nucleotide sequence of the genomic rna of pepper mottle virus

8/3/2019 Nucleotide Sequence of the Genomic RNA of Pepper MOttle Virus

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Journa l o f Genera l V irology (I991), 72 , 2875-2884. Prin ted in Grea t Bri ta in 2 8 7 5

N u c l e o t i d e s e q u e n c e o f t h e g e n o m i c R N A o f p e p p er m i l d m o t t le v i ru s , ar e s i s t a n c e - b r e a k i n g t o b a m o v i r u s i n p e p p e rE . A l o n s o , I . G a r c i a - L u q u e , A . d e l a C r u z , B . W i c k e , M . J . A v i l a - R i n c b n , M . T . S e r r a ,C . C a s t r e s a n a a n d J . R . D i a z - R u i z *U . E . I . F i t o p a t o l o g i a , C e n t r o d e I n v e s t i g a c i o n e s B io l 6 g i c a s , C S I C , V e l h z q u e z , 1 4 4 , 2 8 0 0 6 - M a d r i d , S p a i n

T h e e n t ir e g e n o m i c R N A o f a S p a n i sh i s o la t e o f p e p p e rm i l d m o t t l e v i r u s ( P M M V - S ) , a r e s i s t a n c e - b r e a k i n gv i ru s i n p e p p e r , w a s c lo n e d a n d s e q u e n c e d a n d s h o w nt o b e s i m i l a r t o o t h e r t o b am o v i r u s e s i n i t s g en o m i co r g an i za t i o n . I t co n s i s t ed o f 6 3 5 7 n u c l eo t i d e s ( n t ) an dc o n t a i n e d f o u r o p e n r e ad i n g f r a m e s ( O R F s ) w h i c he n c o d e a 1 2 6 K p r o t e in a n d a r e a d t h r o u g h 1 8 3 K p r o te i n( n t 7 0 t o 4 9 0 8 ) , a 2 8 K p r o t e i n ( n t 4 9 0 9 t o 5 6 8 2 ) an d a

1 7 .5 K co a t p r o t e i n ( n t 5 6 8 5 t o 6 1 5 8 ) . T h i s i s t h e f i r s tt o b a m o v i r u s i n w h i c h n o n e o f th e O R F s o v e r la p . B o t hi t s n u c l e i c ac i d an d p r ed i c t ed p r o t e i n s eq u en ces w er ec o m p a r e d w i t h t h e p re v i o u s l y d e t e r m i n e d s e q u e n c e s o fo t h e r t o b am o v i r u s e s . T h e v a r i a t i o n s an d s i m i l a r i t i e sf o u n d an d t h e i r r e l a t i o n s h i p w i t h t h e p a t h o g en i c i t y o ft h i s v i r u s a r e d i s cu s s ed .

I n t r o d u c t i o nP ep p e r m i l d m o t t l e v ir u s ( P M M V ) i s a m em b er o f t h et o b am o v i r u s g ro u p o f p os i t iv e - s t ran d R N A v i ru s e s. T h eco m p l e t e n u c l eo t i d e s eq u en ce o f t h ree o t h e r t o b a m o v i r -u s e s , t o b acco m o s a i c v i r u s ( T M V ) ( G o e l e t e t a l . , 1982),t o m a t o m o s a i c v i r u s ( T o M V ) ( O h n o e t a l . , 1984) andt o b acco m i l d g r een m o t t le v i r u s ( T M G M V ) ( S ol is &G ar c i a - A r en a l , 1 9 9 0 ) h av e a l r ead y b een r ep o r t ed . T h et o b am o v i r a l R N A en co d es f o u r d i f f e r en t p r o t e i n s :126K, 183K, 30K and 17 .5K, in tha t o rder. The 126Kand 183K pro te ins ar e invo lved in the r ep l ica t ionp r o ces s e s ( Y o u n g e t a l . , 1987; Quad t & Jaspar s , 1989) ,the 30K pro te in par t ic ipa tes in the ce l l - to -ce l l sp read o ft h e v i r u s (D eo m e t a l . , 1987; Meshi e t a l . , 1987) and the17-5K pro te in i s the coat p ro te in . The 126K and 183Kp r o t e i n s a r e d i r ec t l y t r an s l a t ed f r o m t h e v i r a l R N A ,w h er eas t h e 3 0 K p r o t e i n an d co a t p r o te i n a r e t r an s l a tedf r o m s u b g en o m i c R N A s ( P a l u k a i t i s & Z a i t l i n , 1 9 8 6 ) .

P M M V i s o n e o f t h e m o s t d e s t r u c ti v e p a t h o g en s o fp r o t ec t ed p ep p e r c r o p s . I t i s f o u n d i n f ec t i n g p ep p e rcu l t ivar s w i th genet ica l ly incorpora ted r es i s tance toT M V an d T o M V . T h e i n f ec t i o n b y t h is v i ru s p r o d u cesimpor tan t economic losses a l l over the wor ld in cropsgrown under p las t ic o r g lass (Wet ter & Cont i , 1988) .A d d i t i o n a l l y , P M M V i s u n ab l e t o i n f ec t t o m a t o p l an t san d p o s s e s s e s a r ed u ced cap ab i l i t y t o r ep l i ca t e an d / o raccu m u l a t e i n t o b acco p l an t s w h en co m p ar ed t o T M V

Th e n u c l eo t i d e seq u en ce d a t a rep o r t ed i n t h i s p ap e r w i l l ap p ea r i nt h e E M B L , G e n B a n k a n d D D B J n u c l e o ti d e s e q u e n c es d a ta b a s e s u n d e rt h e access i o n n u mb er M8 1 4 1 3 .

a n d T o M V ( W e t t e r e t a l . , 1984; Garc ia-Luque e t a l . ,1990).T o d ev e l o p an u n d e r s t an d i n g o f t h e m ech an i s m ( s )invo lved in these b io log ica l p roper t ies , we have deter -m i n e d t h e n u c le o t id e s e q u en c e o f P M M V - S R N A , aS p an i s h i s o la t e o f P M M V ( A l o n s o e t a l . , 1 9 8 9 ) . T h enucleo t ide sequences o f i t s 5 ' and 3 ' non-cod ing r eg ionsh av e p r ev i o u s l y b een r ep o r t ed ( A v i l a - R i n c6 n e t a l . ,1989). In th i s paper , w e p resen t the c lon ing and com ple ten u c l eo t id e s eq u en ce o f P M M V - S an d t h e an a l y s is o f it sd ed u ced am i n o ac i d s eq u en ces .

M e t h o d sVirus propagation, purification an d R N A extr action . T h e o r i g i n o f

P M M V - S h a s b e e n r e p o r t e d p r ev i o u sl y ( A l o n so e t a l . , 1989). The v i rusw as p u r i f i ed f ro m Nicotiana clevelandii G ray p l an t s a s d e sc r i b ed( G a r c i a - L u q u e e t a l . , 1 99 0) . V i r i o n R N A w a s p r e p a r e d b y c o n v e n t i o n a lSD S-p h en o l ex t rac t i o n a f t e r h ea t i n g o f t h e p a r t i c l e s i n 2 0 raM-so d i u mp h o sp h a t e b u f fe r p H 7 .0 , 0 .5,% SD S fo r 2 0 s a t 1 0 0 C .

eDNA syn thesis and c lon ing , c D N A w a s p r e p a r e d a s d e s c r i b e d b yG u b l e r & H o ffm an (19 8 3) , u s i n g a co mm erc i a l cD N A sy n t h es i s k i t( B o e h r i n g e r - M a n n h e i m ) . P M M V - S R N A w a s 3 ' p o l y a d e n y la t e d invitro w i t h Escherichia coti p o l y (A ) p o l y merase (0 -2 5 u ni t/ ~t g o f R N A ) fo r7 m i n a t 3 7 C , u n d e r t h e c o n d i t io n s r e c o m m e n d e d b y t h e m a n u f a c -t u r e r ( P h a r m a c i a L K B B i o te c h n o lo g y ) , a n d f i r s t- s t r an d c D N Asy n t h es i s w as p r i med w i t h ol i g o (d T) . D o u b l e -s t ra n d ed cD N A w as s i ze -f rac t i o n a t ed i n 0 .8 % ag a ro se g e l s , an d t h e cD N A w as e l u t ed an dl i g a t ed i n t o p l a smi d p U C1 8 d i g es t ed w i t h H i n c I I . I n a n o t h e re x p e r i m e n t , E c o R I l i n k e rs w e r e a d d e d t o d s c D N A t h a t h a d p r e v i o u s lyb een t rea t ed w i t h E c o R I m e t h y l a s e a n d E c o R I - d i g e s t e d p r i o r t o b e i n gs i ze - f rac t i o n a t ed i n ag a ro se ge ls . A f t e r e l u t i o n , t h e cD N A w as c l o n edi n t o E c o R I - d i g e s t e d p U C1 8 . P l a smi d s w e re t e s t ed fo r t h e p re sen ce o fv i r a l c D N A i n s e r ts b y c o l o ny h y b r i d i z a t io n , u s i n g r a n d o m l y p r i m e d

0001-0404 1991 SG M


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riP-labelled cDNA to PMMV-S RNA. Other cDNA clones wereprepared using a primer complementary to nucleotides (nt) 5211 to5228, in which a B a m H I restriction site was created by addition of anextra G at its 5' end. Ds cDNA was restricted with B a m H I and B c i l ,size-fractionated in agarose gels, and the 1200 nt f ragment was elutedand ligated to pU C 18 digested with B a m H I . Anoth er set of clones wasobtained after priming cDNA synthesis with an oligonucleotidecomplementa ry to nt 4021 to 4036. Ds cDN A was restricted with S a i l ,fractionated in a 6% polyacrylamide gel, and the 600 nt frag ment waseluted and cloned into the S a l I - S m a l sites of pUC 18. All recomb inantDNA techniques were as described by Maniatis e t a l . (1982), using E.coli strains JM83 and DH5~.

