Arch Virol (1992) 126:231-238

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Virology © Springer-Verlag 1992 Printed in Austria

On the variability of the 3' terminal sequence of the turnip mosaic virus genome Y. Sano 1, R. van der Vlugt 3, P. de Haan 3, A. Takahashi 4, M. Kawakami4, R. Goldbaeh3, and M. Kojima2 1Graduate School of Science and Technology and 2 Laboratory of Plant Pathology, Faculty of Agriculture, Niigata University, Niigata, Japan 3Department of Virology, Agricultural University Wageningen, Wageningen, The Netherlands 4Department of Molecular Biology, Kitasato University School of Medicine, Kitasato, Japan

Accepted January 24, 1992

Summary. The sequence of the 3'-terminal 1223 nucleotides (nts) of a Japanese isolate of turnip mosaic virus (TuMV-Jap) RNA has been determined. The sequence reveals a single open reading frame (ORF) which terminates at a position 212nts upstream of the 3' poly(A)-tract. Determination of the Nterminal amino acids of TuMV-Jap coat protein (CP) mapped the CP cistron within this ORF and revealed a Glu-Ala dipeptide sequence as the putative cleavage site by which the CP is released from the viral polyprotein. The predicted amino acid sequence of the TuMV-Jap CP shows 97.2% identity with that of a Canadian isolate of TuMV (TuMV-Can) and 99% with a second, Chinese, isolate (TuMV-Chi). However, the Y-terminal non-translated region (NTR) of TuMV-Jap RNA is significantly shorter (212nts) than the 3'-NTR of TuMV-Can RNA (668 nts), but of equal length as the 3'-NTR of the TuMVChi isolate which also measures 212nts. The 3'-NTRs of both the TuMV-Jap and TuMV-Chi RNAs show homology with the first 201 nucleotides of the TuMV-Can RNA 3'-NTR. A search in the EMBL nucleotide sequence database revealed that the 467 nt-long unique extension of the 3"-NTR of' TuMV-Can RNA has 89.8% homology to a part of the chloroplast ribosomal protein 12 gene (rsp 12-gene). Irrespective of the origin of this extra sequence in the reported TuMV-Can sequence, which may have been introduced by a genuine RNA recombination event, it is concluded that the standard TuMV genome has a CP gene of 864 nts and an conserved 3'-NTR of approximately 212 nucleotides in length.

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Y. Sano etal. Introduction

Turnip mosaic virus (TuMV), member of the potyvirus group, is a world wide distributed pathogen infecting many plant species from 20 different dicotyledonous families [ 19]. The virus is sap-transmissible and spread by a considerable number of aphid species [19]. The genomic RNA of potyviruses encodes a single large potyprotein which is subsequently cleaved, by at least two viruscoded proteases, to yield mature viral proteins [3, 4]. Genome mapping and sequence analysis of several potyviral RNAs have revealed that the coat protein (CP) cistron is invariably located directly upstream of the polyadenylated 3' terminus of the genomic RNA. Detailed sequence comparisons of a growing number of potyviral CP genes demonstrate that the internal, core region of the potyviral CP is conserved, whereas both the length and the amino acid sequence of the extended N-terminus may be highly heterogeneous among different potyvirus species [17, 18, 24]. In addition, the 3' non-translated regions (Y-NTRs) of the RNAs of different members of the potyvirus group show no significant homologies [8, 24]. For these reasons both the amino acid sequence of the CP, especially that of the variable N-terminal domain of this protein, as well as the nucleotide sequence of the 3'-NTR have been proposed as molecular criteria for potyvirus classification [8, 22, 24]. Recently the nucleotide sequence of the 3' terminal region, including the CP cistron, has been elucidated for two different TuMV isolates, one originating from Canada (TuMV-Can) and one from China (TuMV-Chi) [20, 10]. These two isolates share a high degree of homology, in both the length and amino acid sequence of their CPs, including the N-terminal region. In contrast to this, their Y-NTRs show a great divergence in length. This observed discrepancy obscures the precise taxonomic relationship between both isolates, and moreover it breaks the generally accepted consensus by which potyviruses are nowadays classified [24]. In order to study the variability among TuMV isolates we decided to determine the 3'-terminal RNA sequence of an additional, third isolate. This paper reports the nucleotide sequence of the CP gene and 3'-NTR of a Japanese isolate of TuMV and its comparison to the isolates from Canada and China. Materials and methods

