Complete nucleotide sequence of the genome of an apple isolate of apple chlorotic leaf spot virus


The complete nucleotide sequence of the genome of an apple isolate of apple chlorotic leaf spot virus (ACLSV-A) was determined. The genome is 7552 nucleotides excluding the poly(A) tail and contains three open reading frames (ORFs 1, 2 and 3), encoding proteins with M r values of 216503 (216.5K), 50453 (50.4K) and 21394 (21.4K), respectively. Nucleotide sequence comparisons between ACLSV-A and the previously sequenced ACLSV from plum (ACLSV-P) showed that the sequence identity at the nucleotide level was 79"8 %. Amino acid sequence identities of ORFs 1 and 2 between both isolates were 88.4% and 79.9%, respectively. The 21.4K protein encoded by ORF 3 of ACLSV-A had an amino acid sequence identity of 88.6 % with the 28.3K protein encoded by ORF 3 of ACLSV-P. Immunoblot analysis of the 21-4K protein expressed in Escherichia coli showed that this protein is the coat protein of ACLSV-A. 

Apple chlorotic leaf spot virus (ACLSV) is distributed worldwide in fruit trees including apple, peach, pear, plum, cherry and apricot (Lister, 1970;N6meth, 1986). In Japan, this virus is one of the causative agents of apple topworking disease and induces lethal decline in apple trees grown on Maruba kaido (Malus prunifolia ringo) rootstocks (Yanase, 1974). ACLSV has very flexuous filamentous particles, approximately 600 to 700 nm in length and contains a polyadenylated plus-sense, ssRNA with an M r of 2"48 x 106 and a single coat protein of 22K (Yoshikawa & Takahashi, 1988). At present, this virus is classified into the closterovirus group based on its particle structure (Francki et al., 1991). Recently, German et al. (1990) reported the nucleotide sequence of the genome of ACLSV isolated from plum (Prunus domestica) (ACLSV-P) in France. 
In this paper, we report the complete nucleotide sequence of the genome of ACLSV isolated from apple (ACLSV-A) in Japan and the comparisons of the nucleotide sequence and the deduced amino acid sequence with those of ACLSV-P. 
ACLSV-A (isolate P-205), originally isolated from an apple tree (Yanase, 1974), was propagated in Chenopodium quinoa and purified as described previously (Yoshikawa & Takahashi, 1988). Viral RNA was extracted from purified virus by dissociation in 2 % SDS The nucleotide sequence reported in this paper will appear in the DDBJ, EMBL and GenBank nucleotide sequence databases under the accession number D14996. for 15min, followed by extraction with phenolchloroform, precipitated from 70% ethanol and suspended in water. First and second strand cDNAs were prepared from 2 tag of ACLSV-A RNA according to Gubler & Hoffmann (1983) using oligo(dT) or random hexanucleotides as primers. The dsDNAs were bluntended using T4 DNA polymerase and ligated into the EcoRV site of Bluescript KS + (BS) or dC-tailed and annealed to dG-tailed, PstI-cut pUC9 (Yoshikawa et al., 1988). The resulting plasmids were used to transform competent Escherichia coli DH5 ~ cells. 
The cDNA clones or subclones produced after the ligation of restriction fragments into BS were digested with exonuclease III and mung bean nuclease (Henikoff, 1984) and the resulting deletion mutants were used for sequencing by the dideoxynucleotide chain termination method (Sanger et al., 1977) using Sequenase version 2.0 (United States Biochemicals). Each base was determined by sequencing at least two independent clones. More than 80% of the genome was sequenced in both directions and dITP was used to resolve ambiguities resulting from compression. 
The sequence of the 5'-terminal region of ACLSV-A RNA was determined by extending a synthetic primer (5' AGGCGTTACGTCAATCTG 3') complementary to nucleotides (nt) 38 to 55 of the viral RNA using reverse transcriptase and terminal deoxynucleotidyl transferase (Deborde et al., 1986). All nucleotide sequence data were collected and analysed using the program GENETYX version 8.0 (Software Development Co. Ltd). 
