Journal of General Virology(1995), 76, 939-949. Printedin Great Britain
939
Sequence polymorphism in the 5'NTR and in the P1 coding region of potato virus Y genomic RNA V. Marie-Jeanne Tordo, 1 A. M . Chachulska, 2 H. Fakhfakh, 3 M . Le Romancer, 4 C. Robaglia 4 and S. Astier-Manifacier 1. 1Laboratoire de Pathologie Vdgrtale, INRA, Route de St-Cyr, 78026 Versailles Cedex, France, 2Institute of Biochemistry and Biophysics, UI. Pawinskiego 5A, 02-106 Warszawa, Poland, 3Laboratoire de Grn~tique, Universit~ de Tunis, Campus universitaire, 1060 Tunis-Belv~dbre, Tunisia and 4 Laboratoire de Biologie cellulaire, INRA, Route de St-Cyr, 78026 Versailles Cedex, France
Potato virus Y (PVY) the type member of the genus Potyvirus, occurs wodd-wide as isolates which differ in host range and the type of symptoms caused. The sequences of a 5' segment of viral RNA overlapping the 5' non-translated region (5'NTR) alone (ten isolates) or the 5'NTR and the adjacent P1 coding region (eight isolates) were established. These data were used to quantify the polymorphism in the Y-terminal part of the PVY genome. Nucleotide sequence identity between isolates ranged from 66-100 % in the 5'NTR and from 70-100% in the P1 coding region. The lowest amino acid sequence similarity between PVY P1 was 77%, illustrating the high variability of this protein in the PVY species. Phylogenetic trees based on either 5'NTR or P1
sequence analyses resulted in the same clustering of the studied isolates into three groups. Group I comprises potato isolates all inducing 'tobacco veinal necrosis' symptoms. Group II contains isolates inducing either 'tobacco veinal necrosis' or mosaic symptoms in tobacco. Group III contains mainly pepper or tomato isolates inducing mosaic symptoms in tobacco and shows a geographical clustering of the Tunisian isolates. This clustering into three groups is discussed in comparison with phylogenetic trees previously obtained from capsid gene or 3'NTR sequence analysis in the PVY species. Multiple sequence alignment indicated conserved motifs potentially involved in viral functions.
Introduction
Nicolas & Lalibertr, 1992; Yeh et al., 1992; Jayaram et al., 1992; Vance et al., 1992; Thole et al., 1993; Palkovics et al., 1993). A single ORF is translated into a polyprotein of more than 3000 amino acids, which is then proteolytically processed by three viral proteases into functional proteins (Carrington & Dougherty, 1987; Carrington et al., 1989; Verchot et al., 1991). Protein sequence alignment of polyproteins from different potyviruses shows that the first protein, the third protein and the Nterminal region of the capsid protein are the most variable regions among the potyviral polyproteins (Shukla et al., 1991). The genus Potyvirus is the largest group of plant viruses, with more than 180 members or possible members (Brunt, 1992). Due to its large size, the taxonomy of this group was not resolved until sequence data, and particularly capsid sequence data, clarified the classification. There is a bimodal distribution of coat protein sequence similarity, with known distinct potyviruses ranging in sequence similarity between 38-71% and known strains of given viruses ranging between 90-99% (Shukla & Ward, 1988). D. D. Shukla and coworkers have shown that comparative sequence analysis
Potato virus Y (PVY) is the type member of the genus Potyvirus in the family Potyviridae; the latter belongs to the picorna-like supergroup comprising animal and plant viruses. Members of this family share similarities in their genome structure, gene arrangement, and mode of protein and R N A synthesis (Atreya, 1992). All of them induce the formation of viral inclusion bodies ('pinwheels') in the cytoplasm of infected cells (Edwardson et al., 1984), the presence of which is a taxonomic criterion (De Bokx & Huttinga, 1981). The flexuous particles contain a single-stranded messenger RNA of about 10000 nucleotides with a viral protein covalently linked to the 5' end and a poly(A) tail at the 3' end (for a review see Riechmann et al., 1992). The complete nucleotide sequence of several members of the group is already known (Allison et al., 1986; Domier et al., 1986; Robaglia et al., 1989; Lain et al., 1989; Maiss et al., 1989; Teycheney et al., 1989; Johansen et al., 1991 ; * Author for correspondence. Fax +33 1 30 83 31 95. e-mail
[email protected] 0001-2803 © 1995SGM
940
V. M . - J . Tordo and others