A Japanese isolate ofTuMV (TuMV-Jap), originally obtained from radish (Raphanus sativus L.) [16], was propagated in turnip (Brassica rapa) by successivesap-inoculation. Virus was purified from infected turnip leaves as described by Choi et al. [5], and genomic RNA was purified according to Brakke and van Pelt [2]. Complementary DNA (cDNA) clones corresponding to regions upstream of the poly (A) tract were constructed using the cDNA synthesis system plus kit (Amersham International PIC) with oligo(dT) as a primer, and subsequently cloned into Sma I-digested pUC 19 [25]. Additional cDNA clones were synthesized using a synthetic oligonucleotide 5'-TGAGCGGCTTGATCGGG-3', whose sequence was derived from obtained nucleotide sequence data. Several cDNA clones from the 3' end of the genomic RNA were obtained. These cDNA clones were subcloned into

The 3' terminal sequence of the turnip mosaic virus genome

233

phage M 13 mp 18 and mp 19 [11] and an ordered set of deletion mutants was generated using the exonuclease-III method [9]. Nucleotide sequences were determined using the dideoxy chain termination method [15]. Data were compiled, stored and analyzed using the DNASIS programs (Hitachi Software Engineering, version 5.0). N-terminal CP amino acid sequence data were obtained using Edman degradation in a gas-phase sequencing apparatus (Applied Biosystems). Results and discussion The sequence of the 1223 Y-terminal nucleotides of TuMV-Jap is presented in Fig. 1. Analysis of the sequence reveals one contiguous open reading frame (ORF) of 1011 nucleotides terminated by an opal stop codon. Comparison of the deduced amino acid sequence with the published TuMV CP sequences [10, 20] predicted that CP release occurs at a Q/A dipeptide sequence (position - 2 8 9 / - 288). In order to verify this, the sequence of the amino-terminal part of the CP was determined by Edman degradation. The obtained sequence of 19 residues (Fig. 1) allowed a proper mapping of the CP N-terminus at the alanine residue at position - 288 and moreover, confirmed the deduced amino acid sequence in this region. Hence, the predicted TuMV-Jap CP consists of 288 amino acid residues, corresponding to a calculated Mr of 33,149, which is in good agreement with the apparent Mr of 33,000 as estimated from PAGE studies [6]. An alignment of the CP sequences of TuMV-Jap, the two other TuMV isolates, and of three other potyviruses is presented in Fig. 2. This alignment shows that the TuMV-Jap CP is 98.6% homologous to that of TuMV-Chi and 96.2% to that of TuMV-Can with most of the amino acid differences located in the N-terminal parts of the TuMV CPs, The alignment furthermore confirms the sequence similarities of the TuMV CPs with other potyviral CPs throughout their central and C-terminal regions and the high variability in length and sequence of the N-terminal parts. The overall amino acid sequence homologies between the CPs of TuMV isolates and other potyviruses range from 50-61%. Hence, based on the amino acid sequence of the CPs, the three TuMV isolates studied all belong to the same potyvirus species. Comparison of their 3'-NTRs, however, reveals a striking difference between the TuMV-Jap and TuMV-Chi isolates on one hand and the TuMV-Can isolate on the other. The 3'-NTR of TuMV-Jap consists of 212nts followed by the poly(A)-tail. Comparison of this region with the corresponding regions from other potyviruses shows limited homology (39 to 56%). Mutual comparisons of the 3'-NTRs of the RNAs of the three TuMV isolates shows a high degree of homology over the first 201 nts (82.8-99.5%; Fig. 3 a). Interestingly, TuMVCan has a additional stretch of 467 nts, lacking any significant homology to other potyviral RNA sequences. A search in the EMBL nucleotide sequence database revealed that this sequence corresponds with the 3' terminal 65 nucleotides of exon I followed by a major part of intron I of the soybean chloroplast ribosomal protein S 12 (rps 12) gene, (89.8% identity in 496 bases overlap; Fig. 3b). Similar levels of homology can be observed with rps 12 genes from

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Fig. 1. The nucleotide sequence of the 1223 bases at the 3'-terminal region of the Japanese strain of TuMV RNA (TuMV-Jap) [16]. The derived amino acid sequence is presented above the nucleotide sequence. Asterisks indicate the UGA (opal) stop codon. The proteolytic cleavage site between the glutamine (Q) and alanine (l%) residues is indicated by an arrow. Boxed amino acids have been confirmed by Edman degradation

other plant species like tobacco and maize (results not shown). It is therefore tempting to assume that this sequence represents an artefact introduced during c D N A cloning o f T u M V - C a n R N A . However, it cannot be ruled out that the unique extension at the 3' end o f the T u M V - C a n R N A originates f r o m a genuine

The 3' terminal sequence of the turnip mosaic virus genome TVMV PVY-N T EV- H A T TuMV-Can TuMV-Chi TuMV-Jap Consensus

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Fig. 2. Comparison of the CP amino acid sequence of TuMV-Jap [16] with those of two other TuMV isolates TuMV-Can [20] and TuMV-Chi [10] and three other potyviruses. PVY-N, potato virus Y (tobacco veinal necrosis strain [20]), TEV-HAT, tobacco etch virus (highly aphid transmissible strain) [1], TVMV, tobacco vein mottling virus [7]. Dots indicate gaps and the derived consensus sequence is shown at the bottom. Amino acid residues deviating from the consensus are printed in lower case

recombination event between the viral R N A and a plant chloroplast transcript. Recently, evidence for such a recombination event, involving the addition of a sequence of 119 nt, from an exon of tobacco chloroplast D N A O R F 196, to the 5'-terminus of the viral R N A , has been reported by Mayo and Jolly [14] to explain the observed discrepancy between the 5'-NTRs o f different potato

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leaf roll virus (PLRV) isolates [13, 23]. In favour of this explanation is indeed the observation that the chloroplast derived sequence in the T u M V - C a n R N A is immediately followed by a poly(A)-tail, which makes a cloning artefact less probable. Irrespective of the origin of this extra sequence in the reported T u M V - C a n sequence, it is concluded that different isolates o f T u M V , originating from distinct geographical regions, c o n f o r m the generally accepted rules for the classification of potyviruses [24], share b o t h a highly conserved C P a m i n o acid sequence (more t h a n 95% h o m o l o g y ) a n d a standard conserved 3 ' - N T R (more t h a n 90% h o m o l o g y with a length of approximately 212 nts).

Acknowledgements We wish to thank Dr. Teruo Sano, Plant Virology, Hokkaidou University, for construction of synthetic oligonucleotides.

References 1. Allison R, Johnston RE, Dougherty WG (1986) The nucleotide sequence of the coding region of tobacco etch virus genomic RNA: evidence for the synthesis of a single polyprotein. Virology 154:9-20 2. Brakke MK, van Pelt N (1970) Properties of infectious ribonucleic acid from wheat streak mosaic virus. Virology 42:699-706 3. Carrington JC, Dougherty WG (1987) Small nuclear inclusion protein encoded by a plant potyvirus genome is a protease. J Virol 61:2540-2548 4. Carrington JC, Cary SM, Parks TD, Dougherty WG (1989) A second proteinase encoded by a plant potyvirus genome. EMBO J 8:365-370 5. Choi JK, Maeda T, Wakimoto S (1977) An improved method for the purification of turnip mosaic virus. Ann Phytopathol Soc Japan 43:440-448 6. Choi JK, Wakimoto S (1979) Characterization of the protein components of turnip mosaic virus. Ann Phytopathol Soc Japan 45:32-39 7. Domier LL, Franklin KM, Shahabuddin M, Hellmann GM, Overmeyer JH, Hiremath ST, Siaw MFE, LomonossoffGP, Shaw JG, Rhoads RE (1986) The nucleotide sequence of tobacco vein mottling virus. Nucleic Acids Res 14:5417-5430 8. Frenkel MJ, Ward CW, Shukla DD (1989) The use of 3' non-coding nucleotide sequences in the taxonomy of potyviruses: application to wate~elon mosaic virus 2 and soybean mosaic virus-N. J Gen Virol 70:2775-2783 9. Henikoff S (1984) Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene 28:351-359 10. Kong L, Fang R, Chen Z, Mang K (1990) Molecular cloning and nucleotide sequence of coat protein gene of turnip mosaic virus. Nucleic Acids Res 18:5555 Fig. 3. a Alignment of the 3'-NTRs of the three TuMV isolates. Dots indicate gaps and only nucleotides differing from the derived consensus sequence are shown. The extended stretch of nucleotides at the 3' end of TuMV-Can is represented by (N455). b Alignment of the sequence of the extended stretch of 467 nt from the 3' end of TuMV-Can [20], represented in the top strand, to the sequence of intron I of the rps 12 gene from soybean (bottom strand, EMBL accession no. X 07675). Gaps are indicated by dots. Numbering is consistent with original references

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Y. Sano et al.: The 3' terminal sequence of the TuMV genome

11. Messing J (1983) New M13 vectors for cloning. Methods Enzymol 101:20-78 12. Maiss E, Timpe U, Brisske A, Jelkmann W, Casper R, Himmler G, Mattanovich D, Katinger HWD (1989) The complete nucleotide sequence of plum pox virus RNA. J Gen ViroI 70:513-524 13. Mayo MA, Robinson D J, Jolly CA, Hyman L (1989) Nucleotide sequence of potato leafroll luteovirus RNA. J Gen Virol 70:1037-1051 14. Mayo MA, Jolly CA (1991) The Y-terminal sequence of potato leafroll virus RNA: evidence of recombination between virus and host RNA. J Gen Virol 72:2591-2595 15. Sanger F, Nicklen S, Coulson AR (t977) DNA sequencing with chainterminating inhibitors. Proc Nat1 Acad Sci USA 74:5463-5467 16. Sano Y, Kojima M (1989) Increase in cucumber mosaic virus concentration in Japanese radish plants co-infected with turnip mosaic virus. Ann Phytopathol Soc Japan 55: 296-302 17. Shukla DD, Ward CW (1988) Amino acid sequence homology of coat proteins as a basis for identification and classification of the potyvirus group. J Gen Virol 69: 27032710 18. Shukla DD, Ward CW (1989) Identification and classification of potyviruses on the basis of coat protein sequence data and serology. Arch Virol 106:171-200 19. Tomlinson JA (1970) Turnip mosaic virus. CMI/AAB Descriptions of Plant Viruses, no 8 20. Tremblay M, Nicolas O, Shinha RC, Lazure C, Lalibert6 J (1990) Sequence of the 3"terminal region of turnip mosaic virus RNA and capsid protein gene. J Gen Virol 71: 2769-2772 21. Van der Vlugt RAA, Allefs S, de Haan P, Goldbach RW (1989) Nucleotide sequence of the 3' terminal region of potato virus yN RNA. J Gen Virol 70:229-233 22. Van der Vlugt RAA (1992) Is PeMV a strain of PVY? Arch Virol (in press) 23. Van der Wilk F, Huisman MJ, Cornelissen BJC, Huttinga H, Goldbach R (1989) Nucleotide sequence and organization of potato leafroll virus genomic RNA. FEBS Lett 245:51-56 24. Ward CW, Shukla DD (1991) Taxonomy of potyviruses: current problems and some solutions. Intervirology 32:269-296 25. Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13 mp 18 and pUC19 vectors. Gene 33: 103-119 Authors' address: R. van der Vlugt, Department of Virology, Agricultural University Wageningen, Binnenhaven 11, NL-6709 PD Wageningen, The Netherlands. Received October 28, 1991

On the variability of the 3' terminal sequence of the turnip mosaic virus genome.

The sequence of the 3'-terminal 1223 nucleotides (nts) of a Japanese isolate of turnip mosaic virus (TuMV-Jap) RNA has been determined. The sequence r...
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