert Shields for critical reading of the manuscript and helpful suggestions. References Adams, M.J., Swaby, A.G. and Macfarlane 1. (1986) The susceptibility of barley cultivars to barley yellow mosaic virus (BaYMV) and its fungal vector, Pofymyxu gram&k. Ann. Appl. Biol. 109, 561-572. Allen, G. (1981) In: T.S. Work and R.H. Burdon (Eds.), Sequencing of Proteins and Polypeptides. Laboratory Techniques in Biochemistry and Molecular Biology. Elsevier, Amsterdam. Barnett, O.W. (1991) Potyviridae, a proposed family of plant viruses. Arch. Viral. 118, 139-141. Batista, M.F., Antoniw, J.F., Swaby, A.G., Jones, P. and Adams, M.J. (1989) RNA/cDNA hybridisation studies of UK isolates of barley yellow mosaic virus. Plant Pathol. 38,2X-229. Chen, J. and Adams, M.J. (1991) Serological relationships between five fungally transmitted cereal viruses and other elongated viruses. Plant Pathol. 40, 226-231. Davis, P.B. and Pearson, C.K. (1978) Characterisation of density gradients prepared by freezing and thawing a sucrose solution. Anal. Biochem. 91, 343-349. Dougherty, W.G., Allison, R.F., Parks, T.D., Johnston, R.E., Field, M.J. and Armstrong, F.B. (1985) Nucleotide sequence at the 3’-terminus of the pepper mottle virus genomic RNA: evidence for an alternative mode of capsid protein gene organisation. Virology 146, 282-291. Ehlers, U. and Paul, H.-L. (1986) Characterisation of the coat proteins of different types of barley yellow mosaic virus by polyacrylamide gel electrophoresis and electro-blot immunoassay, J. Phytopathol. 115, 294-304. Frenkel, M.J., Ward, C.W. and Shukla, D.D. (1989) The use of 3’ non-coding nucleotide sequence in the t~onomy of potyviruses: application to watermelon mosaic virus 2 and soybean mosaic virus N. J. Gen. Virol. 70, 2775-2783.

89

Hay, J.M., Fellowes, A.P. and Timmerman, G.M. 0989) Nucleotide sequence of the coat protein gene of a necrotic strain of potato virus Y from New Zealand. Arch. Viral. 107, 111-122. Holmes, D.S. and Quigley, M. (1981) A rapid boiling method for the preparation of bacterial plasmids. Anal. Biochem. 114, 193-197. Huth, W. and Adams, M.J. (1990) Barley yellow mosaic virus 1BaYMV) and BaYMV-M: two different viruses. Intervirology 31, 38-42. Huth, W., Lesemann, D.-E. and Paul, H.-L. (1984) Barley yellow mosaic virus: purification, electron microscopy, serology, and other properties of two types of the virus. Phytopathol. Z. 111, 37-54. Inouye, T. and Saito, Y. (1975) BaYMV CMI/AAB Descriptions of Plant Viruses no. 143. Kashiwazaki, S., Ogawa, K., Usugi, T., Omura, T. and Tsuchizaki, T. (1989aJ Characterisation of several strains of barley yellow mosaic virus. Arm. Phytopathol. Sot. Jpn. 55, 16-25. Kashiwazaki, S., Hayano, Y., Minobe, Y., Omura, T., Hibino, H. and Tsuchizaki, T. (1989b) Nucleotide sequence of the capsid protein gene of barley yellow mosaic virus. J. Gen. Viral. 70, 3015-3023. Kashiwazaki, S., Minobe, Y., Omura, T. and Hibino, H. (1990) Nucleotide sequence of barley yellow mosaicvirus RNA I: a close evolutionary relationship with potyviruses. J. Gen. Virol. 71,2781-2790. Kashiwazaki, S., Nomora, K., Kuroda, H., Ito, K. and Hibino, H. (1992) Sequence analysis of the 3’ terminal halves of RNA-l of two strains of barley mild mosaic virus. J. Gen. Virol. 72, 2173-2181. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. Maiss, E., Timpe, U., Brisske, A., Jelkman, W., Casper, R., Himmler, G., Mattanovich, D. and Katinger, H.W.D. (1989) The complete nucleotide sequence of plum pox virus RNA. J. Gen. Viral. 70, 513-524. Matsudaira, P. (1987) Sequencing from picomole quantities of proteins electroblotted onto polyvinylidine difluoride membranes. J. Biol. Chem. 262, 10,03510,038. Niblett, C.L., Zagula, K.R., Calvert, L.A., Kendall, T.L., Stark, D.M., Smith, C.E., Beachy, R.N. and Lommel, S.A. (1991) cDNA cloning and nucleotide sequence of the wheat streak mosaic virus capsid protein gene. J. Gen. Virol. 72, 499-504. Prols, M,, Davidson, A., &hell, J. and Steinbiss, H.-H. (1990) In vitro translation studies with cDNA clones corresponding to the RNA’s of barley yellow mosaic and barley mild mosaic viruses. J. Phytopathol. 130, 249-259. Sambrook, _I., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning. A laboratory manual. Second edition. Cold Spring Harbour Press, USA. Sanger, F., Nicklen, S. and Cot&on, A.R. (1977) DNA sequencing with chain-te~ination inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463-5467. Shukla, D.D. and Ward, C.W. (1988) Amino acid sequence homology of coat proteins as a basis for identification and classification of the potyvirus group. J. Gen. Virol. 69, 2703-2710. Shukla, D.D., Strike, P.M., Tracy, S.L., Gough, K.H. and Ward, C.W. (1988) The N and C termini of the coat proteins of potyviruses are surface-located and the N-terminus contains the major virus-specific epitopes. J. Gen. Viral. 69, 1497-1508. Shukla, D.D., Frenkel, M.J. and Ward, C.W. (1991) Structure and function of the potyvirus genome with special reference to the coat protein coding region. Can. J. Plant. Pathol. 13, 178-191. Timmerman, G.M., Calder, V.L. and Bolger, LEA. (1990) Nucleotide sequence of the coat protein of pea seed-borne mosaic potyvirus. J. Gen. Viral. 71, 1869-1872. Usugi, T., Kashiwazaki, S., Omura, T. and Tsuchizaki, T. (1989) Some properties of nucleic acids and coat proteins of soil-borne filamentous viruses. Ann. Phytopathol. Sot. Jpn. 55, 26-31. Ward, C.W. and Shukla, D.D. (1991) Taxonomy of potyviruses: current problems and some solutions. Intervirology 32, 269-296.