of whole coat protein sequences can produce plausible phylogenetic trees of potyviruses (Rybicki & Shukla, 1992). Moreover, molecular phylogeny based upon coat protein sequences has revealed subgroupings of potyviruses. One of these is the ' P V Y subgroup' which contains PVY and pepper mottle virus (PepMoV) (Barnett, 1992). PVY can infect tobacco, potato, pepper and tomato as well as wild species, especially those in the Solanaceae. Serological data obtained with coat protein polyclonal antibodies show a single serotype comprising isolates inducing different diseases. Potato isolates have been separated into three main strains according to the symptoms they induce on potato and tobacco (De Bokx & Huttinga, 1981). The term ' s t r a i n ' defines a collection o f isolates causing the same symptoms (Barnett, 1992). The PVY N strain induces typical ' t o b a c c o veinal necrosis' of Nicotiana tabacum whereas the PVY ° strain induces a non-necrotic mosaic on this host. Some PVY N isolates are the sole cause of potato tuber necrotic disease (Le R o m a n c e r & Kerlan, 1991); these isolates share biological properties that distinguish them from classical PVY N isolates (Le R o m a n c e r et al., 1994). They are referred to as PVY ~TN isolates [ N T N = isolates belonging to the necrotic group (N) of PVY and inducing tuber necrosis (TN)], according to a decision of the European Association of Potato Research Virology Section. The y c strain comprises PVY isolates inducing ' stipple streak' symptoms of potato cultivars bearing the N c gene. In Nicotiana tabacum, these isolates induce symptoms similar to those of the y o strain. This classification of potato isolates cannot be easily extended to other PVY isolates, for which other criteria are used. Tobacco isolates of PVY have been classified in relation to the symptoms they induce on flue-cured tobacco
cultivars resistant or susceptible to the root-knot nematode (Gooding & Tolin, 1973). Strain N N causes necrotic symptoms on both cultivars (Sudarsono et al., 1993). Pepper, tomato and Solanum nigrum isolates have been classified into different pathotypes of increased virulence in pepper cultivars bearing resistant genes ( G r b r r Srlassi~ et al., 1985). Nucleotide sequence analysis of isolates PVY-Fr (Robaglia et al., 1989) and P V Y - H (Thole et al., 1993), which have been completely sequenced, shows that the degree of sequence similarity differs across the genome. The overall nucleotide identity between these two isolates is 88"5 % while the 5' non-translated region (5"NTR) and the adjacent P1 coding region have only 70.3% and 72.6% identity, respectively. C o m p a r e d to the 90"6% nucleotide identity for the capsid gene and to the 80.6-98.3% identity for domains of non-structural proteins, this shows that the Y-terminal segment is the most variable region of the PVY genome. We have thus chosen to quantify sequence polymorphism of PVY isolates in this particular region. As 5 ' N T R and P1 coding region sequences were available for only four PVY isolates (Robaglia et al., 1989; Thole et al., 1993; Thornbury et al., 1990), we determined the nucleotide sequence of the 5 ' N T R for ten PVY isolates and of the P1 coding region for eight isolates. Analysis o f these data allowed a clustering of PVY isolates and highlighted nucleic acid and amino acid motifs which could have functional significance in the 5 ' N T R and in the P1 coding region.
Methods Isolates. The acronyms, geographical origin, references and symptoms caused on Nicotiana tabacum by the PVY isolates studied in this paper are shown in Table 1. We have intentionally used the terms
Table 1. P V Y isolates studied in this paper Virus strain PVY-Lba PVY-H° PVY-Nyc PVY-Wic PVY-Fra PVY-Lw~ PVY-Th* PVY-TuI PVCe PVY-NC78g PVY-LYE84h PVY-SON41~ PVY-PU PVY-P211
Host Potato cv. Lola Potato Potato cv. Nysa Potato cv. Wilga Potato Potato cv. Lipinski Wczesny Potato cv. King Edward Potato Potato Tobacco Tomato Solanumnigrum
Pepper Pepper
Country Lebanon Hungary Poland Poland France Poland England Tunisia Southeastern USA Canary Islands South of France Tunisia Tunisia
Biological Veinal necrosis in classification Nicotiana tabacum PVYsrN PVYsrN PVYs PVYN PVY~ PVY° PVY° PVY° PVC PVY-NN PVY-0 PVY-1-2 PVY-0 PVY-0
+ + + + + + -
* Referencesare givenfor each isolate: a, Le Romancer & Kerlan (1991); b, Thole et al. (1993); c, Chrzanowska (1991); d, Robaglia et al. (1989); e, Thornbury et al. (1990);f, this work; g, Gooding & Tolin (1973); h, K. Grbr6 Srlassi6 (personal communication); i, Grbr6 Srlassi6 et al. (1985).
941
Sequence polymorphism in potato virus Y RNA 50
I
P/Y-H "PVY-Lb "PVY-Ny *PVY-NCTII "PW-Wi *PVY-LW PVY-Fr *PVY-LYE84 PVY-Th
AAAUUAAAAC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . AAUUAAAAC . . . . . . . . .
. . . . .
. .
. AAUUAAAAC
. . . . . . . . . ...... . . .
"PVY-P21
"PVY-Tu "PVY-P2 "PrY-SON41
Corm
. . . . .
AACUCAAUAC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . AACUCAAUAC . . . . . . . .
AACAUAAGAA ACAUAAGAA ACAUACGAA ACAUAAGAA ACAUAAGAA ACAUAAGAA AACAUAAGAA
AAU AAU AAU AAA AAA AAA AAA
ACAUAAGAA AACAUAAGAA
AAA AAA
ACAUAAGAA ACAUAAGAA ACAUAAGAA AACAUAAGAA . . ACAUAAGAA
AAA AAA AAA AAA AAA
. . . . .
. . . . .
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AACUCAAUAC
. . . . . . . . . . . . . . . . . . . . . AAAC . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . AACUCAAUAC . . . . . . . . .
. . . . . .
A
aACAUAaGA
aacucaauac
aauuaaaac
CUCA CUCA CUCA CUCA CUCA CUCA CUCA CUCA CUCG CUUA CUUA CUUA CUUA CUUA
AAaCAACGCA
AAAACACUc
UGUUAAGUUU UGUUAAGUUU UGUUAAGUUU UGCUAAGUUU UGCUAAGUUU UGCUAAGUUU UGCUAAGUUU UGCUAAGUUU UGCUAAGUCU UGCUAAAUUU UGCUAAAUUU UGCUAAAUUU UGCUAAAUUU UUCUAAGUUU
CAAUUUCGAU CA!AUUUCGAU CAiACUUUGAU CA G U U U A A A U CA G U U U A A A U CJ GUUUAAAU CA G U U U A A A U CA G U U U A A A U CA G U U U A A A U CA G C U U U G A U CA G C U U U G A U CA G C U U U G A U CA ~ C U U U U G U CA ~ C U U U A A U
UgcUAAglUuU
CAIguUU
100
51
PVY-H 'PVY-Lb "PVY-Ny "PVY-NC78 *PVY-Wi *PVY-LW PVY-Fr "PVY-LYE84 PVY-Th °PVY-P21 "PVY-Tu "PVY-P2 I=VC
CAIACUCUAAU CAIACUCUAAU CAIAUUCUAAC AUUCUCAC AUUCUCAC AUUCUCAC AUUCUCAC AUUCUCAC AUUCUCAC AUUUUCAC AUUUUCAC AUUUUCAC AUUUUCAC AUUUUUAC
CAAAAGCIUUU CAAAAGCUUU CAAAAGCUUU UAAACGCUU UAAACGCUU UAAACGCUU UAAACGCUC UAAACGCUC UAAACGCUU CAAACACUC CAAACACUC CAAACACUU CAAACGCUC CAAACGCUC
"PVY-SON41
AAAc
Consensus
g CUu.
. , Au UCUc Ac
UCAAg CAAc U
101
CUUC •P ~ - L b
U
cucluuu
C U U C A ~U" ~ ~ ~:~~ ! ; ~~~ ~
°PVY-Ny •PVY-NC7B "PVY-Wi "PW-LW PVY-Fr "PVY-LYE84
CUUCGUCGUA CAUUUCCUUG CAUUUCCUUG CAUUUCeUUG CAUUUCCUUG CAUUUCCUUG CAUUCCAUUG CUUUUUCUUG CUUUUUCUUG CUUUUUCUUG
PVY-~ "PV~P21 "PVY-Tu "PV~P2 PVC °P~-SON41 Consensus
CAIAI-0--U --C--ZlQu
UClUUU
CAIAlUUUCAIGU
CAAACCG]UUU CAAUUCUCUU CAAUUCUCUU CAAUUCUCUU CAAUUCUCUA CAAUUCUCUA CAAUUCUCUU CGAUUCUCUU CGAUUCUCUU CGAUUCUCUU
CAIACUUCAGU AAACAAUAUU AAACAAUAUU AAACAAUAUU GAACAAUAUU GAACAAUAUU AAACAAUAUU UAACGAUAUU UAACGAUAUU UAACGAUAUU UAACGAUAUU UAACGAUAUU
"
'~ C
CUUUUUCUUG
CGAUUCUCUU
CUUUUCUUUG
CCAUUCUCUU
Cu
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Au u c uc
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AAc
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151
PVYM "PVY-~ °PVY-Ny "PVY-NC78 "PW-Wi "PW-LW PVY4r PVV-~ "PW-P21 •PW-Tu "PVY-~ PVC •PVY-SON4t Consensus
a
aU
GUAAGCUAUC ui~ OUA
QCUAUC
GUAAGUUCUC
GGAAACCAIUU
GGAAACCAIUU GGAAACCAIUU GGAAACCAIUU GGAAACCAIUU GGAAACCAIUU GAAAACCGUC
150 GUAAUUCAGU GUAAUUCAGU GUAACUCAAU U C A AIC U
u c • *1c' u c A Ale u~ u c A Ale u! u c A AIC
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ACUCUCUC AAGUAGCAAC U C A C U U U : ~ ~ U U U C U C A C U U U ~ U U U C U C A C U U U ~ ~ U U U C U C A C U U C ~ ~ U U U C
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AGAUCCUCAAUG AGAUCCUCAAUGJ AGAUCCUCAAUG
¢
AGAUCCUCAAUG AUAUUCUCCAMG AUAUUCUCCAUG AUAUUCUCCAMG AUAUUCUCCAUG
AUAUUCACCAUG AgAuccuC¢AUG
Fig. 1. Multiple sequence alignment of the PVY 5'NTR. Isolates labelled with an asterisk were sequenced in this work. The others have been published previously: PVY-Fr (Robaglia et al., 1989), PVY-I-I (Thole et al., 1993), PVY-Th and PVC (Thornbury et al., 1990). Undetermined nucleotides at the site of primer annealing are indicated by dashes. The dots indicate gaps inserted into the sequences to improve sequence alignment. The AUG initiation codons are in italics. Noteworthy motifs are on a grey background (CAA) or are boxed (UUUCA). A consensus sequence is presented in the last line. Nucleotides conserved between all the strains are in upper-case letters.
942
V. M . - J . T o r d o a n d o t h e r s
Fig. 2. Percentage nucleotide sequence identities between the 5'NTR (above diagonal) of 14 PVY isolates. Percentage P1 nucleotide sequence identities (below diagonal, in italics, in bottom-right of each box) and P 1 amino acid sequence similarities (below diagonal, in upper-left of each box) of 12 PVY isolates. Structural similarity groups were defined according to Bordo & Argos (1991). PepMoVC has been added for comparison. Isolates labelled with an asterisk were sequenced in this work.
PVY °, PVY N and PVY c only for potato isolates. However, it is easy to determine for any PVY isolate whether or not it induces veinal necrosis in Nicotiana tabacum and subsequently to refer to ' N ' - or 'O'-type symptomatology restricted to this particular host. Four previously published 5'NTR and P1 coding sequences were used for multiple sequence alignment: PVY-Fr (Robaglia et al., 1989), PVY-H (Thole et al., 1993), and PVC and PVY-Th (Thornbury et al., 1990). Virus maintenance, purification and RNA extraction. Viruses were propagated in Nicotiana tabacum var. Xanthi. Virus purification was as described by Dougherty & Hiebert (1980) with some modifications. The PEG precipitate was resuspended in 20 mM-HEPES pH 7-5 with a tissue grinder, clarified and ultracentrifuged (2 h at l l0000g). The resuspended pellet was subjected to centrifugation on a caesium sulphate gradient (360 mg/ml in 20 mM-HEPES pH 7.5), in an SW 60.1 Beckman rotor at 34000 r.p.m, for 16 h at 15 °C. Viral RNA was extracted by treating resuspended virus with 1% SDS-1 mg/ml proteinase K, followed by phenol-chloroform extraction and ethanol precipitation.
