Cloning and sequence analysis of the coat protein genes of an Australian strain of peanut mottle and an Indonesian ‘blotch’ strain of peanut stripe potyviruses

-

ELSEVIER

V irus Research

Virus Research 31 (1994) 235-244

Cloning and sequence analysis of the coat protein genes of an Australian strain of peanut mottle and an Indonesian ‘blotch’ strain of peanut stripe potyviruses P.Y. Teycheney,

R.G. Dietzgen

*

Department of Primary Industries, Queensland Agricultural Biotechnology Centre, Gehrmann Laboratories, The University of Queensland, St. Lucia, Q4072, Australia

(Received 12 August 1993; revised 25 October 1993; accepted 1 November 1993)

Abstract We have analysed the coat protein gene sequences of two potyviruses infecting peanut. The 3’ terminal 1247 nucleotides (nt) of an Australian strain of peanut mottle virus (Pe~oV-A~) and the 3’ terminal 1388 nt of an Indonesian ‘biotch’ strain of peanut stripe virus (PStV-lb) were cloned and sequenced. Those regions included the 861 and 864 nt encoding the respective putative coat proteins as well as the 285 and 253 nt, respectively of 3’ non-coding sequences. Comparison of the nucleotide sequences of PeMoV-AU and PStV-Ib revealed a sequence similarity of 64.4% for the coat protein gene and 34.6% for the 3’ non-coding region. The deduced amino acid sequences of PeMoV-AU and PStV-Ib coat proteins are 66.7% identical. These rest&s provide further evidence that PeMoV and PStV are distinct viruses. Comparisons of the 3’ terminal sequences of PeMoV-AU and PStV-Ib with those of the genomic RNA of other strains of PeMoV and PStV and with other potyviruses are discussed. Key words: Coat protein; Potyvirus; Nucleotide sequence comparison

1. Introduction Peanut mottle virus (PeMoV) and peanut stripe virus (PStV) are important pathogens of peanut (Arae& ~y~og~e~), PeMoV occurs world~de while PStV occurs mainly in China, South-East Asia and India, and appears to have been introduced into the USA and Africa through peanut germplasm exchange (Demski

* Corresponding

author. Fax: 617-365 4980.

0168-1702/94/$07.~ 0 1994 Etsevier Science B.V. Alf rights reserved SSDI 0168-1702(93)E0090-K

236

P. Y Teychenzy, R. G. Dietzgen / T/irus Research 31 (1994) 235-244

et al., 1984). PStV is considered to be a major threat for peanut breeding worldwide (Dollet and Dubern, 1991). Both viruses can infect species of the family Leguminoseae and have been reported to induce severe losses on soybean (Glycine max). PeMoV is also a major pathogen of pea (Pisum sativum) and bean (PhaseoZus vulgaris) in Australia. Symptoms of PeMoV and PStV on peanuts can be quite similar, which can make it difficult to differentiate between these two viruses. Different strains of PStV may cause a variety of symptoms including mosaic, blotches or stripes on infected leaves and dwarfism of whole plants (Wongkaew and Dollet, 1990). PeMoV generally induces a mild dark green mottle on infected leaves. Both viruses can reduce peanut production dramatically (Demski and Reddy, 1988) and may cause a severe necrosis leading to the death of infected plants. Both viruses belong to the family Potyviridae which represents one quarter of all plant viruses (Francki et al., 1991). Potyviruses are characterised by long, flexuous rod-shaped particles which contain a single-stranded, infectious and polyadenylated genomic RNA of ca 3 X lo6 Da. PeMoV coat protein has an apparent Mr of 32-36 kDa depending on strains (Sherwood, 1984; Rajeshwari et al., 1983) while PStV coat protein has an apparent Mr of 32 kDa (Demski et al., 1984). Recent data suggest that PStV is closely related to other potyviruses infecting legumes (McKern et al., 1992b) and that PeMoV and PStV may be strains of the same virus (Gunasinghe et al., 1992). However, nucleic acid hybridisation experiments involving different strains of PeMoV and PStV did not show the proposed close relationship between PeMoV and PStV (Bijaisoradat and Kuhn, 1988; R.G. Dietzgen, Z. Xu and P.Y. Teycheney, unpublished). To resolve this issue, we have determined the nucleotide sequence of the 3’ termini of the genomic RNAs of an Australian strain of PeMoV (PeMoV-AU) and of an Indonesian ‘blotch’ strain of PStV (PStV-Ib). These nucleotide sequences as well as the deduced amino acid sequences of the coat proteins have been compared to each other as well as to those of other potyviruses. Implications of the results on potyvirus classification are discussed.

2. Materials

and methods

2.1. virus strains, purification and RNA extraction

The Australian strain 133E of PeMoV (PeMoV-AU) originally isolated from peanut was passaged through single local lesions in beans (Phaseolus vulgaris, cv Kerman, J.E. Thomas, personal communication) and purified from infected bean as described by Demski et al. (1984). PStV-Ib was isolated from an infected peanut seed imported from Indonesia and was propagated in Nicotiana benthamiuna. Virus particles were purified using the protocol described by Demski et al. (1984) with minor modifications. These included the addition of 1% (v/v) Triton X-100 to the buffer used for the resuspension of the polyethylene glycol pellet and replacement of the final sucrose gradient step by a Cs,SO, density gradient centrifugation

P.Y Teycheney,R.G. Dietzgen/ virus Research31 (1994) 235-244

237

at 33 500 x g for 24 h. Genomic RNA was isolated from purified virus preparations by incubation in 0.1 mg/ml proteinase K and 0.1% SDS for 30 min at 37°C followed by three phenol-chloroform extractions and ethanol precipitation. 2.2. cDh!A synthesis, cloning and selection of recombinant clones Oligo(dT) primed cDNA was synthesized from total genomic RNA using the method described by Gubler and Hof~~n (1983) modified by Teycheney (1992). The cDNA molecules were blunt-ended by treatment with Sl nuclease (Boehringer Mannheim) and ligated into SmaI-digested and dephosphorylated pBluescript (Stratagene). Recombinant plasmids were used to transform the DHSa: strain of Escherichia coli (GIBCO BRL). Plasmid DNA from recombinant clones was screened by hybridisation with digoxygenin (DIG)-labeled oligo(dT),, (Pharmacia). Briefly, 100 ng of NaOH-denatured plasmid was spotted onto nitrocellulose membrane (~ersham) and UV cross-linked. The DIG-labeling of olig~dT)~s, prehybridisation, hybridisation and post-hybridisation washes of membranes were performed according to the manufacturer’s instructions (Boehringer Mannheim). Polyadenylated inserts were detected using the DIGTM nucleic acid detection kit (Boehringer Mannheim) by incubation with the chemiluminescent substrate Lumigen PPD and autoradiography for one hour. 2.3. DNA sequence dete~inution

and analysis

Recombinant clones containing the the 3’ terminus of PeMoV-AU or PStV-Ib genomic RNAs were sequenced by the dideoxy chain termination method of Sanger et al. (1977) using the Sequenase TM kit (USB). A set of unidirectional nested deletions were generated using the Erase-a-base kit (Promega). Five oligonucleotide primers were used to complete the full length sequence of PeMoV coat protein gene. Each 3’ terminal sequence was determined on both strands. A minimum of 10 overlapping clones (from subcloning and/or primer walking), having overall a two-fold redundant, were sequenced in each direction for both viruses. DNA and deduced protein sequences were compiled and analysed using MacVector 3.5TM and AssemblyLignTM software (IBI). Multiple DNA and protein sequence comparisons were performed using ClustalV, Pileup, Phylip and Treetool programs available through the Australian National Genomic Information Service (ANGIS). The 3’ terminal sequences of the following potyviruses were included in the comparisons: bean common mosaic virus (BCMV) strains NL-3 (H.J. Vetten, personal ~mmunication), NL-4, NL-8 (Vetten et al., 1992); mild strain (M) of PeMoV (U. Gunasinghe and B. Cassidy, personal communication); US ‘blotch’ (USb) isolate of PStV (Cassidy et al., 1993), US ‘stripe’ (USs) isolate of PStV (McKern et al., 1991), pea seed-borne mosaic virus (PSbMV; Bolger et al., 1990), passionfruit woodiness virus (PWV-K, Gough and Shukla, 1992), South African Passiflora Virus (SAPV; Brand et al., 1993); soybean mosaic virus (SMV) strains C (Chu, R., unpublished) and N (Eggenberger et al., 1989).

238

P.Y

3. Results

Teycheney, R.G. Dietzgen / Virus Research 31 (I 994) 235-244

and discussion

3.1. Nucleotide sequence of PeMoV genomic RNA 3’ terminus

A total of 147 recombinant clones were screened by hybridisation using a DIG-labeled oligo(dT) probe. Fifteen positive clones were detected and used to determine the sequence of 1247 nt at the 3’ terminus of PeMoV genomic RNA (Fig. 1). This sequence contained one long open reading frame (ORF) with no start codons as is typical of potyviruses (Riechmann et al., 1992). The ORF terminated

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UGG CGG CAU CAC ACG GAU C poly(A) C%C

1027 1084 1141 1198

1247

Fig. 1. Nucleotide sequence of the 3’ 1247 nucleotides of PeMoV-AU genomic RNA. The predicted amino acid sequence is shown below the nucleotide sequence. The putative cleavage site for coat protein maturation (A) and translational stop codons (*) are indicated.

P.Y. Teycheney, R.G. Dietzgen / Mrus Research 31 (1994) 235-244

239

in a UAG stop codon at position 962 immediately followed by a UAA in frame stop codon. A 285 nt 3’ non-coding sequence was followed by a poly(A) tail. The deduced amino acid sequence was analysed for the presence of a consensus polyprotein cleavage site for the maturation of the coat protein, common to all potyviruses (Riechmann et al., 1992). Three putative cleavage sites were found at positions 10 (Q/S>, 42 (Q/S) and 91 (Q/G) on the polyprotein sequence (Fig. 1). We assumed that the site present at position 42 is the proteolytic cleavage site of the coat protein since (i> it allows the maturation of a coat protein of calculated Mr 31.6 kDa as is consistent with the apparent Mr of PeMoV-AU coat protein on sodiumdodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE; 32 kDa, data not shown) and (ii) this site contains a V residue in -4 position which is conserved among several potyvirus coat protein cleavage sites (Teycheney, 1992). This strain of PeMoV can be expected to be non-transmissible by aphids, because the deduced amino acid sequence of the coat protein contained no DAG triplet in the N terminal region, a sequence which has been shown to be essential for aphid transmission of potyviruses (Atreya et al., 1991). The nucleotide sequence has been deposited in the EMBL nucleic acid database with accession number X73422. 3.2. Nucleotide sequence

qf PStV

genomic RNA 3’ terminus

Out of a total of 78 recombinant clones which were screened by hybridisation using a DIG-labeled oligo(dT1 probe, 38 positive clones were detected and used for sequencing. Subclones were generated from a 1.6 kbp 3’ terminal clone after treatment with exonuclease III, and used to complete the nucleotide sequence on both strands. The 3’ terminal 1388 nt and the deduced amino acid sequence are shown in Fig. 2. The nucleotide sequence contained one long uninterrupted ORF without a start codon, which terminated at position 1134 in a UAG stop codon followed by a second in-frame stop codon at position 1179. A 255 nt long 3’ non-coding region was followed by a poly(A) tail. Two putative Q/S polyprotein cleavage sites were found at positions 90 and 137, respectively. The first site is the most probable, since (i) it would permit the maturation of a coat protein of calculated Mr 32.1 kDa (vs 25.3 kDa for the other cleavage site), which is in good agreement with the Mr of 33.5 kDa observed by SDS-PAGE (Demski et al., 1988) and (ii) this site contains a V residue in -4 position. This strain of PStV can be expected to be aphid transmissible, because the deduced amino acid sequence of the coat protein contained a DAG triplet at position 102-104. The nucleotide sequence has been deposited in the EMBL nucleic acid database with accession number X21700. 3.3. Sequence comparisons between PStV and PeMoV The coat protein sequence of PStV-Ib was more than 98% identical to the respective sequences of two US isolates (Table 1) which cause blotch (PStV-USb, Cassidy et al., 1993) or stripe symptoms (PStV-USs, McKern et al., 1991). The 3’

240

P.‘u: Teycheney, RG. Di~tzgen / Kws Research 31 iI9!?4) 235-244 CAC AGA ACU GAA GCA Auc mu MHRTEAICAAMIEAWGYP

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284

ACA ACA CAA CCA CCA GUU GUG GAU CCU GGC GCG GAU ACU CCC AAG GAC AAG AAA GAA TTQPPVVDAGADTAKDKKE

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AAG AGC AAC AAA EGA AAA GGU CCU GAA AGC GGU GAA GGG UCA GCX AAC AALIAGU CGU KSNKGKGPESGEGSGNNSR

398 132

ACA GAA AAU CAA UCA AUG AGA GAC AAG GAU GUG AAU GCU GUJ UCA AAA GGA AAG T E N Q S M R I) K D" N A G S K G K

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Fig. 2. Nucleotide sequence of the 3’ 1388 nucleotides of PStV-Ib genomic RNA. The predicted amino acid sequence is shown below the nucleotide sequence, The putative cleavage site for coat protein maturation (A 1 and transfationat stop codons (*) are indicated.

non-translated sequences of the two blotch isolates were 89.8% identical. From these results the Indonesian blotch isolate appears to be a typical isolate of PStV. The PeMoV-AU and PStV-lb coat proteins shared 66.7% amino acid sequence similarity (Table I>. This figure was well below the 90% or mare similarity required for classifying two viruses as strains of the same virus (Shukla and Ward, 1.989) and

241

P.Y. Teycheney, R.G. Dietzgen / Eu.s Research 31 (1994) 235-244

Table 1 Pairwise per cent amino acid sequence similarities between coat proteins of potyviruses infecting legumes Virus

Percent sequence identity a 2

3

4

5

6

7

8

9

10

11

12

13

1 BCMV-NL 2 BCMV-NL8 3 SAPV 4 SMV-C 5 SMV-N 6 PStV-USb 7 pstv-USS 8 PeMoV-M 9 PStV Ib 10 BCMV-NL4 11 PWV-K 12 PeMoV-AU 13 PSbMV

399.2

88.9 89.3

83.2 83.9 78.8

84.3 84.7 81.5 98.1

86.6 86.6 77.4 85.5 87.9

86.6 86.6 77.4 85.5 87.9 99.3

86.2 86.6 77.4 85.8 88.3 98.9 98.3

86.2 86.2 77.4 85.5 88.3 98.9 98.3 97.9

84.7 84.7 76.3 83.3 85.7 93.0 92.7 92.0 93.0

83.6 83.5 75.5 81.0 82.6 83.1 82.7 82.7 83.4 81.6

70.6 70.9 69.9 66.5 68.3 66.7 66.7 66.7 66.7 66.7 66.9

70.9 70.1 69.2 68.4 70.2 65.2 64.8 65.2 65.2 66.6 62.0 62.0

a Calculated using Distances (GCG) on sequences aligned using Pileup (GCG). Figure is the fraction of divisions of total sequence by length of the shorter sequence, excluding gaps.

below the range (72-83%) suggested to classify two viruses in the same subgroup (Brand et al., 1993). Dot plot comparison of these two sequences (Fig. 3) showed that most of the dissimilarities were located in the amino terminus which is regarded as the ‘hypervariable region’ in potyvirus coat proteins (Shukla et al., 1988). The nucleotide sequence similarity between 3’ non-coding regions of PStV-Ib and PeMoV-AU was only 34.6% (Table 2), which further highlights the low level of homology between these two viruses. Our results confirm the classification of PStV and PeMoV as distinct members of the family Potyviridue. However, comparisons of coat protein sequences of different strains and isolates of PeMoV and PStV showed that the recently sequenced M strain of PeMoV (Gunasinghe et al., 1992) was more closely related to PStV-USs, PStV-USb and to a lesser extent to PStV-Ib, than to PeMoV-AU (Table 1). Comparisons of the 3’

50

100

150

200

250

PeMoV-AU Fig. 3. Dot plot similarity comparison of the predicted amino acid sequences of the coat proteins of PeMoV-AU and PStV-Ib. A PAM (Acceptable Point Mutations) matrix of 250 was used.

P. Y Teycheney, R. G. Dietzgen / virus Research

242

Table 2 Pairwise per cent sequence Virus (3’-ncr)

Percent

a

(247) (245)

1 BCMV-NL3 2 BCMV-NL8 3 PeMoV-M 4 PStV-USb 5 PStV-Ib 6 SMV-N 7 PWV-K 8 SAPV 9 PSbMV 10 PeMoV-AU

(2571 (2571 (255) (259) (250) (230) (158)

similarities sequence

between identity

potyviral

genomic

31 (1994) 235-244

3’ non-coding

regions

b

2

3

4

5

6

7

8

9

10

96.3

60.7 60.0

59.9 59.6 97.3

59.1 59.2 91.8 89.8

55.9 57.1 62.6 60.7 61.6

51.4 53.9 48.4 48.8 48.8 53.6

28.7 28.3 39.1 39.6 37.0 35.2 28.3

31.0 32.3 38.0 38.0 36.7 31.6 32.9 34.2

30.7 30.2 34.6 34.6 33.3 30.9 31.2 25.2 20.9

a Length of 3’ non-coding regions. b Calculated using Distances (GCGI on sequences aligned using Pileup (GCG). Figures of divisions of total sequence by length of the shorter sequence, excluding gaps.

are the fraction

non-coding regions of these PStV and PeMoV strains (Table 2) support these findings. It appears likely that an isolate of PStV which was a contaminant of the PeMoV-M culture has been inadvertently cloned and sequenced and has been mistaken for PeMoV-M (B. Cassidy, personal communication). 3.4. Sequence comparisons between PeMov PStV and other potyviruses The coat protein sequences and 3’ non-coding regions of strains of PStV and PeMoV were compared with other potyviruses (Tables 1 and 2). These comparisons included potyviruses which have been classified as members of the BCMV subgroup into which PStV has been placed (McKern et al., 1992b). Peptide profile analysis and serological data suggest that this subgroup includes some strains which have been classified as either SMV or BCMV (McKern et al., 1992a,b). We created a phylogenetic tree based on the similarity analysis between coat protein sequences of potyviruses infecting legumes (Fig. 4). We also included the nonlegume infecting potyviruses SAPV (Brand et al., 1993) and PWV-K (Gough and

t

PStV-USb PSW-uss PeMoV-M

Fig. 4. Phylogenetic analysis of relationship between PStV coat proteins and those of other potyviruses infecting legumes. The tree was created using PHYLIP and TREETOOL from coat protein alignments obtained using CLUSTAL V. The bar represents a dissimilarity index of 0.1.

P.Y. Teycheney, R.G. Dietzgen / Krus Research 31 (1994) 235-244

243

Shukla, 1992). The coat protein sequences of PWV strains have been shown to be more than 70% identical to PStV-USs (McKern et al., 1991). In the phylogenetic tree, the three isolates of PStV and PeMoV-M form a tight cluster. The NL-4 strain of BCMV (serogroup B) appears to be most similar to PStV (Fig. 4), whereas strains NL-3 and NL-8 (serogroup A) are more distantly related, which confirms previous reports (McKern et al., 1992a; Vetten et al., 1992). PeMoV-AU is one of the most distantly related viruses to PStV in this analysis (Fig. 4). These observations were confirmed by pairwise similarity analysis (Table 2) and the phylogenetic tree obtained by sequence comparisons of 3’ non-coding regions of the same viruses (data not shown).

Acknowledgements

The authors are indebted to N. Saleh (Malang Research Institute for Food Crops, Indonesia) for providing the PStV-Ib isolate and to B. Cassidy, U.B. Gunasinghe and H.J. Vetten for generously providing unpublished sequence data. We thank A. Reisner and his team for expert advice in the use of ANGIS and G.A. Smith for a critical reading of the manuscript. This work was funded by the Australian Centre for International Agricultural Research as part of project 9017.

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