The genome of ACLSV-A consists of 7552 nt ex- 
I TGATA•TGATA•AGTG•ACACTCACGTCGTGAGTAAACAGAT¥GA•GTAACG•CTCAATCGTGGT•AAGGA••^GcATTTTAATGCTA•AA•GTTA^AT^CAAT••CATTGT^TTCAAGA 121 A~TTTC~GTTTAACTAcGTGAAA~GTTA~AAATGGcTTTCTcTTATAGAACGcCA~AGGAAGAA~TTCTGAG~AGGTTA~cTCAGTcTCAGcAGGAAGCGATcAGCGGATTTCAGTACGA ORF1 /4 A F S Y R T P ~ E E L L 8 R L P Q S q Q E A 1 S G F Q Y It 241 GCGATTTCAAAAGGAGOAAGAGAAGAAGCTGG~AAACTTC TCTTTTTACCTACCGGAGAAGACCAGGGAGTGGTTCACT AAGTCAGGTGTATATCTCTCACCCTTTGCTTATGTCAATCA R F Q l( E It E K K V E N F S F Y L F It g T 11 E N F T K S G V y L $ P F A ¥ V N H 361 TTCTCACCCTGGTT•CAAAACTCTGGAGAACCACCTTTTATTTAA•G••GTA•CTAGTTATATTAGTAAGTATTCTTATGTAGCTTGTCTTAG•ATTAAGTCGAATAAAAT6AGCAAGAT S H P G C K T L It N H L L F N Y V A $ Y ! S K Y S Y V A C L $ [ K S N K b( $ K N 481 GGAACGTTTGGGTTCC•ATTCAGTCAAAACCTATGACATTTTGAACAGATTAG•TACTGC7AAGGACAAGGCTCGATATGGACCTTTGGCCAGGCCTGAGCGGTCTCCATGTCCAA^GAA It It L G S N S V It T Y D I L N 1~ L V T A K D K A 5 Y G P L A $ P It R S P C P K K 501 ~AC~ACATTTTCATTCATGATGA~ATCCATTATTGGTCAAGGGATCAACTTGAGACATTCCTTCAGGTTCACAGACCCAAAAATTTGTGGGCGACTCTTGTTTTTCCCCCTG~GATCTT T N I F I H D It I H Y W $ R 0 Q L It T F L Q V H 11 p It N L W A T L V F P P It I L 721 GGCTGGTTATAAGTCGTCAGTTTTAccATTTCTTTACcAGTTTGAGATTC^TGGAAAAGATTTGGTTTACATGCCTGATGGAGTTAGATCAGAGA~TACACTCAACCTCTTGAGAATGG A G Y l{ S 3 V L P F L Y q F It I ½ G K D L V Y H P D G V R S It :3 Y T Q P L E N G 841 ATTTCTTCTGAGTTcAA~CAGTATACTTGTGAGAAACAAGGCTACTGGAGTTGAAATTAGATATCAGGTTAGTCTTGTGTATTCTCTGGGTTCTCACCATT7GTTCCACATTTACCCCAC F L L $ S $ S I L V R N K k T G V It [ B Y Q v S L v Y S L G $ H It L F H { Y P T 961 GGAGGACCTGATGAAAGAAGAGGTGAGAA~GTTTGGACCTTATGATTTGTTCGATGTGGGTTCATTATTTGTCAAGCCAGTGCGCGTCCcTATCCAAGACTTCCcT~TGAGTGTCTT~AA It D L 14 It E E V R 1t F G P Y D L F 0 V G S L F v K P V R v p I q D F P L 6 v F It 1081 AAAGATATTCATTTATCTTAGTTCATTAAAGAAGCCTGATGTTCAGTCTGCAGTGGCCAAACTCAGACAGTTATCCGATGCCGACATTTCCATTGAG^GTGTTTTCAT•GTGCA•GAGTT K 'l F [ Y L 5 3 L It K P D v Q $ A V A K L R Q L 3 D A D I 3 [ It 3 Y F ~4 v q E F 1201 TGC~AGTAGGGTGGA~AAAA&~GG^G~C~AAGTTGGTCCTGCTCATTTT~G~AGTGCATG^AGGACTG~TTTTTTG~TAAGCTGCCATACAGAGAGGTGCT~GAAAAAATTG~ATTGGC A 5 11 v It K N G 'V G $ W S C $ F M E C R ~. ~, 't,l F F D 11 L P "Y It E V L ~-It I G L A 1321 GAATGA•TTTACCAGGCGTTTGATGAAAAT•AAA•CATTGG•TTTcGATAT•CA•ACAACAGACAGAC•ACTCACAGTCAGGATGGTCATAGA•CAGATTTGGGGAGAGAGGCAAAG•TC N D F T 5 R L M K 1 K P L A F D I H T T D 11 P L T V R H V [ D Q I W G It It Q $ S 1441 TTGTGATGATGTTCCAAATATTATATTTTATGGAAGGAAGGAATGGCTTGAGCATGGGCTATTGC~TAAGGTGAAGAAAGG~TTAG~CAAATTGGTTCCAGGAAGGGAGACTGGTGGAAG C D 0 V P N I I F Y G B g E N L E H O L L P It V K K G L A It L V P G It It T G G 5 1561 TGATTACCCAGA AQAAATCTATTCTGATTTGCTG'rCATCCACCTCA ATTTGGAGGTCTT A'Jr GAT GAGA ATCT AAGGC ACAGGAA AGCCAGTCCTATTGTGATACTT A AGTCCG AAAAGGC D Y P E It 1 Y 3 D L L S S T S [ W 11 3 Y 0 It N L il H 11 K A It P [ V I L It S It K A 1681 ATACTCTGAGGCCC•TGGGTT•AG•TCAAACTGTATCAGCCTGTGTTCTACGCCTTTTGGTGAGGTTATTGAAAGGACCC•TTTTGAGGTGGAAAGGGAAAGGAAGAAAAGAGAGTTGTC Y 8 It A P G F S S N C I S L C S T P F G E V I E 5 T P F It V !~ 11 It R g K 5 It L S 1801 TTTTGGCTGTCTTGATT~'TCACATT AGAAAGATGAAGQTTAAAGATGCTTCTGAACTTTCCATCAAGTTGGATGAGCAGAATAAAGAAGGTTTGAGG^GGCAGAAAAGAAAGGAGAAGGC F G C L D F H 1 R g H K V It D A 3 It L 6 I K L D E Q N K It G L R R q K $ g It K A 1921 G/~G AAAACAAGGATGATTCCTGTGCATCTGCTAGAATTAGGTTCAGATCAAAAGGAAAAGAATTTGGGGcAGGAGGCTTCCAAAGGAAAAGGAATTG^ACAGGAAGAGAGGAGGAAAAG K K T It N I P V H L L l~ L G S D Q It It K N L G q It A 6 K G K G l E q It It [I It K S 2041 TGATGAAGCAAAGTTTGATTCTGGGCCcTc~GGA~TTTGCAGCATAAAAGCG~AAAATCCTGTTGATGCACAAcACATTGCTGAGCcCGTGcCATGTTT~AAATTGAATGATCTGATTGG D It A K F D S G P 3 G V C S [ K A E N P V D k Q H [ A ~* P V P C L It L N 0 L I G 2161 CAAAGAGAA&ATCTGTTCTT~TGGTCTTATCAAAACAGTTG~AATGATTATCT~A~TTTGGCCAGGCAGATAGAGGATAT GCCCCTCGCCCAGTTGAAGAACAGGAAAGCAGCATATTT It E K I C $ S G L | K T V G N D Y L T L A I~ Q I E D ~ P L A ~ L ~ N It K A A Y F 2281 ~TG~ATTGACTACCCTATGGTGTACTTT~TGACAAGATTTCATA~c~A^CTTTTGAAGCTA~GGTGAGATCAAAcAAATCATCATGAGAGCAAGAGACAAATGGGGTGCAAATTTCAA C I D Y P H v Y F H D K [ S Y P T F It A T G It I K Q I l M 11 A 3 D It W G A N F N 2401 CAG•GCTCTAATTCAGGTCTATAATGACGGGTGCAGGTTACCTTTGCATAGCGATAATGAGGAGTGCTATGATGATGAT•AGATACTCACGATCAATGTTGTcGGTGATGCAAAATTTCA $ h L ! Q v Y N D G C 5 L P L H 5 D N g E C ¥ D D D It I L T I N V V G D A K F H 2521 C ACAACCTGtI"CATGGTGAA ATCATAGATCTG AGGC AGG GTGAT G AAAT ACTGATGCCCGGGGGTT ACC AGAAGATGAACAAGCAT GCT GTGCAAGTGGCATCGG A AGGGCG AACT AGTGT T T C It G It 1 1 D L R Q G D E t L ~ P G G Y Q K ~i ~ K ~ A "~ E '4 k It It G It T S V 2641 TACTTTGAGAGTGCACAAAAGGGACTTCAGCTTTGAGTCAAAACTCAGGTTCATTAAAGGAAAATTTGATTGCTTGTTTGTCAGCATCGCGGAAATAATACACAAAAAACCAGAAGAGGT T L It V H K 0 D F 6 F E S K L 11 F I K G K F D C L F V $ I A E l I H g K P It E V 2761 GATGATGTTTATCCCACATATTGT•GACAGATGTGTGAGCAATAGGGGTTGCTCACTTGATGAC•CAAGAGCAATATGTGAGAAATATGAGATCAAGATAGAATGTGAAGG•GACTGTGG H M F I P H ! V D [I C V S N fl G C S L D D A R A I C 11 K Y E I K I It C E G D C G 2881 •CTAGTTGAGTGTGGAACAATTGGTCTGTCTGTTGGGAGGATG•TCCTCAGAGGGAATCATTTTACAGTGGCTTCGGTAAGAAGAT•ATCCATGGACTCCCTTGCCAATTCAT•AAAAGA L V It C G T I G L S V G R H L L It G N H F T V A 3 V R 11 3 S H D It L A N $ $ K E 3001 ACTCAAATCAAATGGAGTCTTGGATCATGTAATGTTTAATTTTCATAAAAGACTTAGATCCGTGGAACCTGATCTGACTGTAGAGGAAATCAAAGTGGATTCTTCAAGAGCGGGGAAGTT L K 3 N G V L D H V 14 F N F H K R L tq 5 V E P D L T V It E I K V D S 8 R A G K L 3121 GCTCAAAAGTTTG ATGGA'[ GGAATGACAGGAAT AGTTTCCCACAACTCAACTCATG AGGGGTGGAGAATGATCAAAG GC ATAA ATAGCACCTCCGAGATGAGATCATTCATGACT ATGAT L 11 S L M D G ~4 T G I V $ H N $ T H E G W R H I ~ O ! N S T 5 E M R S F ~( $ R I 3241 CAAAGGT A&AAGTG &&GAG ACC AGGGGTGACTT ATTTGACAGAGTTCAGG AAC~GAACTTCAT G AAGGTT A AGATCTACGGCATATTTGGGTTTGCTGG ATCAGGGAAGA GTCATGCAAT K G K $ E It T R G D L F D R V Q E L N F N K V K [ Y G I F G F A G S G K $ H A l 3361 TCAAAATCTCATTCAGAcAGAATTCAAAGGATCACAAGGTGTTATGGTGATCTGTCCAAG^AGGTTTTTGGCCAAGGACTGGAGTGAAAAAGGGGTGGATGAGAAGGATATTAAAACCTT Q N L f Q T E F K G 8 Q G V /4 V I C F 11 R F L A K D ~¢ S It K G Y D It K D l K T F 3481 T G AGAGCGCACTGA AGTCAGATGTTAAAGGAA AGAG ATTATTCATACI"I'GATGA A ATCAG TTq'GTTQCCTAAAGGTTTCACTGACCTCTTGATGCTC AAAATGCAT ATGGAAGGGAT TCT It S A L K S D V K G It 11 L F I L D E ! S L L P K G F T D L L H L K M H H E G I L 3601 GAAAA~GT~AAC~ATTGTGTGTAT~rGGTGACCCTCTGCAAGCTGGGTA~TTCTGT~TAAGGATGACAATTATTTGTCTAGGGAGGGAGAKAT~A~GAGGCTTTTCAAAGATG~GGTCAA g K 3 T l V C I G D P L ~ A G Y F C P K D D N Y L S R E G E I K R L F K D G V N 3721 TTACAAATGGTACAGCTACAGGATTAATAAGTTTATTGCAAAGAAGCTGGCCATAGAGACAATGAATGACTTTATTGGCATTGATGAGCAGAATTTAATATACAAAGATATGCCATC~GC Y K ~ y S Y R I N K F l A K g L A I E T 14 N D F I G I D E Q N L I Y K D H P S A 3841 TCACCATTTCATGGAGAACAAAGGGAATCACATTGAAGTGATTCTCGTTCCCAGCATGAT AGAGAAAGAGTTATACTCGAATTACGGCAATGTGATGACTTTTGGCGAGTGACAGGGTCT H H F H It N K G N H 1 K V [ L V p 3 H [ E K 11 L Y 6 N Y G N V H T F G E S Q G L 3961 TACTTTCAACTGTGGGGTCATAGTGCTGTCCGAAGAGGCCAAGCTATGCTCCGATGCTCATATAATGGTGGCGATCACAAGATTTAGGCGAGGTTTCTGCTTTGCACTTGGCAGTAAAGG T F N C G V [ V L S E It A It L C $ D A H 1 ~ V A I T 6 F 11 11 G F C F A L G S K G 4081 GTCAAAGGAGGATTACAT•AGATCCATGAAAAGTGGATTGCTTCAAAGGATGTGCTCCGGGATTGGTGCTTCTAAGGAATTTATTCTAGGATCCTCTTCAGTCA &ATTAATCCTGAGTGA 3 K E D ¥ ~4 11 $ M K S G L L Q [I ~4 C S G [ G A S g E F [ L G 6 S $ V K L ~ L $ It 4201 AAAGGACGTCTCAAAGGGTGCTGGAGTTG ATGA A ATGGACAGAGAAGCCA~GTTGGAAGGAGATGTTTGGCTC AAGAG CATG AT AT ATCTTGGAAAAAGGTACCATATAGTTGAACCGCT K D v 3 K G A G V D K H D B It A E 1, It G D V W L K 5 H I Y L G II It Y H l v E P L 4321 AGGT~AAGT~ATCAAGCTCACTGAGAATGCGATCAAGTGCGACATT~CTGTTT~TTCAAGCCA~ACTCTAGGTCCTGAGTTGGA~AA~ATTCAAG~AAGAGAGTTCAGGG AATTCAAGGG G Q V I K L T E N A I g C H f P v C 3 S ~ T L G P ~ L D N 1 Q A ~ E F R It F g G 4441 CAAGAACGGGTGGTCCAATcAATTCAGGGAAGAGGCTGGTCCAAATTGGAAGCACCCTTAcAGAGTGAATCAGGCAATGAGTTATGAGGCTATATATCCAAGGcACAGGATGGATGATGA K N G W 3 m ~ F R E E A G P N W K H P Y R v N q A 14 S Y E k 1 Y P R H R H D D D 4561 TCT AACCTTTCTTG CTGCCATCAAGA AGAG GCTTCGTTT CG ~TAACGTTGCT AAT AATTATGCAAAGTTTA AAGCAG CAG AG AGTAGAG~CA kGT p, CTT AGCAA ~AGTTTTCTTGCG ACA L T F L A A I It K R L 11 F D N V A N N y A K F K A A E 3 8 G K Y L A K V F L
AA~GUGATATTC~A~TT~CTGGGGTGAAAAGCTCC~AATTTATT~TGATGTGAGTC~TTT~AGGAAGGC~T~TGA~CTTATGATTCATTGG~ATGAATT~GTATTCAAGGTTATGCCAG R G Y -~ N C W G E K L q N L F Q D I P I A G V K S S R I Y 8 D V S P F R K A S l) t M I H W N E F V F K V M P E 6881 AGGATATT GCAGGT GATGGTTTT AOATTGGCATCCATACCTGTTATACCTTCCTC AG AGGTTCAAGCGGTGCTG AGAAAGAGAGAGAGTACCAACTACGT~CATTGGGGAGCACTATCAA D I A G D G F E L A 6 I p V I P S S E V Q A V L E X R E $ T N Y V H ~ G A L S 1 6001 T ATCCATTGACGC ACTGTTCAGAAAGAATGCCGGGGTT TCAGGGTGGTGTT ATGTGTACGACAACA GAT GGG AAACTTTT GAGCAGGCA3ATGCT ACAAAAGTTTCGGTTCAACCTGGAT A s I D A L F R K N A G V S G ~ C ¥ V ¥ D N R W E T F E Q A H L Q K F R F N L D S 6121 GTGGCTCG GCCACATTGGTGACCTCACCC-A ATTTTCCAGTTTCATTGGAT GAT CCTGGT'Fr GTCCAATTCGATCAGTGTAGCGGTGATGTTTG AGA ATCTGAACTTCAAGCTTGAAAGCT G S A T L V T S P N F P V S L D D P G L S N S I $ V A V M F E N L N F K L E S Y 8241 A~CCTATAAGTGTTAGGGTAGG~AACATG~GTAGATTCTTCGACAGCTTCTTGAGTTGCGTCAAGAATAAAGTGGACT~CAA~TTTTTGC7TGAGGCCGCTAATGCAGATC~GCTTGGCG P I S V R V G N N C R F F D S F L S C V Z N K V D S N F t L E A A N A D F L G A 6361 CGGGTG~TTTCGGGTTTG~ACAGGAC~ATCAGGTCTCTGAATTGTTCAATTATATT~A{]ACTGTCC(;AA~CCAGGCA~TTAAATCCAGGGAG~ATGAAGTA~CGAGGGGTTTGTT~GGTA G A F G F E Q D D Q V S E L F N Y I q T V P T Q A 1 K 8 R E H E V P R G L F G M 6461 TG ATGGGA A AAAAGA AGGT CAAATCTTTTGAGTTTGCCTCTGGTTCAAG AAATCTGGGA AGA AGAAAACCGCAACG~GAAGACCTT TGGAAAGATCT GeT AGCCTGAG AGTTGCTCCTG G R K K V K S F E F A S G 6 R N L G R R K P Q R G R P L E ~ S A S L R V A P G 6601 GATTTAGGTCCCAG~ATGAGAGAGTTGEA~ATCAAGGATTA~CGACTGACTCTGATTTTGAAAATTTTCTGAGG~AAGG~AA~GGAAAAG~TGGAACTGAATC~ATTACTT~AG~AGG~ F R 6 Q D E B V E 14 Q G L S T D S D F ~ N F L R K G K G K A G T E ~ I T 6 E G S 8721CATCCTTCGATAACATTTCTGCA~AGA~TTTCAGTTTGCTAGACAAGAT~AGAAGGCGA~GATGGCG~AGTGCTGAACCT~AATTAA~AGTGG&CGCAGATCTGAAAGCGTTCCTG SFDNISAREFQFARQDQKAEDGGSAEPPIKSGRRSESVFG O~NAAVLNLQLKVDADLKAFL 6841 G~CGCAGAAGG~AGA~CCCTTCATGGAA~GACAG~GGCAATTCTGGAACAGACACT~AGGCCATCTTCG~G~ACATAGCGATACA~GGACCTCGGAACAAACAGAGTTCCTGGACGTG RRRQTPSWKDRGNSGTDTGGHLREHSDTGDLGTNRVPGRA AAEGRPLHGKTGAILEQTLEAIFANIAiQGTSEQTEFLDV 6961CT~GTGGAGGTGAAATCCATGGAGGATCAGAAGGTGGTGGGGTCATTCAATCTGAAGGAGGTAGT~GGTTTGATCAAAATATTCAGGACTA~ATCTT~GGACCCGAATATAAGCAGCATG GGG81HGGSEGGGVIQSEGGSRFDQNIQDYIFGPEyKQHD LVEVKSHEDQKVVGSFNLKEVVGLIKIFRTTS$DPN~SSM 7081A~CTT~CGCCAAGTGTGTGAGGCATTTGC~CGGAGGCAAGAAAT~GGCTCGTAAAGCTAAAGTACAAAGGGGTCTTCA~AA~CTCTTTTCTACTAC~CTGAAGTGGGGGGGAAGTAT LPPSV TFRQVCEAFAPEARNGLVKLKYKGVFTNLFSTTPEVGGKY 7201C~GGAGCTCATGTTTGACTT~AATA~GGCCTAAATATGTT~ATAATGAA~AAAGCCcAGCAGAAAGTGATAACTAATATGAACC~CGT~TTTTACAGA~TGAATTTGCAAAGAG~GAA P E L M F D F N K G L N M F ] N N K A Q q K V I T N M N R R L L Q T E F A K $ E 7321 AAT~AGGC~AAAATaTCGTCTGTTACAACTGATCTTTGCGTTTACTCTCATAAGGAA~TTT~G~TTAATAAATAAAG~AATAAATAGTGTGCATTTGGTTTAATGTCAATTGTGTGTAT N E A g M S S V T T D L C V 7441 •TAATTAACGTCAAAC7CTTGAACCCATGAGAGAGTATAAACAGTCA•GGTATCTAATTGGAGTGTTTAGACTTATAA••AATAGCCTGTAGACTTTTAAATATTTTACTAC pOl~(A)
Fig. 1. The complete nucleotide sequence of the ACLSV-A genome and the amino acid sequences of three ORFs. The amino acid sequences of the conserved motifs associated with the helicase and the RNA polymerase are underlined. 
cluding the 3' poly(A) tail (Fig. 1), three nt fewer than that of ACLSV-P reported by German et al. (1990) (Table 1). Analysis of the putative open reading frames (ORFs) of the nucleotide sequence in both positive and negative strands showed that the genome of ACLSV-A contains three ORFs in the positive strand (Fig. 1). 

Table 1. Comparisons between A C L S V -A and A C L S V -P genomes

ACLSV-A ACLSV-P Identity , respectively (Fig. 1). The overall similarity of the O R F 1 protein between ACLSV-A and ACLSV-P is 88"4% (Table 1). However, the regions surrounding the above two motifs were highly conserved between both isolates and their identities were 97.6% (positions 1058 to 1305 for ACLSV-A and 1057 to 1304 for ACLSV-P) and 97"7% (positions 1637 to 1767 for ACLSV-A and 1636 to 1766 for ACLSV-P), respectively. O R F 2 begins at A U G (positions 5727 to 5729) and terminates at U G A (positions 7098 to 7100) to yield a protein with an M r of 50453 (50.4K) (Fig. 1). The position of the initiation codon of the 50.4K protein is six nt downstream from the corresponding position in ACLSV-P (Fig. 2). This location of the initiation codon was the same in the three different c D N A clones sequenced. The 50.4K protein of ACLSV-A, as did that of ACLSV-P, showed an amino acid similarity of 22.4 % with the gene I protein of cauliflower mosaic virus (Franck et al., 1980), and the identity of the O R F 2 protein between ACLSV-A and ACLSV-P was 79"9 % (Table 1). O R F 3 contains an A U G (positions 6784 to 6786) and ends at U G A (positions 7363 to 7365) encoding a protein with an M r of 21394 (21"4K) which is dissimilar to the 28.3K protein encoded by O R F 3 of ACLSV-P. This discrepancy is explained as follows. The initiation codon (AUG, positions 6616 to 6618) is located in the ACLSV-A genome at the almost same position as the A U G codon of the 28.3K protein of ACLSV-P, but the termination codon (UGA, positions 6670 to 6672) exists between the first A U G and the second A U G (positions 6784 to 6786) which is the initiation codon of the 21.4K protein (Fig. 3). The identity of O R F 3 proteins between both isolates was 88-6 % (Table 1). 
In in vitro translation experiments reported previously (Yoshikawa & Takahashi, 1989), ACLSV-A RNA directed the synthesis of 105K and 51K proteins as major products and several minor proteins of which the 23K protein was immunoprecipitated with ACLSV-A antiserum. The products (51K and 23K proteins) detected in translation experiments may be synthesized from fragmented RNAs of ACLSV-A RNA because the proteins encoded by ORFs 2 and 3 are thought to be translated from subgenomic RNAs (German et al., 1992). The M r (21.4K) of the protein encoded by ORF 3 of ACLSV-A agrees with that of the ACLSV-A coat protein M r estimated by PAGE (Yoshikawa & Takahashi, 1988). To examine whether the 21-4K protein is indeed the coat protein, the ORF 3 region was amplified by PCR using pBCLS38 containing the 3"8 kb insert of the T-terminal region, a synthetic oligonucleotide (5' AAGAATTCAGGATGGCGGCAGTGCTG 3' corresponding to nt positions 6781 to 6798 and containing an EcoRI site) and a universal primer (5' AACAG-CTATGACCATG 3') located in the multicloning site of the BS plasmid. The PCR product was digested with EcoRI and KpnI and ligated to a pTrc99A expression vector restricted with the same enzymes (designated pTrc99A-CP) and the product was used to transform E. coli JM105. Proteins were then prepared from the cultures grown in the presence of IPTG and analysed by immunoblotting using ACLSV-A antiserum as described previously (Yoshikawa et al., 1992). Fig. 4 shows the result of immunoblot analysis, in which the protein expressed in E. coli containing pTrc99A-CP reacted with the antiserum against purified ACLSV-A. This protein is slightly larger than coat protein from purified virus and infected C. quinoa leaves because the expressed protein contains four additional amino acids derived from the vector plasmid and ACLSV-A cDNA. These results demonstrate that the 21-4K protein encoded by ORF 3 is the coat protein of ACLSV-A, supporting the results obtained before following the determination of the partial amino acid sequence of the ACLSV-P coat protein (German et al., 1990). 
At present, ACLSV is classified into the closterovirus group, based mainly on its particle structure (Francki et al., 1991). This group is composed of heterogeneous virus species with different particle lengths, transmission modes and tissue localization of the virus particles in infected material. The genome organization of ACLSV is quite different from that of the 3"-terminal genomic region of beet yellows virus (Agranovsky et al., 1991), the type member of the closterovirus group, indicating that 

Fig. 3 .

 3 Fig. 2. The nucleotide sequences surrounding the ORF 2 initiation codons of ACLSV-A and ACLSV-P. Identical nucleotides are shown by asterisks. Second ATG codons within ORF 2 are underlined. 


Fig. 4 .

 4 Fig. 4. Immunoblot analysis of the proteins expressed in E. coli containing pTrc99A-CP. Lane 1, proteins from E. coli cells containing pTrc99A grown in the presence of IPTG; lane 2, proteins from E. coli containing pTrc99A-CP without IPTG; lane 3, proteins from E. coli containing pTrc99A-CP induced by IPTG; lane 4, coat protein from purified ACLSV-A; lane 5, proteins from C. quinoa leaves infected with ACLSV-A; lane 6, proteins from healthy C. quinoa leaves. An arrowhead indicates the position of the coat protein of ACLSV-A. 


ACLSV should be classified into a new virus group. Further investigations on the genome organization of closteroviruses will provide valuable information on the classification of this group. 
This work was supported in part by a Grant-in-Aid from the Ministry of Education, Science and Culture, Japan.
K Sato, N Yoshikawa, T Takahashi
Complete nucleotide sequence of the genome of an apple isolate of apple chlorotic leaf spot virus