The nucleotide sequence of RNA-2 of raspberry ringspot ... - CiteSeerX

Journal of General Virology (1992), 73, 2189-2194. Printed in Great Britain

2189

The nucleotide sequence of RNA-2 of raspberry ringspot nepovirus V. C. Blok, 1 J. WardeH, 1 C. A. Jolly, 1 A. M a n o u k i a n , ~ D. J. Robinson, t M . L. Edwards 2 and M . A. M a y o 1. 1Scottish Crop Research Institute, Invergowrie, Dundee D D 2 5 D A and 2N E R C Institute o f Virology and Environmental Microbiology, Mansfield Road, Oxford OX1 3SR, U.K.

The nucleotide sequence of raspberry ringspot nepovirus (RRV) RNA-2 consists o f 3928 nucleotides and a poly(A) tract at the 3' end. RNA-2 contains one open reading frame which encodes a polypeptide of Mr 123508 (123K). E d m a n degradation located the N terminus o f the coat protein 514 residues from the Cterminal end o f the 123K protein, which suggests that the coat protein is released from the polyprotein by cleavage o f a C - A bond. T h e R R V coat protein has

some sequence similarities with the coat proteins o f other nepoviruses, but is no more like any one nepovirus than another. In contrast, the portion o f the 123K protein to the N-terminal side o f the coat protein is similar in sequence to the corresponding parts o f the polyproteins of t o m a t o black ring and grapevine c h r o m e mosaic nepoviruses, though not to those o f other nepoviruses.

Introduction

(Meyer et al., 1986), grapevine chrome mosaic ( G C M V ; Brault et al., 1989), grapevine fanleaf ( G F L V ; Serghini et al., 1990) and (in part) arabis mosaic (ArMV; Bertioli et al., 1991), and for the RNA-2 molecule of the subgroup 2 virus tomato ringspot (TomRV; Rott et al., 1991). As a first step towards identifying the gene products responsible for the characteristics assigned to RNA-2, and to examine the relationships among RRV and other nepoviruses, we have determined the nucleotide sequence of RRV RNA-2.

Raspberry ringspot virus (RRV), a nepovirus, can cause economically significant disease losses in raspberry crops (Murant, 1978). It has a bipartite genome (Harrison et al., 1972) composed of an 8 kb (RNA-1) and a 4 kb (RNA-2) R N A molecule (Murant et al., 1981), each of which is polyadenylated (Mayo et al., 1979). RNA-2 carries determinants for the serological specificity of the coat protein, for nematode transmissibility and for a yellowing symptom in infected Petunia hybrida; RNA-1 carries determinants for other types of symptom (Harrison et al., 1974). The Mr of the RRV coat protein has been estimated to be 54000 (Mayo et al., 1971) or 57000 (Acosta & Mayo, 1990b) by gel electrophoresis in different gel systems, but in vitro translation of a mixture of RRV R N A species yields mainly larger polypeptides and none that comigrates with protein from virus particles (Jones et al., 1985). As with other nepoviruses such as tomato black ring virus (TBRV; Meyer et al., 1986; Demangeat et al., 199t), it is likely that RRV coat protein is released from a polyprotein by the action of a virus-encoded protease. Nepoviruses can be divided into several subgroups on the basis of serological relationships (Murant, 1981), or, more simply, into two subgroups according to whether the RNA-2 molecule is smaller (subgroup 1) or larger (subgroup 2) than 5.4 kb (Francki et al., 1985); RRV belongs to subgroup 1. Sequences are known for the RNA-2 molecules of the subgroup 1 viruses TBRV 0001-0883 © 1992 SGM

Methods Virus propagation and purification. An inoculum of the S strain of RRV (Harrison, 1958)was obtained from a single local lesion (Acosta & Mayo, 1990a),and the virus was propagated in Nicotiana clevelandii. Virus particleswerepurifiedfrom infectedplants as describedby Mayo et al. (1982). cDNA synthesis and cloning. RNA was extracted from virus particles by treatment with phenol and SDS (Mayoet aL, 1982),and cDNA was prepared from it by reverse transcription, essentiallyas described by Gubler & Hoffman(1983). Primers were oligo(dT),5"GGACATCCTGACCTTTCCAG (primer A; complementaryto nucleotides2701 to 2720), 5" AGACATCCGTGGCAGGTT (primer B; complementary to nucleotides 1245 to 1262) or 5' TTAATATTTGTCACCAGAAAGA (primer C; complementaryto nucleotides94 to 111). The cDNA was ligated into SmaI-cut pUCI9 and cloned in Escherichia coli JM101 or DH5~ as described (Mayo et al., 1989). The RNA species from which each oligo(dT)-primedclonedeDNA had been transcribed was determinedby probing Northern blots of purifiedRRV RNA with labelled cloned cDNA (Sambrook et al., 1989).