Nucleot ide sequence de terminat ion and analysis . The nucleotidesequence ofc DNA clones was determined by the chemical degradationprocedure (Maxam & Gilbert, 1980). Subclones used for sequencingwere generated by deletions with nuclease Bal 31 or r estriction enzymedigestion. Sequences were analysed using the DNASTAR computerprograms.

Results and Discuss ionSequen ce de terminat ion an d termina l non-coding regionsFig. 1 shows the strategy used to determine the sequenceof the genomic RNA of PMMV-S from a set ofoverlapping cDNA clones. They contain sequencesrepresenting all but the first 34 nucleotides located at the5' end of PMMV-S RNA. Most of the sequence wasobtained from at least two independent cDNA clones.

The nucleotides of the 5' and 3' non-coding regions ofPMMV-S RNA have previously been sequenced directlyon the viral RNA (Avila-Rinc6n e t a l . , 1989). As withother tobamoviruses, PMMV-S possesses a 69 nt leadersequence, devoid of G residues, termed the ~) fragment(Richards e t a l . , 1977; Avila-Rinc6n e t a l . , 1989). Its 3'non-coding region is 199 nt long. It was previouslyproposed that some structural features in the tRNA-likeconformation of PMMV-S RNA such as two unpairednucleotides connecting the aminoacyl and anticodonarms could be related to its lower replicability observedin tobacco plants (Avila-Rinc6n e t a l . , 1989) as describedfor certain chimeric tobamoviruses (Ishikawa e t a l . ,1988).

The determined nucleotide sequence of the cDNAclones coincides with that of the RNA except for a t;inglebase transition at position 6181 which would change theC/G pair (6197/6181) situated at the beginning of the Vstem in the proposed secondary structure (Avila-Rinc6net al . , 1989) to a C/A pair. This nucleotide substilutionwas present in three of four sequenced cDNA clones. Inother parts of the genome no sequence heterogeneity wasfound in the clones analysed. The only nucleotidedifference was found in clone 4, in which the insertion ofa T between nt 5385 and 5386 could lead to a truncatedprotein.

N u c l e o ti d e se q u en c e o f P M M V - S a n d c om p ar is on w i thother tobamovirusesFig. 2 shows the sequence of PMMV-S RN A and tha t ofits deduced amino acid. The genome of PMMV-S is 6357nt long. It shares an overall sequence identity of 69.4%with the RNA of ToMV (Ohno e t a l . , 1984), 68.5% withthat of TMV (Goelet e t a l . , 1982) and 64% wi thTMG MV (Solis & Garcia-Arenal, 1990), the othermembers of the tobamovirus group whose entire RNAsequences are already known. As shown in Table 1, also,PMMV-S shares a higher degree of amino acid sequenceidentity with ToMV than with TMV and TMGMV.

Organ i za t i on o f t he 126K/183K ge ne se que nc eThe first open reading frame (ORF) of PMMV-S RN Abegins at nt 70, in the first AU G encountered from the 5'end, and extends to nt 3423, encoding a protein of 1117amino acids (126K), with a calculated Mr of 126 304. Thereadthrough of the amber codon (UAG), possibly byinsertion o f tyrosine (Beier e t a l . , 1984), results in a 183Kprotein which termina tes at position 4908. It is composedof 1612 amino acids with a predicted Mr of 183340. Thenucleotide and amino acid sequences in the readthroughpart of the 183K protein (nt 3421 to 4908, amino acids1118 to 1612) (Fig. 2 and 3) are the most highly-conserved in all the genome, with only 14 and 15 non-conservative amino acid substitutions with respect to thecorresponding proteins of ToMV and TMV,respectively.

The 126K and 183K proteins are thought to beinvolved in viral replication because they have beendetected in partially purified preparations of the viralpolymerase complex and because they contain severalsequence motifs which are conserved in proteins knownto act in replicative processes of plant and animal viruses(Young e t a l . , 1987; Goldbach & Wellink, 1988; Strauss& Strauss, 1988; Quadt & Jaspars, 1989). The al ignmentof the 126K/183K proteins of PMMV-S with those fromthe more closely related tobamoviruses (ToMV andTMV) shows that the sequence is well conserved along allthe protein (Fig. 3), except for three stretches (aminoacids 155 to 191,623 to 669 and 768 to 791) in which non-conservative substitutions as well as deletions andinsertions occur. Other regions of weaker amino acidsequence ident ity correspond to positions 382 to 388,537to 555 and 991 to 1001 (Fig. 3).Based upon the existence of conserved motifs betweenthe tobamoviral 126K protein and those from otherRNA viruses, two functional domains have beendefined. The first one, in the amino part of the protein,has homology with the nsP1 pro tein of alphaviruses andwith the amino part of other proteins implicated in the


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1 2 6 K I 1 8 3 K 3 o K IV - w e - 1

5 rE H AI I I I

1000~ D j

NsBABSa K, , f ' f f (2 0 ' 0 0

EC-8d t

AE A BS BcNsBc B KI t I I I I ' l l I I l I

I I

3000 4000

75

H-92

U-11;17

PH . B s N Sc, 1 I f f , r 3 ,

5000 600085

174

BKB-8;3

Fig. 1. Genomic organization, partial restriction map and sequencing strategy for PMMV-S cDNA. Open boxes drawn approximatelyto scale represent the coding regions for the 126K, 183K, 30K and coat protein (CP) gene products. Arrows represent the strategyfollowed to determine the sequence of the overlapping cDNA clones used (EC-8, 75, H-92, U-I 1, U-17, BKB-8, BKB-3, 85, 4 and 174).Abbreviations for restriction sites are : A, A v a I ; B , B g l l I ; Bc,B c l I ; E , E c o R V ; H , H i n d l I I ; K , K p n I ; N , N s i I ; S , S a l I ; Sa, S a c I ; Sc, S a c l I .

Table 1. P e r c e n t a g e s o f s e q u e n c e i d e n t i t y b e t w e e n t h e P M M V - S g e n e s a n d t ho s e f r o mo t h e r t o b a m o v i r u s e s

Gene126K 183Kt 30K CP

Virus* N:~ A~ N A N A N AToMV 68.8 74.7 73-7 82.0 65.8 64-5 67.5 73.9TMV 67.9 73.3 73.0 80.0 63.4 67.4 65.6 72.0TMGMV a 62.4 62.0 68.4 73-2 63-1 61.5 65.6 70.1TMGMVb - - - - 64.7 65.0 65.6 70.1CGMMV-W . . . . 46.0 33-5 48.1 36.5S HM V . . . . 38.5 26-7 46-4 40.8

* Data are from Ohno e t a l . (1984), Goelet e t a t . (1982), Meshi e t a t . (1981, 1982), Saito e t a L (1988) andMeshie t a l . (1983), for ToMV, TMV, SHMV (30K and coat protein), CGMMV 30K and CGMMV-W CP,respectively. Data reported for two isolates of TMGMV (TMGMVa and TMGMVb) are from Solis &Garcia-Arenal (1990) and Nejidat e t a l . (1991), respectively.t The sequences analysed correspond only to the readthrough part of the 183K protein.:~ N and A, nucleotide and amino acid sequence homologies, respectively. No sequence data are available for comparison.

repl icat ion of RNA viruses with a monopar t i te ordivided genome (Ahlquis t e t a l . , 1985). In this region,Rozanov e t a l . (1990) have identified two conservedsequence motifs defined by the presence of an invariantHis in the firs t motif and the sequence AspoX-X-Arg inthe second one, that are located at amino acid positions76 to 81 and 134 to 138 in the 126K/183K protein ofTMV (Fig. 3), respectively. By analogy with the nsP1protein of Sindbis virus (Mi e t a l . , 1989), this domain

may be responsible for the methyltransferase activitynecessary for the cap formation of the genomic andsubgenomic RNAs. Of the two amino acid sequencemotifs described by Rozanov e t a l . (1990), the predicted126K/183K protein from PMMV-S possesses both,except for a conservative substitution lie to Val atposition 135 (Fig. 3). This domain is the best conservedwith respect to TMGMV.