On the PVY-Fr genome, the P1 coding region ends at nucleotide 1036 (Mavankal & Rhoads, 1991 ; Robaglia et al., 1989). First-strand cDNAs were synthesized with avian myeloblastosis virus reverse transcriptase (Boehringer), using purified viral RNA as the template, and three different 3" oligonucleotides depending on the isolates. For isolate PVY-Lb, the reaction was primed with oligonucleotide no. 1 (5' TTTTGGATCCTCACTGAGTAACTCTAGAAC 3') which is complementary to nucleotides 994-1010 of the PVY-Fr genome (nucleotides not complementary to the viral sequence are underlined). For isolates PVY-Ny, PVY-NC78, PVY-Wi, PVY-LW, PVY-LYE84 and PVY-SON41, the reactions were primed with oligonucleotide no. 2 (5' TTGGATCCCTAAAACTGGATCATTGATTTAAA 3') which is complementary to nucleotides 1015~1036 of the PVY-Fr genome. For the three Tunisian isolates, the reactions were primed with oligonucleotide no. 3 (5' CGAATTCAAACCATCTCTTGC 3') which is complementary to nucleotides 1262-1279 of the PVY-Fr genome. The cDNAs were amplified using Taq polymerase (Promega), one of the three 3' primers described above, and a 5' primer corresponding to the 5' terminal 20 nucleotides of the PVY-Fr genome
Sequence polymorphism in potato virus Y R N A
(a) --
PVY-H
(b)
943
PVY-H 100
PVY-Lb
100
PVY-Ny
PVY-Ny ,- PVY-LW PVY-NN 100 57
, PVY-Wi
PVY-Wi PVY-NN
65
PVY-LW
100 ,. PVY-Th
PVY-Th
98
' PVY-Fr
PVY-LYE84 55 -
-
PVY-Fr
PVY-P2
79 PVY-P21 100 90
PVY-Tu 74
72
PVY-Tu
PVY-P2 91
65
" PVY-P21
PVC
PVC PVY-SON41
PVY-SON41
PepMov-C
PepMov-C
Fig. 3. Phylogenetic trees obtained with PAUP 3.1 based on 5'NTR sequences (a) or Pl-coding region amino acid sequences (b). Numbers above the branches indicate the percentages of bootstrap analyses which support the grouping at each node.
(5" TTTTGGATCCAATTAAAACAACTCAATACA 3"). These oligonucleotides amplified the 5'NTR and the entire P1 coding region, except for isolate PVY-Lb, where the last nine amino acids are missing. PCR was done using a thermocycler (Hybaid) programmed for 31 cycles: two cycles of 94 °C for 1 min, 37 °C for 1 min and 72 °C for 3 min, 28 cyclesof 94 °C for 1 min, 50 °C for 1 min and 72 °C for 3 min, and one final cycle of 72 °C for 10 min. The PCR fragments were cloned into the plasmid vector pBluescript (Stratagene) using the procedure of Marchuk et al. (1990). Alternatively, the Pharmacia Sure-Cloning kit with Sinai-cut pUC18 vector was used.
Nucleic acid sequencing. PCR-derived clones were used to determine the nucleotide sequence of the 5'NTR and P1 cistron. All sequences were read on both strands, in one or severalclones. Sequential deletions were performed using Exonuclease III followed by mung bean nuclease treatment according to the method described by Steggles (1989), or with S1 nuclease (Promega). Nucleotide sequences were determined by the dideoxynucleotide chain termination method (Sanger et al., 1977) using an Applied Biosystems DNA Sequencer 370A with dye primers (Universal-21M13 and M13 reverse primers from Applied Biosystems) and either Taq polymerase (Promega) or T7 DNA polymerase (USB), or alternatively a Pharmacia ALF DNA Sequencer with fluorescent primers and T7 DNA polymerase (Pharmacia).
Sequence analysis. Sequence data were analysed by two methods: a distance matrix analysis, UPGMA (unweighted pair group method using arithmetic average) using the PileUp program from the Genetics Computer Group (Devereux et aL, 1984), and a maximum parsimony method, with the program PAUP (Phylogenetic Analysis Using Parsimony) version 3.1 (Swofford, 1992).
Results 5"NTR sequence comparisons I n order to evaluate sequence p o l y m o r p h i s m in the 5"NTR, detailed sequence c o m p a r i s o n s were m a d e u s i n g o u r results a n d previously reported sequence data. The multiple 5 ' N T R sequence a l i g n m e n t o b t a i n e d with P i l e U p is presented in Fig. 1. The length of the 5 ' N T R o f different P V Y isolates shows little variation. Two insertions, one o f three nucleotides at position 60 a n d the other o f one nucleotide at p o s i t i o n 162 were observed in three isolates: P V Y - H , P V Y - L b a n d PVYNy. The v a r i a t i o n s are n o t u n i f o r m l y distributed
944
V. M.-J. Tordo and others
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Fig. 4. Multipleamino acid sequencealignmentof PVY P1 protein. Isolateslabelledwith an asterisk have been sequencedin this work. The consensus sequencewas obtained with the PRETTY program from GCG. Dots indicate residues identical to the consensus and lower-case letters indicate residues which differ from the consensus sequence.The alternation of acidic and basic amino acid residues and the 'LFIVRG' motif have been boxed. Residues forming the proteolyticdomain of PI are indicated by an arrowhead. At the 3' end of the sequences of some isolates, dashes indicate undetermined amino acids correspondingto the 3' amplificationprimer. throughout the 5'NTR, being more abundant in the second half of the sequences. PileUp analysis provided a pairwise comparison of the PVY 5 ' N T R sequences. Fig. 2 presents the pairwise percentage nucleotide identities. The comparisons begin at the nucleotide where all the strains can be aligned; the sequence of the amplification primer has not been taken into account. PVY isolates appear to be clustered in three groups designated I, II and III. The intragroup identity is greater than 83 % in group I, greater than 94 % in group II and greater than 86 % in group III; the intergroup identity ranges from 65-81%. In contrast, the sequence identity of the 5 ' N T R comparing PepMoV-C (Vance et al., 1992) and any isolate of PVY is about 40 % (Fig. 2). This value clearly indicates the limit of the PVY species. Analysis of the matrix of aligned sequences with P A U P resulted in three most parsimonious trees with minor differences. Trees were rooted by including PepMoV-C as an outgroup. A bootstrap 50 % majorityrule analysis enabled us to define a consensus tree and to associate a percentage occurrence of each node, as shown in Fig. 3 (a). This consensus tree represents graphically the clustering presented in Fig. 2, and provides statistical support to the definition of groups I, II and III.
P1 sequence comparisons Multiple sequence alignments were obtained using PileUp, with either P1 nucleotide (data not shown) or amino acid sequences (Fig. 4). PVY P1 is 284 amino acids long, taking into account the cleavage site determined on tobacco vein mottling virus (TVMV) and as deduced from multiple alignment of other potyviral polyproteins (Mavankal & Roads, 1991). The percentage nucleotide sequence identities and the percentage amino acid similarities for each pair of PVY isolates studied in the P1 coding region are presented in Fig. 2. Differences
were observed in the PVY-Wi sequence at amino acid position 102 (E in one of three clones). The isolates can be classified into three groups according to the global identity or similarity of their sequences. These groups are the same as those previously defined with the 5 ' N T R sequences. The nucleotide sequence identity is 91% in group I; in group II it ranges from 95-100% and in group III it ranges from 89-99-8%. The intergroup sequence identity is about 70% between group I and either group II or group III, and is about 80 % between groups II and III. The percentage amino acid sequence similarity is higher than the percentage nucleotide identity in each group. At the nucleotide level, the percentage sequence identities are in the same range for either the 5'NTR or the P1 coding region. Although the percentage similarities are higher at the amino acid level, this study further demonstrates that P1 is a highly variable protein encoded by the PVY genome. Multiple sequence alignments were used as entry data matrices for the P A U P program. The dendrogram deduced from the PileUp alignment (data not shown) and the trees produced by P A U P have the same pattern. PepMoV-C was used to define the outgroup of the PVY cluster. Fig. 3 (b) shows the bootstrap 50 % majority rule consensus tree obtained with PAUP. It must be emphasized that the clustering obtained with P1 sequences is the same as the one obtained with the contiguous 5'NTR, at least for the 12 isolates sequenced in both regions. Group I is the most genetically distant from the other two. This group includes two isolates inducing tuber necrosis that have identical 5 ' N T R sequences and a reference isolate from Poland, PVY-Ny. As already mentioned, these three isolates have two insertions in common in their 5'NTR. PVY isolates inducing tuber necrosis have been described relatively recently (Beczner et al., 1984; Weidemann, 1985; Le Romancer & Kerlan, 1991); their recent emergence as well as tuber exchange can explain their identical 5 ' N T R sequences.
946
V. M.-J. Tordo and others
Groups II and III are more closely related to each other. Group II isolates have the highest intragroup sequence identities. It is the largest group, containing isolates from different hosts (tobacco, tomato and potato) and from distant geographical regions. It includes potato isolates belonging either to the PVY ~ (PVY-Fr, PVY-Wi) or to the PVY ° (PVY-LW, PVY-Th) strains. The PVY-Fr isolate has been described as belonging unambiguously to the PVY ~ strain (PVY ring test carried out in Braunschweig, Germany, 1991). However, Van der Vlugt et al. (1993) found that it clustered together with PVY ° isolates on the basis of its capsid sequence. The tobacco isolate PVY-NC78 also belongs to group II and causes typical 'tobacco veinal necrosis' symptoms (McDonald & Kristjansson, 1993). In Group III, the three Tunisian isolates, originally isolated from pepper (PVY-P21 and PVY-P2) and from potato (PVY-Tu) form a tight cluster, indicating a common origin. Conserved motifs and structures within the P V Y 5 ' N T R
The genomic RNA of potyviruses functions as messenger RNA. No clear mechanism has been described for the translation initiation process of this RNA, which is devoid of a cap structure and is covalently linked to a viral protein (VPg) at its 5' extremity. Translational enhancement mediated by a potyviral 5'NTR has been described for the tobacco etch virus (TEV) (Carrington & Freed, 1990), for the pea seed-borne mosaic virus (Nicolaisen et al., 1992) and for PVY-Fr (Mazier et al., 1995). This effect on translation was found to be equivalent to the enhancing effect mediated by the tobacco mosaic virus (TMV) ~ ' sequence, a wellcharacterized translational enhancer (Gallic et al., 1987). The multiple alignment of PVY 5'NTR sequences revealed the presence of conserved and repeated motifs. These motifs have already been identified in other viral leader sequences, where in some cases their functional significance has been established. It has been shown (Gallic & Walbot, 1992) that the core regulatory element of the translational enhancement conferred by the TVM leader, ~', consists of a direct repeat motif together with a (CAA), motif of 25 nucleotides. Moreover, specific binding of a wheat germ protein factor to fl' and specifically to the (CAA), motif has been recorded; this association could mediate the translational enhancement associated with f~' (Gallic, 1993). Duplicate or triplicate CAA triplets with one or two nucleotides inserted between this motif are present throughout the 5'NTR of PVY isolates (Fig. 1). This kind of motif is also present in all other known potyviral 5'NTR sequences. The CAA motifs present in the first 80 nucleotides of PVY RNA are strictly conserved in all the
isolates and are gathered in two blocks (Fig. 1). They are located in positions 3~46 and 72-78. In the second half of the 5'NTR sequence, the distribution of CAA motifs depends on the group of isolates. U U U C A (or UUUCC) are conserved motifs within animal picornavirus leader sequences. These motifs, located 20-24 nucleotides upstream of an AUG, are necessary for efficient translation initiation (Pilipenko et al., 1992). Levis et al. (1992) showed that hybridization with an antisense oligonucleotide overlapping the U U U C A motif located 28 nucleotides upstream from the A U G initiation codon on the PVY-Fr R N A results in a 75 % decrease in translation of a reporter gene. This U U U C A motif is almost conserved in all the PVY isolates sequenced to date. This motif was sought in the multiple sequence alignment presented in Fig. 1. Two U U U C A motifs are highly conserved among all the PVY isolates: they are located at positions 88-92 and 157-161, the latter being changed to UUCCA in the three Tunisian isolates which have another U U U C A motif at position 167-171. The extreme 5' segment contains the motif 'ACAACAU', termed 'box A' by Turpen (1989) and present in the 5'NTR of most potyviral RNAs. A stem-loop structure similar to the folding predicted for the 5'NTR of turnip mosaic virus (Nicolas & Lalibertr, 1992) can be found within the first 150 nucleotides of all PVY isolates (data not shown). Further work is necessary to investigate the relevance of these nucleic acid motifs and structures in the functioning of potyvirus 5'NTRs. Amino acid motifs in the P1 protein
In infected cells, genomic potyviral R N A is translated to give a polyprotein which is further processed by viral proteases resulting in at least eight mature products. The first two proteases to be identified were NIa (Carrington & Dougherty, 1987; Hellmann et al., 1988) and the helper component (Carrington et al., 1989). P1 is the third viral protease and catalyses cleavage at its own C terminus. By site-directed mutagenesis, amino acids involved in the autoproteolytic activity of TEV P1 have been determined (Verchot et al., 1991, 1992). It appears that P1 proteinase contains an active site similar to that of serine-type proteinases. The identity and positions of the catalytic residues of PVY-Fr as deduced from the multiple potyviral P1 sequence alignment are H (192), D (201), S (235) and D (268). These residues and the FIVRG motif at position 256-260, whose involvement in the proteolytic activity of TEV P1 has been established, are strictly conserved between all the PVY isolates (Fig.
4). P1 is a very basic protein with a high isoelectric point (pI = 10-9 for PVY-Fr); an RNA-binding activity has been demonstrated for the P1 protein of TVMV
Sequence polymorphism in potato virus Y R N A
(Brantley & Hunt, 1993). Sequence motifs common to many RNA-binding proteins have been identified (for a review see Tan et al., 1993). The LFIVRG motif (amino acids 255-260) is similar to the RNP2 motif found in the RNP-CS-type binding domain (Bandzilius et al., 1989). Some proteins containing this motif interact with RNA during pre-mRNA processing. A short sequence of ten amino acids located at position 80-90 is an alternation of acidic and basic residues (KKREEREREE) and occurs in an a-helix region [according to the prediction of Chou & Fasman (1978) - data not shown]. It recalls the heptad periodicity of basic and acidic residues in the nucleocapsid VP2 of rotavirus (Ernst & Duhl, 1989). In the VP2 protein, these residues occur over adjacent turns of a helical structure that includes a double-stranded RNA binding domain (McCormack et al. 1992).
Discussion In order to improve understanding of genetic diversity within the PVY species, we determined the sequences of the 5'NTR (ten isolates) and the P1 coding region (eight isolates) of PVY. The lowest percentage sequence identity between two PVY isolates in these two regions is about 70 %, illustrating the high degree of sequence polymorphism in the Y-terminal part of the PVY genome. However, a comparative analysis of four plum pox virus sequences (Palkovics et al., 1993) has shown that the non-coding regions and the P1 coding region exhibit 6"6 % change while the overall sequence diversity between isolates is 11%. Thus a high degree of sequence variability within the N-terminal protein is not a general feature in the genus potyvirus. Previous sequence analysis of the capsid protein gene of 21 PVY isolates (among which PVY-Fr and PVY-H are common with our study) indicates a subdivision of the isolates into two groups, in agreement with the historical classification into y o and yN strains based on biological criteria (Van der Vlugt et al., 1993). In contrast, sequence comparison of the 5'NTR (14 isolates) and of the P1 coding region (12 isolates) resulted in a clustering of PVY isolates into three groups. Group I contains only potato isolates inducing 'tobacco veinal necrosis', including PVY ~TN isolates. Group II contains isolates that cause PVY °- or PVYN-type symptoms on tobacco. This indicates a subdivision of isolates inducing 'tobacco veinal necrosis' into two phylogenetically distinct groups. PVY-Fr, which belongs to group II, has been classified by Van der Vlugt et al. (1993) in the PVY ° group according to its capsid sequence, and in the PVY N group according to its 3'NTR sequence. Group III contains all the pepper isolates, the Solanum nigrum isolate and two potato isolates, all showing 'O-type' symptoms on tobacco. Geographical clustering
947
associated with the Tunisian isolates can be observed within this group, suggesting a recent common origin. Using the 3'NTR as a taxonomic criterion, Van der Vlugt (1993) found that the 'N-type' PVY isolates in his study clustered into the same group. In contrast, the PVY 'N-type' isolates studied in this paper are found either in group I or in group II. In particular, the necrotic isolate PVY-Fr was found in our study to belong to group II whereas PVY-H belongs to group I; these two isolates are clearly classified together based on their 3'NTR sequences. This observation raises the question of potential recombination events involved in the PVY species. An example of RNA recombination as an evolutionary factor has been described for another potyvirus (Cervera et al., 1993). The similar phylogenetic trees obtained for the 5'NTR and the P1 region suggest, however, that these two adjacent regions have evolved in a coordinate manner. M. Chrzanowska, K. Grbrr-Srlassir, G. Marchoux, C. Kerlan and G. V. Gooding are gratefully acknowledged for providing isolates of PVY. We also thank H. Willigsecker for greenhouse assistance; H. Chiapello and C. Drevet for help using computer programs; Y. Drouaud and T. Desprez for running sequencing gels, Y. Brygoo and M. Tepfer for helpful discussion, and C. Young and P. Tordo for useful comments on the manuscript. V. Marie-Jeanne was a recipient of fellowship from the Ministbre de la Recherche et de la Technologie, A. Chachulska was a recipient of a fellowship from a joint program of the Centre National de la Recherche Scientifique and the Institute of Biochemistry and Biophysics in Warszava, and the EEC program 'Capital Humain et Mobilit4'. H. Fakhfakh was a recipient of a joint program of the Centre National de la Recherche Scientifique and the University of Tunis.
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(Received 27 July 1994; Accepted 25 November 1994)