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Nucleotide sequencing. Sequences were determined by dideoxynucleotide chain termination (Sanger et al., 1977) essentially as described by Mayo et al. (1989). The T-terminal sequencesof RNA-1 and RNA-2 were likely to be very similar, and therefore they were determined only from eDNA clones that also possessed sequence unique to either RNA species. The sequence of the Y-terminal 13 nucleotides was obtained by primer extension from primer C as describedby Mayoet al. (1991).Sequenceswere assembledby usingthe programs DBUTIL and DBAUTO (Staden, 1982), and analysed by using ANALYSEQ (Staden, 1984), COMPARE and DOTPLOT (GCG packageversion; Devereuxet al., 1984).The percentageidentity and similaritybetween amino acid sequenceswere computed by using GAP with the default settings (GCG package version; Devereuxet al., 1984). Determination of the N-terminal amino acid of RR V coat protein.

B component particles of RRV were purified by centrifugation to equilibrium in CsCI in 10 raM-phosphatebufferpH 7 (density 1.5 g/ml) for 16 h at 4 °C, and protein was extracted from them by heating in 1% SDS. Coat protein was separated from contaminatingprotein by SDSPAGE (Acosta & Mayo, 1990b), transferred to a Immobilon P membrane (Millipore) and subjected to Edman degradation using an Applied Biosystems470A protein sequencer (Hewick et al., 1981).

Results Sequence o f R R V R N A - 2

The complete nucleotide sequence of RRV RNA-2 is shown in Fig. 1. Except for the 13 5'-terminal nucleotides and the 27 T-terminal nucleotides, all the sequence was determined by sequencing D N A in both orientations. The molecule contains 3928 nucleotides [excluding the poly(A) sequence of unknown length at the 3' end]. More than 90 % of the sequence was obtained from more than one cloned eDNA. Four variations were found from the sequence shown in Fig. 1 : G replacing U at position 18 (non-coding), C replacing U at position 1427 (no coding effect), C replacing U at position 2552 (no coding effect) and A replacing G at position 3300 (an Ala codon changed to a Thr codon). The sequence contains one large open reading frame (ORF) which encodes a polypeptide of Mr 123508 (123K). The only other ORF of more than 300 nucleotides in length is in the negative sense, commencing at nucleotide 2392 and ending at nucleotide 2021 (numbering as in Fig. 1); there is no analogous O R F in RNA-2 of other nepoviruses. The sequence deduced for the 5' non-coding region of RRV RNA-2, like those of other nepovirus RNAs (Rott et al., 1991), contains relatively few G + C residues and abundant U residues. CU dinucleotides are especially abundant near the 5' end, and UC, CU and U U dinucleotides account for 91 of the 205 dinucleotides. The 3' non-coding region is 397 nucleotides long with a composition of 23% A, 22% C, 23% G and 32% U and substantially the same sequence as RNA-1 (Fig. 1).

Oligonucleotide sequences have been shown to be shared among the Y non-coding sequences of nepovirus RNAs (Serghini et al., 1990) and some sequences have been found in the non-coding regions of both TBRV R N A and cowpea mosaic virus R N A (Meyer et al., 1986); none of these sequences were found by A N A L Y S E Q in RRV RNA-2. Proteins encoded by R R V R N A - 2

The unambiguous sequence of the N-terminal amino acids of isolated coat protein obtained from three analyses was A Y E V D P L H L L Y Y E S V . Its position in the 123K polypeptide is shown in Fig. 1. Therefore the coat protein arises by proteolysis of the cysteinyl-alanine bond between residues 593 and 594. Assuming this cleavage is the primary processing event, the polyprotein sequence on the 5' side of the coat protein would yield a 66K protein or, if cleaved further, proteins with a combined size of 66K. There are three other CA dipeptides in the 123K protein, but only that at residues 407 and 408 has an adjacent sequence (MCAFEV) similar to the sequence around the site of cleavage of the coat protein (GCAYEV), suggesting recognition by the RRV protease of a sequence to the C-terminal side of the cleaved peptide bond; cleavage between residues 407 and 408 would yield products of about 46K (N-terminal part) and 20K (C-terminal part). However, cleavage of the polyprotein of tobacco etch potyvirus by a virusencoded protease is determined by sequence to the Nterminal side of the cleavage site, and does not involve sequence on the C-terminal side (Dougherty & