Weg 10, D-5000 Kbln 30, Germany. The complete nucleotide sequence of .... 270. B N. W I V. R F. T V. D L H S S L K S A L E V D. D T. G G. N A V L 0 L L. 309.
Journal of General Virology (1991), 72, 989-993. Printedin Great Britain
989
The nucleotide sequence of RNA 2 of barley yellow mosaic virus A. D. Davidson,* M. Pr01s, J. Schell and H.-H. Steinbiss Max-Planck-Institut f~r Ziichtungsforschung, Abteilung Genetische Grundlagen der Pflanzenzftchtung, Carl-von-Linne Weg 10, D-5000 Kbln 30, Germany
The complete nucleotide sequence of RNA 2 of a German isolate of barley yellow mosaic virus (BaYMV) has been determined. The RNA is 3585 nucleotides in length excluding a 3'-terminal poly(A) tail. The viral plus and minus strands in all three reading frames contained only one large open reading frame which started at positions 156 to 158 and terminated with a UAG codon at positions 2828 to 2830, thus encoding
an Mr 98000 polypeptide. Comparisons with sequences of other viruses revealed that the amino terminus of the polypeptide has homology with the proteolytic domain of the helper component proteinase of several potyviruses. It was determined that the 98K protein is a polyprotein composed of an aminoterminal 28K protein and a 70K protein.
Barley yellow mosaic virus (BaYMV) is a bipartite, positive-sense, single-stranded R N A virus (RNA 1 approx. 7-7 kb, RNA 2 approx. 3.6 kb) and is a causal agent of a serious disease of increasing agronomic importance of winter barley cultivars in European and Asiatic countries (Inouye & Saito, 1975; Huth et al., 1984; Huth, 1988). The virus is transmitted by the soilborne fungus Polymyxa graminis (Adams et al., 1989), hence control measures are difficult. BaYMV was originally classified as a potyvirus as it has several properties in common with these viruses (Hollings & Brunt, 1981; Shukla & Ward, 1989). However, it has recently been proposed that BaYMV be classified separately in the bymovirus group due to the accumulating evidence of differences between BaYMV, and other similar fungus-borne viruses, and the classical, generally aphid-borne potyviruses (Kashiwazaki et al., 1989; Usugi et al., 1989). The isolation of cDNA clones corresponding to the genomes of BaYMV and barley mild mosaic virus (previously BaYMV-NM and BaYMV-M types) is leading to a greater understanding of the genomic structure of these viruses and their relationship to other viruses (Kashiwazaki et al., 1989; Pr61s et al., 1990). In order to understand better the molecular biology of BaYMV we have constructed and sequenced a cDNA clone corresponding to RNA 2 of this virus. Results similar to those presented in this manuscript have been obtained during parallel work by Kashiwazaki etal. (1991) using a Japanese strain (II-1) of BaYMV. The almost full-length cDNA clone psY35, represent-