The second funct ional domai n in the 126K/183K


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2878 E , Alonso and others

~ 12~ 1 8 3K 1 GT~.I.~.I.I.I C ~ C ~ C ~ C ~ C A C A A A C ~ C ~ C ~ C A ~ A C CAAAATAC~ TAC~T C~ACA CAC~C ~GCTA CC~C GCC CA~AGC~GTACTCTCC~M A Y T Q Q A T N A A L A S T L R

121 G G ~ T ~ C C C C ~ G G T G ~ C ~ T C ~ G C T ~ T C G G A ~ C T G T A C G ~ T C AG C G G T C G ~ c ~ T G C~ T G c A C A T G A C CG C A G G C C C ~ G G ~ I' I ' I ' I ~ G G T C ~T ~ G C ~ GG N N P L V N D L A N R R L Y E S A V E Q C N A H D R R P K V N F L R S I S E E241 CA~CGC~ATCGC~CT~GGCCTACCCTGAG~CC~TCACG~CTAC~CACGCAG~CGCTGTGCACAGTCTCGCAGGTGGAC~CGGTC~GG~CTAG~TAC~GATGATGQ T L A T K A Y P E F Q T F Y N T Q N A V H S L A G G L R S L E L E Y L M M361 CAGATCCCCTACGG~C~CGACATATGATATCGGGGG~ TTTGCTGCTC ACATG~GGTCG TGACTACG~CA ~GCTGCATGCC T~CATG~C~AC GT~CGT~TGCG TQ I P Y G S T T Y D I G G F A A H M F K G B D Y V H C C M P N M D L R D V M R481 CAC~TGCTCAAAAGGATAGCA~G~CTGTACC~C~GC~GCGCAAAAGAAAAAGGT~TACCGCCATATCAAAAGCCATGC~GAT~TACACGGAC~TCCGC~T~AH N A Q K D S I E L Y L S K L A Q K K K V I P P Y Q K P C F D K Y T D D P Q S V

601 GTGTGCTCG~CC.~-~-~.CAGCACTGCG~GG~G~CGcACTG~ACGGAT~GTATACGCTGT~GC~GCACAG~ATAC~CA~CcAGCAGATG~GGGG~G~C~TGV C S K P F Q H C E G V S H C T D K V Y A V A H S L Y D I P A D F G A A L L721 AGGAG~TG~CATGTCTGCTATGCTGCC~CCACTTTTCTGAG~TC~CTTTTAG~GA~CGTATGTCAGTC~GACGACATAGGCGC~C~CTCGA~GAGGGCGATATG~GR R N V H V C Y A A F H F S E N L L L E D S Y V S L D D I G A F F R E G D M L8 4 1 ~ C . F ~ - ~ C T T T T G T A G C A G A G A G T A C ~ A T A C T C A ~ C C T A T A G T ~ T G T G C ~ G T A T G T G T G T ~ G A C ~ A C ~ C C C C G C ~ C T A G T A G A G ~ G T G T A C A T G ~ G G A G ~N F S V A E S T L N Y T H S y S N V L K Y V K T Y F P A S S R v Y M K E F961 ~GGT~CTAGGGT~TAC~GGTTTTGT~G~TC~GG~AGATACC~TGTACTATATAGAGGTGTATACCACAGAGGTGTAGAC~GGAGCAA~ACAGTGC~TGG~ATL V T R V N T W F C K F S R L D T F V L Y R G V Y H R G V D K E Q F Y S A M E D

1081 G C ~ G G C A ~ A C A A A A A G A C ~ G G C ~ T G A T G ~ T A G C G ~ G ~ T C C T C ~ A G A G G A ~ C A T c G T C T G ~ A ~ G G ~ C C ~ G ~ T A T G G T ~ T A G T A C C ~ G ~ CA W H Y K K T L A M M N S E R I L L E D S S S V N Y W F P K M K D M V I V P L F1 2 01 G A C G T A T C ~ A C A G ~ C G A G G G G A A A A G G ~ A GC ~ G ~ G G A G G T C A T G G T C A G C ~ G G A C ~ C G ~ AT A C T G T G C ~ T C A T A ~ C G C A C A T A c C A G T C G ~G C G c ~ A C ~ A C

D V S L Q N E G K R L A R K E V M V S K D F V Y T V L N H I R T Y Q S K A L T YGC~TGTA~ATCG~CG~GAGTC~AT~GATC~GAGTGAT~TC~TGGGGTGACTGCGcGCTCAGAGTGGGATGTGGAT~GGC~G~GCAGTCCCTGTC~TGAc~zT~.~C1321 A N V L S F V E S I R S R V I I N G V T A R S E W D V D K A L Q S L S M T F F1441 ~ G C A G A C c ~ G G C C A T G C T C ~ G G A T G A C c T C G T G G T T C A G ~ C C ~ G T G C A T T C C ~ T C G C T C A C T G ~ T A T G T C T G G G A T G A G A ~ A C T G C T G C T T T T ~ c ~ G ~ i ~ i ~ i .L Q T K L A M L K D D L V V Q K F Q V H S K S L T E Y V W D E I T A A F H N C F1561 C C T A ~ T C ~ G G A G A G G ~ C ~ G ~ C T C A T ~ C T G ~ T C G G A A A A G G C T C ~ G ~ G T A C C T G A ~ G T A T G T ~ C ~ C C A C G A T A ~ G G ~ G G A G T A C ~ GP T I K E R L I N K K L I T V S E K A L E I K V P D L Y V T F H D R L V K E Y K1 68 1 T c ~ C G G T G G ~ T G C c G G T A C T G G A C G ~ A A A A A G A G C ~ G G ~ G ~ G C A G ~ G T G A T G T A C ~ T G C T ~ G T C A G ~ T C T C ~ C ~ G A c A G T G A C ~ G ~ G A T G ~ T G ~S S V M P V L D V K K S L E E A E V M Y N A L S E I S I L K D S K F D V D V1801 . ~ . ~ . ~ W C C C G ~ T G T G T ~ T A C A ~ AG G C G T A G A T c C A ~ G G T G G c A G C ~ G G T ~ TG G T A G C T G T G G ~ C ~ T G A G A G T G G ~ G A CC ~ c G ~ G A ~ G G c C T A C C ~ G C A A AF S R M C N T L G V D P L V A A K V M V A V V N E S G L T L T F R P T E A N1 9 2 1 G T C G C A C ~ G C A ~ G C ~ C C G A C ~ A C A T C ~ G G A G G ~ G G ~ c G ~ G ~ G A ~ G T G T C G T C A G A C G T A G ~ T G A G T C C T C ~ T c ~ G G ~ G T G G ~ C G A A A A T C A ~ G A ~ C T A T GV A L A L Q P T I T S K E E G S L K I V S S D V G E S S I K E V V R K S E I S M

. . . 2041 ~GGTCT~CAGGC~CACAGTGTCCGATGAG~CC~G~GTACAG~TCGAGTCG~GCAGCAG~CATATGGTATCCACAGAGACGA~ATCCGT~CAGATGCATGCGATGL G L T G N T V S D E F Q R S T E I E S L Q Q F H M V S T E T I I R K Q M H A M2161 G T G T A T A C T G G TC C G C T A A A A G ~ C ~c ~ G c ~ G ~ c T A ~ TA G A c A G c c T G G T AG C C T c G C T c T c T Gc T G c G G T A T c ~ cc T G ~ G A T ~ T c ~ G Ac A C A G C T G C T A T A~V Y T G P L K V Q Q C K N Y L D S L V A S L S A A V S N L K K I I K D T A A I D2281 C T c G A ~ C ~ G G A A A A A ~ G G A G T C T A C G A C G T G T G C C ~ G ~ T G G T T G G T G ~ c c T c T A T c A ~ A G G A C A T G C ~ G G G G T G T G G T G A T G G A C T ~ G A C T A T ~ G T G C ~ G ~L E T K E K F G V Y D V C L K K W L V K P L S K G H A W G V V M D S D Y K C F V2401 GCGC~CTCACATAC~TGGC~G~CA~GTGTGCGGAGAGACATGGCGTAGAGTCGCAGTGAGcTCCG~TC~GGTGTA~CAGATATGGGG~GAT~GAGcTATACGCTcTGTGA L L T Y D G E N I V C G E T W R R V A V S S E S L V Y S D M G K I R A I R S V2 5 2 1 C ~ G A C G G T G ~ C C C C A T A T ~ G C A G T G C ~ G G ~ A C A C ~ G T T G A T G G T G ~ C C T G G ~ G C G G ~ G A C ~ G G A G A ~ C ~ C G A G G G T C ~ C ~ G A C G ~ G A T C T A G ~ C T GL K D G E P H I S S A K V T L V D G V P G C G K T K E I L S R V N F D E D L V L