ing the entire coding region and 128 nucleotides of the 5' non-coding region of R N A 2 of a German isolate of BaYMV was isolated as described previously (Pr61s et aL, 1990). The dideoxynucleotide chain-termination method (Sanger et al., 1977) was used to sequence both strands of subclones derived from psY35 by using both restriction enzyme fragments and exonuclease IIIgenerated nested deletions (Henikoff, 1987). Thus the complete nucleotide sequence of psY35 was determined for both strands of the DNA. For primer extension analysis, 20 pmol of the oligonucleotide 5' CACAGTCAAAGAATAACCG 3', which is complementary to nucleotides 174 to 192 of R N A 2, was added to 1 ~tg of BaYMV R N A (total volume 8 ktl in H20) isolated as described previously (Pr/51s et al., 1990). The mixture was heated to 65 °C for 3 min and then incubated at room temperature for 10 min. Reagents for cDNA synthesis were added (Pr/51s et al., 1990) and the reaction was incubated for 1 h at 42 °C. The size of the extended cDNA was determined by electrophoresis in a 6 ~ sequencing gel and comparing the mobility of visible bands to that of a plasmid sequencing reaction using the same primer. The remaining primerextended cDNA was purified from the oligonucleotide primer using a Centricon 10 ultrafiltration device and was poly(A)-tailed (Sambrook et al., 1989) using terminal deoxynucleotide transferase (BRL). After hydrolysis of the R N A template the cDNA was amplified in a polymerase chain reaction using Taq polymerase (Perkin Elmer Cetus) and the primers 5' GAGATATCGCGGCCGCATCGA(T)ls 3' and
0000-9936 © 1991 SGM
990
Short communication
1 ~T~Mcc~T~c~m~c~c~A~AT~G~A~`b~cT~AGG~A~M~AcT÷GTAGAGT~h-r~TGT~AT~cTAcTGTTT~T~GcT~T~
12o
121 AG~T~cTcG~T~G~÷~T~TT~÷~A~ATGT~TA~TT~AGb~A~GGrfA÷T~TTTc~T~TGGcAGT~T÷GATTGG~T~G~T÷ATrrGG~GA&~A~cAE 240 M S T S S S R L
F D C G 5 L D W P
K 5 L F 6 D P T T
29
241 GAGATGTGATGGACGAACACATTTCTAGCACGTGGAATGCGGTTATTAGGAGGCACATGT [GGCTCCCAACGCCGACGCCGAAACCATATTGGGCCGTGATGGTTTACCATCTGCTCAAT 30 D V M D E H I S S T W N A V I R R H M A P N A D A T I G R D G L P S A Q
360 69
361 T~GACGCGTATGGAGCCATGCTAcCTAGCTTTATCCAAGcTcTAAAcGCACCAAcAACGCGTCTTCC~ATCAGCGCACCGCTGTCTACCGCCGAGTCCATTTTGTGCGCTGATGCATCTC 70 D A Y G A M L P S I Q A L N A P T T R L R I S A P S T E S L C A D A S H
480 109
481 ACGCTcCTTGGTTGTACATGGcAAACAGTGTGTGCGCATAcGAGGcAACTCATTTGcAGCCTGTACAAACTTTcATCGcCTTTAACTTcGCACATGGTTATTGCTACCTcAGTCTTTTCA 110 A P W L Y M A N S V C A Y E A T H L Q P V Q T F I A N F H G Y C Y L $ L F
600 149
601 TACCACTAAGCTTTCGCATTACCCCCGAGAATGCTCGAAGTTTCAGTCGGTTCCTTGAGCAGCTTCCCGACATTTTAGGTGCGTATCCAACATTGGCCTCACTATATAA~CAATGTTAT 150 P L F R I T P E A R F S R F L E Q L P D I L G Y P L A L Y K T M L F
720 189
721 TTGCTGTTAGGCTTTTCCCAGAGGTGCTACAAGCTCCAATCCCCATTATTGCAAAAAGGCCTGGTGTGCTACAATTCCATGTTAGTGATGCCAGAGGACTGCCACCTTCGTGGTTCCCCA 190 A V L F P E V L A P P I I A K R P G V L Q F H S D R G P P S W F P M
840 229
841 TGAAGTGcGGCAGTGTAGCATcTTTcATAGCGCTTATTAccAACAAccTTAACAGcGATTTGcTTAATGG~ATTGTTGGTTCAAACGGTGATGGTGAGCAcTACAcAAATTGGAAcTcTG 230 K C G 5 V A S F I A L I T N N L N S D L L N G I V G N G G E Y T N W N S G
960 269
961 GGCATAATCACTGGATTGTGAATCGATTTATAACAGTCAAGGATTTACATAGCAGTTTAAAATCAGCTCTAGAAGTTGATTTGGACACAGAAGGTGGGCGA~ACGCTG TTCTCC~I-FTGC 1080 270 B N W I V R F T V D L H S S L K S A L E V D D T G G N A V L 0 L L 309
ioai TTcTAGATcTTGGAGTCAcTAATcTTGTTCGAAGAGAGAAAcGTTTccckGcATATTTCCAGGGAGcTG~AGCGTATACCTGCTTCTTTCATGTGAAAGAGTTGGGAAEGAGcT12oo GGTGG 310
L D L G V T
L V
R E K R F P A Y F Q G A E S V Y L L L
C E
V G N E L V A
349
1201 CTGTGCAAGATGCTCTTCAGGAACCATTAGCTAATTATACTGGAAAGGATTTGAGAGcTCTCATCATCAATCTTGGcGGTTTACCAAGCAGACAccCTGAGATTTGTTAcACACGTAACA 350 V Q D A L Q P L N Y G K D R A I I N L G G P S H P I C Y R N
1320 389
1321 TCTTTGAA~TGATAATCACCTGGTTTGGAATTTTGAGTTCTACcGAATAGCCTcAATTACGAAGAATGCACAAATTGATAGAGATGTCcTTAGcTcATcCATGGCGAAcTTATTTAGTG 390 F E N D N H V W F E Y R I S I K N A Q I D R D V S S M A N F S D
1440 429
1441 ATTTTGTTTCAGAGTCATCAAATGG TGAGTACAGAGTTAAGGAACC/b~GACCTGTTACTCAGTACAGGGTTGAGCATGATGAACCAG TTGCTAGTGGTGCCCCATCTGCTTGGTGGCAAG 1560 430 F V S E S S G E R V K E P R V T Y R V E H D E P V S G A P S A W Q 469
1561 TTCTTGTTGGCATCACCACCGCCATTCTGGGCGcAATAAl.ATTcTTTCTTTGGAGGTGTTTcTTGCGTGCTAAGCGTGTcAAATTccAGGCAAAGGATTccTTCccATGGTTTAccACGT 1680 470
L V G I
T T A I
L
A I
I
F F L
R C
L R A K R V K F Q A K D S F P W
T T
509
1681 CTGGTGATGATGACCTCcCGCCCccCcCTGGTGATTccCCGTCAcGcCCTCCTGGGcGCAGcccGGATCGAGTTCTTccAcGTACAGTTGTTCGAGATCTCAGCTTcAATGATGATGATG 510 G D D D L P P P P D S P S R P G R P D R V L P R T V V R D L S F N D D
1800 549
1801 ATTTAcACAGTGTTGATcTTAATGAAGCTGGGTCGCGTT~GGGGAGGTTGTCTcTTTAATTGCAAGGGGAAATcTCCGCGAGCTAGCTGGTGCGATTCCTGAATcTCTTAGCAAcTTAA 650 L H S V D L N E A 5 R F G E V S L A R G N L R E L A G A I P E S L N L
lg20 589
1921 CTCTCCTTCJ~AC~GTGCAAGTG~TCTGGCTTTTACACTkTGG-fGGCTCTGTACCTTGCCACTT~AGGG~TGCT~iEACT$CAlT£CA~GCA~GCTTCACC~GCCAC~ 2040 590
L L Q
T
S k
S G
S G
F Y
T M V k
Y L
T
L
D A
I T A
F H E H N D A
S P A
T
629
2041 TTCAATCACTGCGCACGCTCGAGCTTCAGCTTGAAGCCCGTGGTTTGCGTTTCAAT~GCTGGCACACCCGC~TC T(IATTCAG/kGGGGCGTTAAATCTTCI-GTTGGTA~GCACTTG 630 Q S L R T L E L Q L E A R G L R F N E G T A N L I Q R G V K S S V G A L
2160 669
2161 TGCGACTMCACAC~a,GTGCTC TTCTGGCC~CTGGTG~G~4u~CTTTCGGAC~CGCATGGC~GCCACACTTGAN~£~ua`TTGC.FGCTGAGCGCCTCAAC~CGTl-AACCGCCTATGATCAGCGAG 670 R L T Q S A L L A T G E N F R T R M A T L R I A A E R L N T L T A V Q R
2280
TcA~c~Aic~c~c~ccTA~cA6c~Tc~AET~c~cTT~T~`c~cA~cTcAc`rccAcA~cTcGc~c~cc~G~Tc~Gc`;-Tc~TTA~c~TcTcT
2400
2281 710
I
E H I
T E L L k A I
K T A L E V E R
E L T P H L
N A
k L L Q V Y N N L
709 749
2401 TTAGcAcCGATTATGCTTcAGCATCGCTGCTTGcTCTl~AGACGcGAGATGATTCTTAGGAGTGcTGAAGGGCGTGTTGGAGAAcAGccAAcTAGTGcTTcTGATGcGGCTAATGAGGAAT 750 S T D Y A S A S L L A L R R E M I L R A E G R V G Q P S A S D A A N E E
2520 789