. . 2 64 1 G T A C C A G G A A A A C A G G CT G C T G ~ T G A T ~ G ~ G ~ G G G C ~C A G ~ T G G ~ T C G T G G ~ G A C C ~G G A G ~ T G T ~ G G A C G G T A G AC T C ~ C ~ T G ~ A C G G T C ~ G G TV P G K Q A A E M I R R R A N S S G L I V A T K E N V R T V D S F L M N Y G R G2 7 61 C C G T G C C ~ TA C A A A A G G C TG ~ C T G G A T ~ GG T C T ~ T G ~ A C AC C ~ T G G ~ G T G ~ T T T T C T G G ~ G G C A T G T C T C T A T G C T C C G A G G C ' I " F ~ G ~ A T G ~ c C C A G ~ Gp C Q Y K R L F L D E G L M L H P G C V N F L V G M S L C S E A F V Y G D T Q Q2 8 8 1 A ~ C C ~ A C A T C ~ C A ~ G ~ G ~ C T T T T C C C T A T C C T ~ G C A ~ G A G T C ~ C T C G A G G T C G A T G C T G ~ G ~ C T C G C A G ~ C ~ C G ~ G C G G T G T C ~ G C T ~ T A T C A C C ~ C ~ CI P Y N R V A T F P Y P H L S Q L E V D A V E T R R T T L R C P A D I T F F3 00 1 ~ T C A G ~ A C G ~ G G G C ~ G ~ A T G T G C A C A T C ~ G T G ~ A C A C G C T C G G T G T C A C A C G A G G T C A T C C ~ G G T G C A G C G G T ~ T ~ T C C A G T G T C T ~ C C A C ~ G G G ~ GL N Q K Y E G Q V M C T S V T R S V S H E V Q G A A V M N P V K P L K G K3 1 2 1 G T G A ~ A C A ~ C A C T C A G T ~ C ~ G T C A ~ G C T G C T C T C G A G G G G ~ A C G ~ G A T G T G C A T A C C G ~ C A T G A G G T G C ~ G G G G ~ C G ~ G ~ G A C G T C T C A c T A G T ~ G G ~ C GV I T T Q S D K S L L L R G Y E D V H T V E V Q G E T F E D V S L V R L T3 2 4 1 C C ~ C A C C C G T G G G ~ T ~ C A A A G C A G A G T C C G C A C C T G ~ G G T C T C A ~ G T C T A G G C A T A C ~ G G T C G A T C ~ T A ~ A C A C A G ~ G T G C T A G A T G C A G T C G ~ G T G ~ A ~P T P V G I I S K Q S P H L L V S L S R H T R I K Y Y T V V L D A V V S V L R3361 ~T~G~GTGTGT~GTAG~ACCTG~AGATATGTAC~G~GATGTGTCGACTC~TAG~ACAGATAG~TCGGTGTAC~GGTG~CC.~.~.~.~.CGTCGCAG~c~AAAAD L E V S S Y L L D M Y K V D V S T Q * Q L I E S V Y K G V N L F V A A P K3481 ACAGGAGATG~CTGACATGC~TA~A~ACGAC~GTGT~GCCGGG~CAGTACTATACTC~TGAGTATGATGCTGT~CTATGcAAATACG~AG~TAG~G~TGTC~GT G D V S D M Q Y Y Y D K C L P G N S T I L N E Y D A V T M Q I R E N S L N V K

. . .3601 GA~GT~G~GGATATGTCG~TCGGTGCCTC~CCGAGAG~TCTGAGACGACA~G~CCTGTGATCAGGACTGCTGCTGAAAAACCTCGAAAACCTG~G~GGAAAA~GD C V L D M S K S V P L P R E S E T T L K P V R T A A E K P R K P G L L E N L . . 3 72 1 G T C G C G A T G A T C A A A A G ~ C ~ C T C T C C C G ~ A G T A G G G G T T G ~ G A C A T C G ~ G A C A C C G C ~ C T C T A G T A G T A G A T ~ G ~ G A T G C A T A C ~ G A A A A G A A AV A M I K R N F N S P E L V G V V D I E D T A S L V V D K F F D A Y L I K E K K


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PMMV-S RNA sequence 2 8 7 9

3 8 4 1 A A A C C A A A A A A T A T A ~ C T ~ T G ~ T T T C ~ G G G C G A G ~ G G A A A G A T G G A T C G A A A A G ~ G A G ~ G T ~ C ~ G G C C A ~ G G C T G A T T T T G A C T T T A ~ G A ~ A C C ~ G C C G ~ G A TK P K N I P L L S R A S L E R W I E K Q E K S T I G Q L A D F D F D L P A V D3961 ~ T A C A G G C A C A T G A T C ~ G C A G C A G C ~ G A A A C A G ~ G ~ G G A T ~ A G T A ~ A A A C T G ~ T A C C c G G C ~ G ~ A A A C T A ~ G T G T A T C A T A G C ~ G A A A A T C ~ T G C G C . ~ - ~ - ~ . ~ . ~ . G G TQ Y R H M I K Q Q P K Q R L D L S I Q T E Y P A L Q T I V Y H S K K I N A L F G4 0 81 C C T G T A ' I ' I ' I ' I ' C A ~ 6 ~ C A G C T G C T A ~ G A C ~ G A C A G ~ 6 ~ G A ~ C A T G T T T T A T A C ~ G G A A AA C GC C TA C AC A GA T CG ~ G~ ' I' I ~ ' I~ CT C A~ T CT G GA C TC T ~TP V F E L T R Q L L E T D S S R F M F Y T R K T P T Q I E E F F S D L D S N

. . 4 20 1 G ~ C C T A T G G A C A T A ~ A ~ G C T A G A C A ~ C C ~ G T A T G Ac A A A T C A C A G ~ C G ~ C A ~ G T G C A G T C G A G T A T G A G A~ G G A A A A G G ~ A G G C ~ A G A C G A ~ C ~ G G C T G ~V P M D I L E L D I S K Y K S Q N E F H C A V E Y E I W K R L G L D D F L A E

4 32 1 G ~ G G A A A C A C G G G C A T ~ G G ~ G A C ~ C G ~ G A A A G A C T A C A C A G C C G G ~ T A A A A A C G T G ~ G T G G T A ~ C A G A G G A A A A G C G G T G A T G T C A C ~ A C A ~ G G A A A C A C G A T C A ~V W K H G H R K T T L K D T A G I K T C L W Q R K S G D V T T I G N T I I4 4 4 1 A ~ G C T G C A T G T C T G T C C TC T A T G C T A C C G A T G G A GA G A ~ G ~ A A A G G T G C C T T TT G T G G T G A T G A T A G T A T A~ A T A C ~ C C A A A G G G C A CT G A ~ C C C C ~ T A ~ C ~ C A G GG CI A A L S S M L P M E R I K G A F C G D D I L Y F P K G T D P D I Q Q G4 56 1 G C A A A C C ~ C T C T G G ~ T T T ~ G ~ G C C ~ G ~ G ~ A G G ~ G A G A T A T G G ~ A C T T T T G C G G T A G G T A C A T ~ C A C C A T G A c A G A G G C T G T A ~ G T A T A ~ A T G A c C 6 T C T A A A A ~ GA N L W N F E A K L F R K R Y G Y F C G R Y I H H D R G C I V y D p L K L4 68 1 A T C T C G A A A C T C G G T G C A A A AC A C A T C ~ G ~ T A G A G ~ C A ~ A G AG G ~ A G G A C C T C T C ~ G T G A T G ~ GC T G G G T C G ~ G ~ C ~ G T G C G T A CT A T A ~ C A ~ C G A CI S K G A K H I K N R E H L E E F R T S L C V A G S L N N C A Y Y T H L N D~3OK4 8 0 1 GCTGTCGGTGAGG~A~GACCGCACCTC~GG~CG~T~TATAGAGCA~AGTT~GTAC~GTGTGATA~GG~A~CAAACA~Gr~2"~GGAGT~TGGCG~AGTAA V G V I K T A P L G S F V Y R A L V K Y L D K R L F Q T L F L E * M A L V4 92 1 G T C ~ G G A C G A C G ~ G A ~ C T G A G ~ C A T C ~ G T C T G C C G C T G A G ~ C ~ C T G C T G ~ A T G A C ~ C G G T C ~ G A c G G T A C G ~ T T T C G A A A G ~ G A C A A A G T G A ~ G C AV K D V K I S E F I N L S A A E K F L P A V M T S V K T V R I S K V D K V I A5 0 4 1 A T G G A A A A C G A ~ C G ~ A T C C G A T G T G ~ G C ~ A A A G G T G T A A A G C T T G T T ~ G G A T G G ~ A T G T G T G ~ A G c A G G G ~ A G ~ G T G T C C G G G G A G T G G ~ C C T A c C C G A C ~ C T G CM E N D S L S D V N L L K G V K L V K D G Y V C L A G L V V S G E W N L P D N C5 1 61 A G A G G T G G A G T ~ G C G ~ GT T T G G ~ G A T ~ G A G ~ T G C AA A G A G A T G A C G ~ G C ~ C A C ~G G A T C ~ A T A G ~ C C A G T G C AG C T ~ G A A A C G A T T T G C ~ C AA A ~ G A T C C C G ~ TR G G V S V C L V D K R M Q R D D E A T L G S Y R T S A A K K R F A F K L I P N5 28 1 T A T A G C A ~ A C T A C C G CC G A T G C T G A G A G A A A A G TT T G G C ~ G T T T T A G ~ T A ~ A~ G G T G ~ G C C A T G G A A A AG G G T T T C T G T C C T ~ A T C TT T G G A G T T T G T C T C A G ~G T A ~Y S I T T A D A E R K V W Q V L V N I R G V A M E K G F C P L S L E F V S V C I5 4 0 1 G T A C A C A A A T C C ~ T A T A A A A ~ A G G C ~ G A G A G A G A A A A T T A C T A G T G T G T C A G ~ G ~ G G A C C C G ~ G ~ C ~ A C A G ~ G C A G T C G ~ G A T G A G ~ C A T C G ~ T C A G ~ C C ~ T G G C TV H K S N I K L G L R E K T S V S E G G P V E L T E A V V D E F I E S V P M A5 52 1 G A C A ~ A C G T A A A T T T C G C ~ T C ~ T C T ~ G A A A G G ~ G T ~ T ~ G T A T G T A G G T ~ G A G A A A T G A T ~ T ~ G G G ~ G ~ T ~ G G ~ G G G ~ G C T G T T T G A T ~ G G ~ A G ~ G G GD R L R K F R N Q S K K G S N K Y V G K R N D N K G L N K E G K L F D K V R I G~ C P -5 64 1 C A G A A C T C G G A G T C A T C G G A C G C C ~ G T c ~ C ~ C G T T ~ F ~ C T A T G G C ~ A C A C A G ~ C C A G T G C C ~ T C ~ A G T G T A ~ A G G ~ C T G T A T G G G C T G A T C C A ~ A ~ G ~ A c ~Q N S E S S D A E S S S F * M A Y T V S A N Q L V Y L G V W A D P L E L Q A5 76 1 A T C T G T G T A C ~ C G G C G ~ A G GC ~ T C A G ~ C A A A C A C ~ C A G G C T A G~ C T A C G G ~ C ~ C A G ~ G ~ C T C T G A T GT G T G G ~ G A C T A ~ C C G A C C G C T A CA G ~ A G A ~ C C T G c T AL C T S A L G N Q F Q T Q Q A R T T V G Q Q F S D V W K T P T A T V R F P A5 8 81 C T G G T T T C A A A G T T T T C C G A T A T ~ T G C ~ G T G C T A G A ~ C T C T A G T G T C G G C A C ~ C T C G G A G C C ~ G A T A C T A G ~ C A G G A T ~ T A G ~ G ~ G A A A A T C C G C A A A A T C C T A C ~ C T GG F K V F R Y N A V L D S L V S A L L G A F D T R N R I I E V E N P Q N P T T6 0 01 C C ~ G A C G C ~ T G C G A C ~ G GC G G G T A G A C G A T G C G A C G GT G G C C ~ A G G G C C A G T A T ~ GT ~ C C T C A T G ~ T G A G ~ A G ~C G T G G C A C G G G ~ T G T A C ~ TC ~ G C T C T G ~ C GE T L D A T R R V D D A T V A I R A S I S N L M N E L V R G T G M Y N Q A L F6121 A G A G C G C ~ G T G G A C T C A C C T G G G C T A C ~ T C ~ A A A C A T G A T G G C A T A A A T ~ G ~ G ~ C G ~ A ~ A A A C G T C C G T G G ~ G A G T A C G A T ~ C T C G T A G TQ 'I 'I ~I 'I 'I 'C CC TC CA C~S A S G L T W A T T P *6 24 1 A T C G A A G G G ~ G T C G ~ G G G A T G G ~ C G C ~ A ~ A T A C A T G T G T G A C G T G T A ~ G C G ~ C G A C G T ~ A ~ i . i . f ~ C A G G G G ~ C G ~ T C C ~ C ~ C C G ~ C G C G G G T A G C G G ~ C C A ~ H

Fig . 2 . T he ent ire nuc leot ide sequence o f the PMM V-S genome , shown as D NA , and deduced amino ac id sequences o f the ORFs.Arrows indicate init iat ion codons. Terminationcodons and the amber readthroughcodon are ind icated by an asterisk. The predictedposit ion ~r the origin of assembly is underl ined.

p r o t e i n h a s b e e n m a p p e d t o a m i n o a c i d s 8 3 3 t o 1 08 6 , a n di s k n o w n a s t h e h e l ic a s e d o m a i n ( H o d g m a n , 1 9 8 8 ;G o r b a l e n y a & K o o n i n , 1 9 8 9; H a b i l i & S y m o n s , 1 9 89 ). I ti s i m p l i c a t e d i n n u c l e i c a c i d u n w i n d i n g , a n d p o s s i b l y i no t h e r p r o c e s se s s u c h a s r e c o m b i n a t i o n a n d t r a n s c r i p t io n .O f t h e s ix c o n s e r v e d m o t if s c o m m o n t o S in d b i s - li k ev i ru s e s i n w h i c h t h e t o b a m o v i r u s g r o u p h a s b e e nc la s si fi ed ( G o l d b a c h & W e l li n k , 1 9 88 ), t h e N T P - b i n d i n ga c t i v i ty h a s b e e n a s c r i b e d t o d o m a i n s I a n d I I ( a m i n oa c i d s 8 3 3 t o 8 5 0 a n d 9 0 2 t o 91 3 , r e s p e c t iv e l y ) . T h ed o m a i n I m o t i f i s s t r i c t l y c o n s e r v e d i n t h e 1 2 6 K p r o t e i no f P M M V - S w i t h re sp e ct to t h o se o f T o M V a n d T M V ,b u t d i ff er s fr o m t h a t o f T M G M V i n w h i c h t h e c o n s e n s u sT h r a t p o s i t i o n 8 4 2 h a s b e e n s u b s t i t u t e d b y T y r , a n d t h ef o l l o w i n g G l u - I le - L e u - S e r s e q u e n c e h a s e v o l v e d t o G l y -A s p - P h e - G l u , t h e fi rs t s u b s t i t u t i o n b e i n g o f t h e s e m i -

c o n s e r v a t i v e t y p e a n d t h e o t h e r t h r e e o f t h e n o n -c o n s e r v a t i v e t y p e . I t i s t h e r e f or e p o s s i b l e t h a t t h e a m i n oa c id e x c h a n g e s o c c u r r in g i n t h i s d o m a i n i n T M G M Vc o u l d r e s u l t i n i t s l o w e r r e p l i c a b i l i t y , i n c o m p a r i s o n w i t hT M V o r T o M V ( W e t te r , 1 9 8 6 ). I n th e d o m a i n I I t o Vm o t i f s ( a m i n o a c i d p o s i t i o n s 9 0 2 t o 9 1 3 , 9 3 0 t o 9 4 0 , 9 6 6 t o9 7 4 , 1 0 3 8 t o 1 0 5 5 a n d 1 0 7 0 t o 1 0 8 5 , r e s p e c t i v e l y ) , a l l o ft h e a m i n o a c id s u b s t it u t io n s t h a t oc c u r i n th e P M M V - Sp r o t ei n w i th r e s p e ct t o t h e T o M V a n d T M V o n e s a re o ft h e c o n s e r v a t i v e t y p e : I l e to L e u ( p o s i t i o n 9 0 6 ) , T y r toP h e ( p o s i t i o n 9 3 0 ) , a l t o l le ( p o s i t i o n 9 7 4 ) , T y r t o P h e( p o s i t i o n 1 0 4 8 ), A l a t o G l u ( p o s i t i o n 1 0 4 9 ), V a l t o L e u( p o s i t i o n 1 0 7 0 ), L y s t o A r g ( p o s i t i o n 1 0 7 9 ) a n d L e u t o I le( p o s i t i o n 1 0 8 1 ). T h e s a m e o c c u r s w i t h t h e a m i n o a c i ds u b s t it u t io n s i n T M G M V ( F i g. 3 ).

T h e c a r b o x y l e n d o f t h e 1 8 3 K p r o t e i n r e s u lt s f r o m


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2880 E . A l o n s o a n d o t h e r s

. . . . I - I P M M V - S m A Y T Q Q A T N A A L A S T L R G N N P L V N D L A N R R L Y E S A V E Q C N A H D R R P K V N F L R S I E E Q T L I A T K A Y P E F Q I T F Y ~ Q N A V ~ S A G G L R S L E L E Y L M M Q I P 1 0 0ToMV T SS LE V T K DT DEF R SKVV I IT M V T TS LD V S K DT EF R SKV R I I~ m s n s . L E S V S X T ~ , D ~ E F R s z ~ ~ v s . i I A , VP M M V - S Y G S % ~ Y D I G N F A A H M F K G R D Y V H C C M P N M D L ~ - - - - ~ A Q K D S I L Y L S K L A Q K K K V I P P Y Q K P C FD K ~ ' T D D PQ S V V C K P F Q H - C E G V S H C - T D K V Y 1 9 7T o M V L S L A L V I I ~G R E R G N H V N F E A R A E M N E H D T T - R - H S Q E Y G RT M V L S L A L V I I ~G R E R G G T V N F E A R A E I E D A H N T T M - R - H Q P M Q Q S G RT M G M V P L L I i I ~G M R S R S N E F R E A N R A E A N E C T D- R I H P P E N - S G R RP M M V - S A V A L H S L Y D I A D E F G A A L L R R ~ d V C Y A A F H F S E N L L L E D S Y V S L D D I A F F S R E D M L N F S F V A E S T L N Y T H S Y S N V L K Y V C K T Y F P A S R E V Y M K E F 2 9 7To MV I I K H N E N C Q D R T AS S I NTMV I I K T N E N C D KT AS I NTMGMV VH ISK I SILA A DQTE T NE T K DVS F AD K K I H V S I FP M M V - S L V T R V N T W F C F R L D T F V L R G V Y H R G V D Ed F Y S A M E D A W H Y K K T L A M M N E R I L E D S S V N Y W F P K M K D M V I V P L F D V S L Q N E G K R L A R K E V M V S K 3 9 7ToM V I L K A K S K C R I ET- S -T LT M V I L K A KS S T C R I E T - S - T LTMGMV TKV YI KS RQV C SD E FA F T A I F R T A F E G I - T - S K M T S I N RP M M V -S D F V Y T V L N H I R T Y Q S K A L T Y A N V L S F V E S R S R V I I N G V T A R S E W DV D K A L L Q S L S M T F F L Q T K L A M L K D D L W Q K F Q V H S K S L T E Y W W D E I T A A F H NC F 4 97ToMV A S S H V LIS ALGP TVSQ H SL G ATMV F A S Y H V LIS SLG TVCQ H SL G ATMGMV A Q I P A Q I MG RCLD TTS LI VGKF G VP M M V - S P T I E R L I K K L T V S E K A L E I V P D L Y V T F H D R L V K E Y K S S V E M P V L D V K K S L E E A E V M Y N A L S E I I K D S D K F D V D V F S R M C N T L G V D P L V A A K V M V 5 9 7ToM V S R KIT N R S M D IR KM T E L V N Q QS E MT ITMV SV L R R AGD R T A D A IR KM T L V RE Q QS E MT ITMG MV VS R ILD N K I WK F A TK E L H I D Q D L GA N IAK KD KA D S D R I

P M M V - S A V V S N E S G L T L T F E R P T E A N V A L A L Q P T I S K E E G S L K I V S D V G E S I E V V R K S E I M L L T G N T V S D E F Q R S T E I E L Q Q F H M V S T E T I K Q M H A M 6 9 7ToMV M Q DSEK ASD- A VVT R E P GSMARG LQLA S DVPE SSYT E E ATAS SL H CSIT M V M D Q E K A S - A V V T R E E P M G S M A R G L Q L A A D H P E S S Y S K N E E A T A D S L S S IT M G M V A E R D K E K K S - A E A V V C E P T E E N V N K F S - I A E G R L P V C A - E S H G L T N A N L E L Q L N D K A C V D S V T A S VP M M V - S V Y T G P L K V Q Q C K N Y L D S L V A S L S A A V S N L K K I I D T A A I D L E T K E K F G V Y D V C L K K W L V K P L S K G H A W G V V M D S D Y K C F V A L L T Y D G E N I V C G E T W R R V A 7 9 7ToMV M FI V L RQ L AS R SA N ETHA R YH EH EFG ITCDNTMV I M FI V L RQ L ASR I TA S ETHA R YH E EQGV TCDDTMG MV S M V A T C L EVGY SDS R V W T L AA S L YKG M T S E DR M TES DP M M V - S V S S E S L V Y S D M G K I R A I R S V L K 6 G E P H I S A K V T ~ V D G V P G C G K T K E I ~ V N F D E D L V L V P G K Q A A E M I R R A N S S G L I V A T K E N V R T V D S F L M N Y G R G 8 9 7ToM V V A L TL RL V V -- E I R A I D KTMV V A L TL RL RN V V | I I D K M F KST M G M V D T M I A L Q N L K T M R E P T M V Y G D F ~ F D L I A R M D L H - P K -P M M V -S P - C Q ~ F L D E G L M L + G C V N F L V G M S L C S ~ V Y G D T Q Q I ~ N R V A T F P Y P K HL S Q L E V D A V E T R R TT L R C P A D I F F L N Q K Y E G Q V M C T S S V T R S V 9 9 6ToM V AR F I I ~ E DI Y | TG A FAK E V H R H EKETMV TR F i I ~ A EIi Y | SG A FAK E V HY RB F S KKTMG MV R-S H I I ~ LI G DID YI ~ QN FEK Q E M G VN QS A TT T QP M M V - S S H E V I Q G A A V M N P V S K P LK ~ K V I T F T Q S D K S L L L S R GY E D ~ T V H E V Q G E T F E D V S~ u v * 1 u T P T P V G I I S K Q S &L L V S L S R H T R S I K Y ~ ~ V V L D A V V S V L 1 0 9 6ToMV Q MVS SI IL EA A| YA | S ARD I V K L M PL IITMV Q MVG I ~I H IL EA S| YS | S AGD |V A C L M PL IITMG MV S M G KG L S IV A FE EEK K~ N I ] A LTL S |V A T K F PL QIIP M M V -S R D L E C V S S Y L L D M Y K V D V S T Q * Q L Q I E S V Y K G V N L F V A A P K T G D VS D M Q Y Y Y D K C L P G N S T I L N E Y D A V T M Q I R E N S L N V K D C V ~ D M S K SV P L P R ES E T T 11 9 6ToMV R AG * VD F NF I F L N KLTDI AA KDVKPTMV KL AG * D F S I F MM NF RLTDI AA KDQIK PTMG MV S SSLL LF E M EAG SR* MD A F H T S FP L F V K RL D F I M K VKP CP M M V - S L K P V I R T A A E K P R K P G L L E N L V A M I K R N F N S P E L V G V V D I E D T A S L V V D K F F D A Y L I K E K K K P - K N I P - L L S R A S L E R W I E K Q E K S T I G Q L A D F D F I D L P 1 2 9 4ToM V I MV M QT S N S L R N FS- F E N A QV VT M V I M V M Q T A S I I N S L R N VS- F E N L QV VTMGMV E L P AA A D T TI S V S F KE YT- AGVM TKD MM L NRKE VLLD D NYN T

. . . . . . I . P M M V - S A V D Q Y R H M I K Q Q P K Q R L D L S I Q T E Y P A L Q T I V Y H S K K I A L F G P V F S E L T R Q L L E T I S S R F M F Y T R K T P T Q I E F F S D L D S ~ P D I L E L D I S K Y D K ~ 1 3 9 4To MV A K I L DS L F A D G ~ V VT M V A K T I L DS V L F A D G ~ VT M G M V I K A K N Q GI-L AG L AF KE L F E Q ~ VP M M V -S N E F H C A V E Y E I W K R L G L D D F L A E V W K H G H R K T T L K 6 Y T A G + T C L W Y Q R K S G D V T T F I G N T I I I A A C + S M L P M E R L I ~ ~ - F C G DD S I L F P K G T6 F P D I 1 4 94T o M V R Z Q | v s ~ z '. CE YT M V R FE G Q | I V ~ KI L CE VT M G M V N E Q I | V ~ KV V L

P M M V - S L W N F E A K L Y G Y F C G R Y I I H D R G C I V Y Y D P L K I I S K L G A K H I N P ~ H L E E F R T S L C D V A G S L N N - C A Y Y T H L N D A V G E V I T A P L G S F V Y 1 59 3T OM V ~ M ~ Q V D WD R E - Q D H PT M V H ~ M ~ Q V D W R V - QD W H PTMG MV ~C M ~ K A DYD L V C G W LGF PQ A IK H ID AFP m 4 v - s m ~ v z ~ c D Z ~ F Q ~ Z ( 16 1 21T o m , K S s v a s D G S S C ( 1 6 1 6 1T M V K S S V R E I D G S S C ( 1616 )TMG~4V NCV N F F R NGC (1610)

F i g . 3 . A l i g n m e n t o f th e d e d u c e d a m i n o a c i d s e q u e n c e s o f t h e 1 2 6 / 1 83 K p r o t e i n s f r o m d i f f e r e n t t o b a m o v i r u s e s . S o u r c e o f a m i n o a c i ds e q u e n c e s a s i n T a b l e 1 . O n l y a m i n o a c i d e x c h a n g e s a r e in d i c a t e d . G a p s a r e i n d i c a te d b y ( - ) . N u m b e r i n g c o r r e s p o n d s t o t h a t f r o mP M M V - S . N u m b e r s i n p a r e n t h e s e s i n d i c a t e th e t o t a l l e n g t h o f e a c h p r o t e i n . T h e s e q u e n c e m o t i f s d e f i n ed b y G o r b a l e n y a & K o o n i n(1989), Poch e t a l . (1989) , a nd Roz a nov e t a l . (1990) a re boxed.


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P M M V - S R N A s e q u en c e 2881

readthrough of the UAG stop codon at nt 3421 to 3423.The suppression of a termination codon is a widespreadphenomenon among animal and plant RNA viruses, andis related to the regulation of the expression of thedifferent components of the viral RNA polymerase(Ishikawa e t a l . , 1986; Strauss e t a l . , 1988). The fact thatthe surrounding nucleotide sequences (ATAGCAAT-TACAG) at positions 3420 to 3432 (Fig. 2) are strictlyconserved in all the tobamoviruses reveals the funct ionalimportance of this particular region in their genomes, asit also occurs in alphaviruses (Strauss e t a l . , 1988). Theso-called polymerase module is found in the carboxyportion of the protein, in which four domains (A to D)have been defined (Poch e t a l . , 1989). This module iscommon to all of the DNA- and RNA-dependent RNApolymerases, and it is known to be involved in theelongation of pre-existing chains (Quadt & Jaspars,1989). The Gly-Asp-Asp mot if first identified by Kamer& Argos (1984) is found in the C domain surrounded byhydrophobic residues. The alignment of the PMMV-S183K protein wi th those from other tobamoviruses (Fig.3) also shows that all the amino acid substitutions inthese domains are of the conservative type.

The only non-conservative differences betweenTMGMV and PMMV-S occur in the D domain, Cys toGly (position 1496) and Asn to Leu (position 1500). Thehigher sequence variability in this readthrough part ofthe 183K protein is found in the region located at aminoacids 1250 to 1291. There is also lower amino acidsequence identity in both the N- and C-terminalsegments (Fig. 3), a feature common to other RNAviruses (Haseloff e t a l . , 1984; Allison e t a l . , 1989).

It remains to be determined whether the attenuatedbiological behaviour of PMMV-S in tobacco, in com-parison with TMV and ToMV, could be ascribed toregions of maximum sequence heterogeneity or tosegments of the non-highly conserved sequence of the126K/183K protein, as previously described for theattenuated L11A strain ofToMV (Nishiguchi e t a l . , 1985)in which the amino acid substitutions responsible for thischaracteristic have been mapped in regions, not highlyconserved, of the 126K protein (amino acid residues 348,759 and 894). It is also unknown whether the ability ofPMMV-S to break the resistance against tobamovirusesconferred by the E 1 and L 2 genes in pepper (Boukema e ta l . , 1980; Garcia-Luque e t a l . , 1990) is due to any of theamino acid changes which take place in this protein, asdescribed for the Ltal strain of ToMV, in which twoamino acid substitutions (Glu to Gin and Tyr to His) atpositions 979 and 984, respectively (980 and 985 in thePMMV-S protein) have been identified as those respon-sible for the ability of this strain to break the Tm-lresistance gene in tomato (Meshi e t a l . , 1988). However,since none of the resistance conferred by the L 1 and L zgenes in pepper is expressed in protoplasts (unpublished

results), it is plausible to consider that other regions ofthe PMMV-S genome may be implicated also.