2521 TAGTCCAAcGTTCcATGACAAAGTTGGATAAGGAA~TTGAGTTATTccAAGcAcAAATTGATAGccAGCGTCGcGCTGTcAccATCACTGAAGCATcCAATCTCAGAGAAAACATATTGC 790 V Q R S M T K L D K E I E L F Q A Q I S Q R R A V I T A S N L R E N I L Q
2640 829
264~ AGccGATc~cAc~TT~`c~TATT~cc~TG~T~A~TTTTcT~c~G~Gc~cGcGccATc~cA~GccAGGGATAccA~AT~TG~cT~cGcccAT~Tcc~ccc÷TTTc~A~cTT 2760 830
P I
N T V A N I
A M A G A F L R G G A
H R M P G I
D V
A P M S N P F R A F
869
2~61 TcTcAG~GG~cATTckcTcAc~c~`cT~TG~TG~TcTTrr~cGcccTc~TTTA~TiTTTccATcATAGcA~rATTTAcmccGccGcc~TAGcA~TTTA~Tc2880 870
S G R G H S L
T T T
R G A G L
F R R P R V *
2881 AA~ATTATCCATTTCTT~Cl~CCTTT~AC~ATCTG~ACACCG~TG~ATU~ATATGGTAGAGTTAGCGGTTCATCCCCCGCTACGCTCAA~ATCTCCAT~TTAGAT~A~CTGCAAA~ACA 3000
3001 T~TAcc~TAccA~TTATcATTc÷~'cTc~Gc~ccAGcAT~÷~TTAccc~c~TTGcAT÷ATc~T~T~&ATc~Tk~GG~c~T~cATc~cATETGcTc~cAT~ 3120 3121 ATTGGGI'GT/~,TGGGGCCAGi'GTTBTTTTGiIGTATGGAAA~GATTBGT~GAATTG~a,TTC TTC TCACCATGAGTTTTCC,a,GGGGAT~GC'FCGGl'GCTA6AGCTAGCGB6TGTATCCTTi;
3240
3241 ~T~ccc6AcAccAc~;~CAcCAcT~wrA~T~AGcc~rrr6~TATccc~CTGc~GcAc~TAG6ATT~ATcT÷cA~T~"~cc~TTAGA6Tc~cTGGcA6
3360
~36~ ~T~TGGGcT~CAGGA~cAET~T~cTG~TcTGTCA~c~TcTGTC~T6~C~TcT~T~TcTAGCA~Tkc~6T~&CAcGTCTTT~TcCCCAG~ATGmACThAT~TcTGE 3,~o 3481 ATA~A/~J~AC6TCACATCTGi~GATACAAA/Vk~ATGC~TA6GTGCCCATC/kTTGGTCGCG(iGTTGCAGTCiGACGAAC~(~:MCCTTT~TACACAGG~a`AATGT~AC(A) n"
Fig. 1. Nucleotide sequence of BaYMV RNA 2 and the amino acid sequence derived from the single large ORF starting at nucleotide 156.
Short communication
5' CACAGTCAAAGAATAACCG 3' following the method of Frohman et al. (1988). The resulting products were cloned into the NotI/AccI sites of psY35AC (psY35AC was constructed by digesting psY35 with AccI and religating the 3.1 kb restriction fragment which contains pBSK- and the first 140 nucleotides of psY35). cDNA clones having appropriately sized inserts were sequenced. Oligonucleotides used for this work were made in an Applied Biosystems model 380B DNA synthesizer. To facilitate investigation of the organization and maturation of protein products encoded by RNA 2, the clone psY35AH was constructed. A 393 bp HindlII fragment located between nucleotides 195 and 588 of psY35 was deleted by partial restriction enzyme digestion and the remaining fragment was religated. This deletion conserved the reading frame of the encoded protein. In vitro transcription and translation reactions and the subsequent detection of in vitro translation products were as described previously (Pr61s et al., 1990). Nucleotide sequence data was compiled and analysed using a Vax 220 computer and the software package of Devereux et al. (1984). Primer extension analysis resulted in the detection of two oligonucleotides differing by a single nucleotide (data not shown) which extended psY35 by 19 or 20 nucleotides. Sequencing of several cDNA clones demonstrated that both primer extension products had been isolated as cDNA clones. As no other bands were detected by primer extension it is likely that the sequence shown in