O r g a n i z a t i o n o f t h e 3 0 K p r o te i n g e n eThe third ORF of PMMV-S encodes the 30K protein(Fig. 2). Translation initiates at nt 4909 and terminates atnt 5682; thus the coding region for this protein overlapswith neither the coding region for the 183K protein norwith that for the coat protein. Its putative translationproduct is 257 amino acids long with a calculated Mr of28 347. Therefore, this is the first tobamovirus in whichnone of the reading frames overlap, since in ToMV,TMV, cucumber green mottle mosaic virus (CGMMV)and TMGMV the 5' end of the genes encoding the 30Kprotein overlap with the 3' end of those encoding the183K proteins (Goelet e t a l . , 1982; Ohno e t a l . , 1984;Saito e t a l . , 1988; Solis & Garcia-Arenal, 1990), and insunn-hemp mosaic virus (SHMV) as well as in CGMMV(Meshi e t a l . , 1982; Saito e t a l . , 1988) their 30K-codingregions overlap at the 3' end with their coat proteingenes.The 30K proteins of tobamoviruses are responsible forcell-to-cell spread of the viral infection (Deom e t a l . ,1987; Meshi e t a l . , 1987), by modify ing the plasmodes-mata (Wolf e t a l . , 1989). Although the exact mechanismof action is unknown (Hull, 1989), a domain responsiblefor binding to nucleic acids which maps between aminoacid positions 65 and 87 has been defined (Citovsky e ta l . , 1990). As with the consensus sequences in the126K/183K proteins, the amino acid substitutions thattake place in this region of the PMMV-S 30K protein areof the conservative type with respect to TMV, ToMVand TMGMV, except for the semi-conservative ex-change Ala to Val (position 70) in the ToMV 30K protein(Fig. 4).As in other tobamoviruses (Ohno e t a l . , 1984; Solis &Garcia-Arenal, 1990) the PMMV-S 30K protein isencoded in the least conserved part of the entire genome,both at the nucleotide and amino acid levels (Table 1). Itsalignment with those from the most closely relatedtobamoviruses (Fig. 4) shows that the PMMV-S 30Kprotein shares a higher degree of amino acid sequenceidentity with that of TMV th an with those of ToMV orTMGMV, in contrast to other proteins encoded byPMMV-S. It conta ins two well conserved regions locatedat amino acid positions 46 to 125 and 151 to 204. In thefirst one, only one non-conservat ive amino acid substitu-tion takes place in the PMMV-S 30K protein withrespect to ToMV and TMV (Arg to Tyr, both at position109) and TMGMV (Ala to Cys at position 113). In thesecond well-conserved region, all of the amino acidchanges among the TMV, ToMV and PMMV-S 30Kproteins are of the conservative type, but it is lessconserved compared to TMGMV, with three non-


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2 8 8 2 E . A l o n s o a n d o t h e r s

P M M V - ST o M VT M VT M G M V aT M G M V b



G K N N D K S L S M F P S M V I M V H E E I E ~ V M ] E AG K N N D T K M I S M F P S M C I M V H E E I D S I T | E A

V S L R T D K Q D E I F K S T I M V K D ~ D I | K Sv s L ~ T ~ Q D E I F ~ s IMvK D ~ i ] , ~ s

Y A Q VV G K KNI KN K SA Y ~ N V ~D M S E M N IY A Q V V A Q M K N N K S A ~ R N N ~ D M E M D [K A HA P C N S SE KHP K E Y I V I N VR K R L ~D S I K E VDE I

N A H A P c , ~ s s E ~ P K E ~ i v I N V K R L ~ F ~ S ~ ~ ~ ~ I

~ V A . . . . . . . . . . T K S R P - K N N N L G R S G G P K - - - P S F ~ E V E E ~ L - E D E A T V D D Y ] ( 2 6 4)~ I a . . . . . . . . . . S R T G S D V R K G N S S N D R S V P N K N Y R N V D ~ G G M S F K K ~ L - D D D A T V D 1 ( 2 68 )I V K E . . . . . . . . . . V P E N - K E M - - - V G N N N N . . . . K K I - - - N S g K G F I - ~ E I E D N V S D D E I S T I ( 25 6 )[ V K E R F R K T K K G K K R K K E K - K R V - - - V G N S N N . . . . . K K I - - - N S G ~ G L V - E ~ I E D N V S D D E I S T J (266)

Fig. 4. A lignment of the amino acid sequence of the 30K p rotein of PMM V-S with those from the most related tobamoviruses.Numbering, symbolsand sourceof the amino acid sequencedata as in Fig. 3 and Table 1. The amino acid sequencedom ains definedbySaito et al. (1988) are boxed.

i 0 0

2 0 0

c o n s e r v a t i v e s u b s t i t u t i o n s ( L e u t o L y s , T h r t o L e u a n dA l a t o L y s a t p o s i t i o n s 1 7 2, 1 79 a n d 1 92 , r e s p e c t i v e l y )( F i g . 4 ) . S e v e r a l a m i n o a c i d c h a n g e s i n t h e c e n t r a ls e g m e n t o f t h e t o b a m o v i r a l 3 0 K p r o t e i n s h a v e b e e ni d e n t if i e d i n t e m p e r a t u r e - s e n s i t i v e m u t a n t s d e f e c t i v e i nc e ll -t o -c e ll m o v e m e n t ( O h n o e t a l . , 1 9 8 3 ; Z i m m e r n &H u n t e r , 1 9 83 ) a s w e l l a s i n t h e L t b l s t r a i n o f T o M V ,k n o w n t o o v e r c o m e t h e r e s i st a n c e c o n f e r r e d b y t h e T m - 2g e n e i n t o m a t o p l a n t s ( M e s h i e t a l . , 1 98 9 ). T h i s c a p a b i l i t yr e s i d e s i n t w o a m i n o a c i d s u b s t i t u t i o n s , a t p o s i t i o n s 6 8a n d 1 33 ( C y s t o P h e a n d G l u t o L y s , r e s p e c t i v e l y ) . I n t h i sr e g i o n o f t h e P M M V - S 3 0 K p r o t e i n ( F ig . 4 ), th e r e a r et h r e e n o n - c o n s e r v a t i v e a m i n o a c i d c h a n g e s w i t h r e s p e c tt o t h e p ro t e i n s o f T M V ( G l u t o M e t , A l a t o L y s a n d L y st o A l a a t p o s i t i o n s 1 3 3, 1 4 7 a n d 1 5 0, r e s p e c t i v e l y ) a n d o fT o M V ( A l a t o L y s, A l a t o L y s a n d L y s t o A l a a t p o s i t io n s1 3 0, 1 4 7 a n d 1 5 0) b u t o n l y o n e ( V a l t o P r o a t p o s i t i o n 1 3 6 )w i t h re s p e c t t o T M G M V . A l t h o u g h s o m e o f t h e ses u b s t i t u ti o n s m a y b e o f a c o m p e n s a t o r y t y p e , t h e y c o u l db e r e s p o n s i b l e f o r t h e a b i l i t y o f P M M V - S t o o v e r c o m et h e p e p p e r L 1 a n d L 2 r e si s t a n c e g e n e s . T h e c a r b o x yr e g i o n o f th e t o b a m o v i r a l 3 0 K p r o t e i n s a r e t h e m o s tv a r i a b l e i n t e r m s o f le n g t h o r a m i n o a c i d s e q u e n c e .H o w e v e r , S a i t o e t a l . ( 1 9 8 8 ) f o u n d t h a t a ll o f t h e s ep r o t e i n s h a v e a p a r t i c u l a r c h a r g e d i s t r i b u t i o n w i t h ab a s i c d o m a i n f l a n k e d b y t w o a c i d i c d o m a i n s . I n t h i ss e n se , t h e c o n t e n t o f a c id i c a m i n o a c i d s ( G l u , A s p ) i n t h ee x t r e m e C t e r m i n u s o f th e 3 0 K p r o t e i n o f P M M V - S i sl o w e r (f o u r) t h a n t h a t o f T M V a n d T o M V ( si x a n d s e v e n ,r e s p e c t i v e l y ) . T h e s e c h a n g e s c o u l d b e i n v o l v e d i n t h ea d a p t a b i l i t y o f P M M V - S t o it s p e p p e r h o s t, a l t h o u g ht h e y m a y o n l y r e p r e s e n t , a s s t a t e d a b o v e , t h e h i g h d e g r e eo f v a r i a b i li t y i n t h is a r e a o f t h e t o b a m o v i r a l 3 0 Kp r o t e i n s .