Fig. I corresponds to the full-length sequence of RNA 2 (nucleotides 1 to 20 correspond to the larger primer extension product and nucleotides 21 to 3585 to psY35). However it cannot be excluded that extra nucleotides may be present at the 5' terminus as the reverse transcriptase may terminate prematurely due either to a protein bound to the RNA or to the secondary structure of the RNA. Thus the sequence of RNA 2 would appear to be 3585 nucleotides in length excluding a poly(A)tail (Fig. 1). The first AUG codon in RNA 2 is at positions 156 to 158 in the sequence ACCAUGU which is in good agreement with the consensus sequence for initiation of translation of eukaryotic mRNAs (Kozak, 1986; Liitcke et al., 1987). The open reading frame (ORF) starting at position 156 continues until a UAG codon at positions 2826 to 2828. The 3' non-coding sequence is 757 nucleotides long. There are no other ORFs coding for proteins > 8K in any frame of either RNA strand. The Mr of the putative translation product is 98K. Recently, Maiss et al. (1989) have identified the sequence UCAACACAACAU in the 5' non-coding region of several potyviruses. The sequence is not in the 5' non-coding region of BaYMV RNA 2 which, like
TEV TVMV PPV PVY BaYMV
609 559 612 586 103
TEV TVMV PPV PVY BaYMV
656 607 660 634 150
TEV TVMV P?V PVY BaYMV
703 653 706 680 195
TEV TVMV PPV ?VY
750 700 753 727 242
SaY,IV
+*+++++ + + +****+++*+ YSDLKHPTKRHLVIGNSGDSKYLDLPVLNEEKMYIANE~MNIFF YSELKMPTKNHLVIGNSGDPKYLDLPGEISNLMYIAKZ~iii I NIFL ESEIISPTKNHLVVGNSGDSKYVDLPTAKGGAMFIAK NIFL ESTFYPPTKKHLVIGNSGDQKFVDLPKGDSEMLYIAK NVFL LCADASHAPWLYMANSVCAYEATHLQPVQTFIAFNFAHY~C~LSLFI I ++ ++++ ++*++*÷+++ +++ + **++ *++ ++ ++ * + ++*
ALLVNVKEEDAKDFTKFIRDTIVPKLGAWPTMQDVATACYLLSILYP AMLVNVDEANAKDFTKRVRDESVQKLGKWPSLIDVATECALLSTYYP AMLININEDEAKSFTKTVRDTLVPKLGTWPSM~4DLATACHFLAVLYP AMLINISEEDAKDFTKKVRDMCVPKLGTWPTMMDLATTCAQMRIFYP PLSFRITPENARSFSRFL EQLPDILGAYPTLASLYKTMLFAVRLFP ++++* +* + +++ +** *+ * +++++ +* ++++ ++++ DVLSAELPRILVDHDNKTM~LDSYGSRTTGYHMLKMNTTSQLIEFV AAASAELPRLLVDHAQKTI~V~DSYGSLNTGYHILKANTVSQLEKFA
ETRNAELPRILVDHEAKIF~DSFGSLSTGMHVLKANTINQLISFA DVHDAELPRILVDHDTQTC~/~DSFGSQTTGYHILKASSVSQLILFA EVLQAPIPIIAKRPGVLQF~DARG-LPPSWFPMKCGSVASFIALI 2 +++*+*++ + +** + HSGLESEMKTY~-~--~ SNTLESPMAQY~VGG 1 SDTLDSNMKTYI~VGGI NDELESDIKHY~ TNNLNSDLLNG 3
991
655 606 659 633 149 702 652 705 679 194 749 699 752 726 241
?64 714 767 741 256
Fig. 2. Comparisonof aminoacids 103 to 256 of BaYMVRNA 2 with the proteolyticdomainsof HC-Pro of TEV, TVMV, PPV and PVY. The alignmentof the potyvirusaminoacid sequencesand identificationofconservedactivesitedomains(boxes 1and 2) are takenfromOh & Carrington(1989). Box 3 is the predictedcleavagesite for HC-Pro. Amino acids that are invariant between BaYMV and the four potyviruses (*) and either identical to at least one of the four potyvirusesor functionallysimilarto the potyviralaminoacids(+) are marked.
many other plant RNA viruses, is A/U-rich and G-poor (10~). There is little 3' sequence similarity between the RNA 2 3' non-coding region and that of RNA 1 (Kashiwazaki et al., 1989; our unpublished data) with the exception of the sequence U U A U G U U C located 109 nucleotides upstream of the poly(A) tail which contains the putative polyadenylation signal UAUGU found in several potyviral genomes (Maiss et al., 1989). Comparison of the amino acid sequence of the 98K protein with protein databases revealed similarities only between the amino-terminal region and part of the helper component proteinase (HC-Pro) of several potyviruses. The sequence between amino acids 125 and 256 of the 98K protein can be aligned with regions in the polyproteins of four potyviruses that correspond to the proteolytic domain of the proteinase HC-Pro which is autocatalytically cleaved from the potyviral polyproteins (Fig. 2) (Carrington et al., 1989). Although there is less homology between BaYMV and tobacco etch virus (TEV), tobacco vein mottling virus (TVMV), plum pox virus (PPV) and potato virus Y (PVY) than among the four potyviruses in this region, there is strict amino acid conservation around the two active site amino acids, a cysteine and a histidine that were found to be essential for the autocatalytic processing of the HC-Pro (Oh & Carrington, 1989). There is also amino acid conservation at the predicted cleavage site in the potyvirus sequences except that the G/G cleavage site of TEV, TVMV, PPV and PVY is replaced with a G/S dipeptide in BaYMV (Fig. 2, box 3). However serine is functionally similar to
992
Short communication
1
2
9
97-4K
"~-'-66.2K
"-'-- 42.7K
?
"~'--31K
-,~---21.5K Fig. 3. Comparison of in vitro translation products of transcript RNA corresponding to psY35 (lane 1) and psY35AH (lane 2). Differences between the translation profiles of the corresponding RNAs are indicated. The Mrs of a set of protein markers (Bio-Rad) are shown.
glycine and has been found at cleavage sites in other viral polyproteins (Kr/iusslich & Wimmer, 1988). In addition, the conservation of functional domains and structural similarity at the secondary and tertiary levels of proteins is often a more important criterion for protein homology than the overall amino acid composition (Gorbalenya et al., 1989). In vitro translation of R N A 2 (Pr61s et al., 1990) yielded several translation products smaller than 98K. The 28K product had the size expected for a protein cleaved from the 98K protein at the G/S dipeptide. Thus it appears that the 98K protein is a polyprotein that is subsequently processed; however, the possibilities of either premature termination or endogenous proteolytic degradation occurring in vitro could not be dismissed. If the 28K protein lies at the amino terminus of the 98K protein and has autocatalytic activity analogous to the HC-Pro, then deletion of one of the putative active sites would lead to a loss of autocatalytic activity and the production of an unprocessed but correspondingly deleted 98K protein. To test this hypothesis the clone psY35AH was constructed, psY35AH encodes an 84K protein which has amino acids 21 to 151 of the 98K protein, encompassing the putative active site sequence, GCYC, removed. As demonstrated in Fig. 3, analysis of the in vitro translation products of transcript R N A corresponding to psY35 and psY35AH confirmed that the 98K protein is a polyprotein. The 28K protein was
absent from the in vitro products corresponding to psY35AH, demonstrating that this protein lies at the amino terminus of R N A 2 and that cleavage of the 98K polyprotein is likely to occur at the G/S dipeptide. The 74K protein which probably corresponds to the 70K protein predicted from the sequence data was also absent but an 88K protein appeared. This product is the size expected if the remaining 14K of the 28K protein could not be cleaved from the 74K (70K) protein. However, it is apparent that several other products are produced upon in vitro translation of R N A 2 which are not affected by the 14K deletion. Thus these products cannot originate from the initial A U G codon and are not generated as a result of proteolytic activity of the 28K protein. It is not known whether these products are artefacts of the in vitro system, i.e. products arising from translational initiation at downstream A U G codons, or arise from a novel translational strategy of this virus. Although the 28K protein has homology with the potyvirus HC-Pro, this homology is only in the domain displaying proteolytic activity, The domain corresponding to that in HC-Pro which is involved in aphid transmission is quite distinct. Thus from this study there is no evidence that the 28K protein has a role in the fungal transmission of BaYMV. In conclusion, R N A 2 of BaYMV encodes a 98K polyprotein, of which a region at the amino terminus appears to have an autocatalytic activity analogous to that of HC-Pro, allowing the generation of two protein products of 28K and 70K. Thanks to W. Schmalenbach for the synthesis of oligonucleotides used in this work and R. Walden for critical reading of this manuscript. This work was financed in part by Gebriider Dippe Saatzucht GmbH, Bad Salzuflen, Germany. A. Davidson was supported by the Alexander von Humboldt Foundation.
References ADAMS, M. J., BATISTA,M. DE F., SWABY,A. G. & ANTONIW, J. F. (1989). Fungally-transmitted viruses of cereals in the UK. EPPO Bulletin 19, 573-577. CARRINGTON,J. C., CARY,S. M., PARKS,T. D. & DOUGHERTY,W. G. (1989). A second proteinase encoded by a plant potyvirus genome. EMBO Journal 8, 365-370. DEVEREUX,J., HAEBERLI,P. & SMITHIES,O. (1984). A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Research 12, 387-397. FROHMAN,A., DUSH,K. M. & MARTIN,G. R. (1988). Rapid production of full length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proceedings of the National Academy of Sciences, U.S.A. 85, 8998-9002. GORBALENYA,A. E., DONCHENKO,A. P., BLINOV, V. M. & KOONIN, E. V. (1989). Cysteine proteases of positive strand RNA viruses and chymotrypsin-like serine proteases. FEBS Letters 243, 103-114. HENIKOFF,S. (1987). Unidirectional digestion with exonuclease III in DNA sequence analysis. Methods in Enzymology 155, 156-165. HOLLINGS, M. & BRUNT, A. A. (1981). Potyviruses. In Handbook of Plant Virus Infections: Comparative Diagnosis, pp. 731-807. Edited by E. Kurstak. Amsterdam: Elsevier.
Short communication
HtrrH, W. (1988). Barley yellow mosaic - a disease in Europe caused by two different viruses. In Developments in Applied Biology: Viruses with Fungal Vectors, vol. 2, pp. 61-70. Edited by J. I. Cooper & M. J. C. Asher. Wellesbourne: Association of Applied Biologists. Hua~d, W., LESEI~AI~rN,D.-E. & PAUL, H. L. (1984). Barley yellow mosaic virus: purification, electron microscopy, serology, and other properties of two types of the virus. Phytopathologische Zeitschrift 111, 37-54. Ir~otJvE, T. & S~rro, Y. (1975). Barley yellow mosaic virus. CMI/AAB Descriptions of Plant Viruses, no. 143 KASmWAZArd,S., HAYANO,Y., MINOBE,Y., OMURA,T., HIB1NO,H. & TsucmzAKI, T. (1989). Nucleotide sequence of the capsid protein gene of barley yellow mosaic virus. Journal of General Virology 70, 3015-3023. KASI-HWAZAKI, S., MINOBE, Y. & HIBINO, H. (1991). Nucleotide sequence of barley yellow mosaic virus RNA 2. Journal of General Virology 72, 995-999. KOZAK, M. (1986). Point mutations define a sequence flanking the A U G initiator codon of eucaryotic ribosomes. Cell 44, 283-292. K~USSLICn, H.-G. & WIMI~g, E. (1988). Viral proteinases. Annual Review of Biochemistry 57, 701-754. Lf.rrCKE, H. A., CHow, K. C., MICKEL,F. S., MOSS,K. A., KERN, H. F. & SCHEELE,G. A. (1987). Selection of AUG initiation codons differs in plants and animals. EMBO Journal 6, 43-48. MAtss, E., TIMpI~, U., BRISSKE, A., JELKMANN, W., CASPER, R., H~ILER, G., MATrANowc-~d,D. & KA~NGER, H. W. D. (1989). The
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complete nucleotide sequence of plum pox virus RNA. Journal of General Virology 70, 513-524. OH, C.-S. & CARRmGTON, J. C. (1989). Identification of essential residues in potyvirus proteinase HC-Pro by site directed mutagenesis. Virology 173, 692-699. PROLS, M., DAVIDSON,A., SCHELL,J. & STEINBISS,H.-H. (1990). In vitro translation studies with cDNA clones corresponding to the RNAs of barley yellow mosaic and barley mild mosaic virus. Journal of Phytopathology 130, 249-259. S~BROOK, J., FRn~SCH, E. F. & MA~TtS, T. (1989). Molecular Cloning: A Laboratory Manual, 2rid Edn. New York: Cold Spring Harbor Laboratory. 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. SnUKLA, D. D. & WARD, C. W. (1989). Structure of potyvirus coat proteins and its application in the taxonomy of the potyvirus group. Advances in Virus Research 36, 273-314. Usu~I, T., KAsmwAzAKI, S., O ~ , T. & TsucmzAKI, T. (1989). Some properties of nucleic acids and coat proteins of soil-borne filamentous viruses. Annals of the Phytopathological Society of Japan 55, 26-31.
(Received 24 September 1990, Accepted 9 January 1991)