C o a t p r o t e i n g e n eT h e f o u r t h O R F o f P M M V - S e n c o d e s t h e c o a t p r o te i n . Itr a n g e s f r o m n t 5 6 8 5 to 6 1 5 8, w i t h a n i n t e r g e n i c r e g i o n o ft w o n u c l e o t id e s b e t w e e n t h e 3 0 K a n d c o a t p r o t e i n O R F s( F i g . 2 ) . T h e r e s u l t i n g p r o t e i n c o n s i s t s o f 1 5 6 a m i n oa c i d s , w i t h a c a l c u l a t e d M r o f 1 7 1 1 0 . T h i s v a l u e d i f f e r sf r o m t h e p r e v i o u s r e p o r t o f 1 5 8 a m i n o a c i d s f o r t h e c o a tp r o t e in o f a n I t a li a n i so l at e o f P M M V ( W e t t e r e t a l . ,1 9 84 ), d e t e r m i n e d b y a m i n o a c i d a n a l ys i s. A l t h o u g hP M M V - S a n d P M M V a r e d if f er e n t i so l at es , w h i c h c a nb e d i s t i n g u i s h e d b y t h e i r r e s p o n s e s i n C a p s i c u m s p p . w i t hd i f f e re n t re s i s t a n c e g e n e s a n d t h e r e f o r e h a v e b e e ni d e n t i f i e d a s d i f f e r e n t p a t h o t y p e s ( G a r c i a - L u q u e e t a l . ,1 9 90 ), s e q u e n c i n g o f t h e I t a li a n P M M V c o a t p r o t e i ng e n e h a s s h o w n t h a t i t a l s o c o n s i s t s o f 1 5 6 a m i n o a c i d s( M . L . F e r r e r o , I . G a r c i a - L u q u e , E . A l o n s o , A . d e l aC r u z , J . F . R o d r i g u e z , M . T . S e r r a & J . R . D i a z - R u i z ,u n p u b l i s h e d r e s u l t s ) .

T h e a l i g n m e n t o f th e d e d u c e d a m i n o s e q u e n c e f or th ec o a t p r o te i n g e n e o f P M M V - S w i t h t h o s e o f o t h e rt o b a m o v i r u s e s ( F i g . 5 ) s h o w s t h a t t h e r e i s a s t r i c tc o n s e r v a t i o n o f t h o s e a m i n o a c i d s e q u e n c e m o t i f s (3 6 to4 1 , 8 8 t o 9 4 , 1 1 3 t o 1 2 0 ) w h i c h c o r r e p o n d t o t h e R N A -b i n d i n g s i t e i n t h e c o a t p r o t e i n ( A l t s c h u h e t a l . , 1987).T h e l o w e r a m i n o a c i d s e q u e n c e i d e n t i t i e s a re l o c a t e d a tt h e N , C a n d c e n t r a l r e g i o n s o f t h e p r o t e i n s ( F i g . 5 ). I tr e m a i n s a n o p e n q u e s t i o n a s t o w h e t h e r a n y o f th e s ec h a n g e s a l s o af f e c t t h e a b i l i ty o f P M M V - S t o o v e r c o m et h e r e s i s t a n c e c o n f e r r e d b y t h e L 1 a n d L 2 g e n e s i n p e p p e rp l a n t s , a s d e s c r i b e d f o r s e v e r a l m u t a n t s t r a in s o f T M Va n d T o M V i n w h i c h c h a n g e s i n t h e i r c o a t p r o t e i n sm a k e t h e s e m u t a n t s e i t h e r a b le t o b e l o c a li z e d b y th e N 'g e n e i n N . s y h , e s t r i s o r t o e s c a p e i t s a c t i o n ( K n o r r &


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P M M V - 8 A Y T V s s A N Q L X / Y L G S V W A D P L E L Q N L C T S A L G N Q Q Q Q F S D V W K T I P T A T V R F P A T G F K V F R Y N A V L D S L V S A L L G V E N P QToMV S SIT PS F F S I L V NS ] I E PF QS GDVY Y P T I I QT M V S S I T T P S F F S A I I N I I V R E P S Q V D S D Y P T I ] Q A~ G ~ P I N P S F S A~ : V Q I . L [ A A p v sM s D YY s~ ~ I F ~ , s ~ D Q PP M M V - S s N P T T A E T L D A ~ [ R A S I S N L MN E L V R G T G M YN Q A L F E S A S GL T W A T T P ( 15 6)~ o ~ ~ s A N v ,. ~ M V ~ S A A S 1 15 81T M V S A N I V I S R S S S V T S G A T ( 1 5 8)T M G M V A N T I V N ~ N A F G T V T A T ( 1 5 8 )

F i g . 5 . A l i g n m e n t o f t h e c o a t p r o t e i n s e q u e n c e o f P M M V - S w i t h t h o s e f r o m t h e m o r e c l os e ly r e l a te d t o b a m o v i r u s e s . N u m b e r i n g a n ds o u r ce o f a m i n o a c i d s e q u e n c e s a s i n F i g. 3 a n d T a b l e 1 . T h e R N A - b i n d i n g d o m a i n s a r e b o x e d .

i00

D a w s o n , 1 9 8 8 ; S a i t o e t a l . , 1 9 8 8 , 1 9 8 9 ; Cu l v er &D a w s o n , 1 9 8 9 ) .

B a s e d u p o n n u c l e o t i d e s e q u e n c e h o m o l o g y w i t h t h eT M V o r i g i n o f a s s e m b l y ( Z i m m e r n , 1 9 7 7) , t h e p r e d i c t e dp o s i t io n o f t h is r e g io n i n P M M V - S i s l o c a te d b e t w e e n n t5 4 5 8 a n d 5 5 1 7 , i n a c c o r d a n c e w i t h t h e a b s e n c e o fe n c a p s i d a t io n o f i ts c o a t p r o te i n m R N A a s s h o w n b ye l e c t ro n m i c r o s c o p y o b s e r v a t i o n ( W e t t e r e t a l . , 1 9 8 4 ) a n de l ec tro p h o ret i c a n a l y s i s o f th e v i r i o n p a rt i c l e s (Ga rc i a -L u q u e e t a l . , 1990).

F u n c t i o n a l a n d e v o l u t io n a r y c o n s i d e r a ti o n sT h e d e t e r m i n a t i o n o f t h e n u c l e o t id e s e q u e n c e o fP M M V - S R N A h a s a ll o w e d u s t o co n f ir m t h a t t h e e n ti r eg e n o m e o f t h i s v i r u s h a s d i v e r g e d f r o m o t h e r r e l a t e dt o b a m o v i r u s e s a t a s i m i l a r r a t e . T h e g r o u p i n g o f t h et o b a m o v i r u s e s b a s e d o n t h e a m i n o a c i d c o m p o s i t i o n o ft h e ir c o a t p r o t e i n s ( G i b b s , 1 9 8 6 ) a n d o n t h e b a s i s o f t h ep e p t i d e p a t t e r n o f t h e 1 2 6 K p r o t e i n s ( F r a il e & G a r c i a -A r e n a l , 1 9 9 0 ) c o r r e s p o n d s w e l l t o w h a t i s d e d u c e d f r o mt h e e n t i r e g e n o m e , i . e . P M M V i s l o c a t e d i n t h e s a m ec lu st er a s T M V , T o M V a n d T M G M V , b e in g m o r ec l o s e l y r e l at e d to T o M V a n d T M V . T h e s e d a t a a l s oc o n f ir m th e r e la t io n s h ip b e t w e e n T o M a n d P M M V - Sp r e v i o u s l y f o u n d b y s e r o l o g i c a l a n a l y s i s ( W e t t e r e t a l . ,1 9 8 4 ; A l o n s o e t a l . , 1989).

T h e p o s s e ss i on b y P M M V - S R N A o f al l t h e co n s e rv e ds e q u e n c e m o t i f s n e c e s s a r y f o r r e p l i c a t i o n a n d v i r i o ns ta b i l i ty , i t s b i o l o l g i ca l p ro p ert i es su ch a s th e d i m i n i sh edc a p a b i l i t y t o r e p l i c a t e a n d / o r a c c u m u l a t e i n t o b a c c op l a n t s, i t s a b i li t y t o o v e r c o m e t h e t o b a m o v i r a l r e s i s t a n c eg e n e s i n p e p p e r a n d i t s i n a b i li t y t o i n f e c t t o m a t o p l a n t s ,s h o u l d b e a s c r i b e d t o t h e r e q u i r e m e n t s f o r t h e e s t a b l i s h -m e n t o f a f u n c t i o n a l i n t e r a c t i o n ( s ) w i t h t h e h o s t f a c t o r (s )k n o w n t o b e n e c e s s a r y f o r t h e e f f ic i e n t m u l t i p l ic a t i o n o ft h e v i r u s e s i n t h e i r h o s t p l a n t s . W h e t h e r t h e s e r e q u i r e -m e n t s a r e r e l a t e d t o c h a n g e s a t t h e a m i n o a c i d l e v e l( p o l y m e r a s e , 3 0 K a n d / o r c o a t p r o t e i n s ) o r a t t h en u c l e o t i d e l e v e l ( 3 ' n o n - c o d i n g r e g i o n ) i s u n d e r c u r r e n ts tu d y .

T h e a u t h o r s t h a n k M . V . L a f i t a fo r t y p in g t h e m a n u s c r i p t . B . W . a n dE . A . w e r e s u p p o r te d b y f e l lo w s h i p s f r o m M E C a n d F u n d a c i b n R a m 6 nA reces , re sp ec t i v e l y . Th e w o rk w as su p p o r t ed b y g ran t s f ro mP L A N I C Y T ( A G R 8 8 -0 0 8 2) a n d F u n d a c i 6 n R a m 6 n A r e c e s.

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(Received 3 June 1991; A ccepted 20 Augu st 1991)

nucleotide sequence of the genomic rna of pepper mottle virus

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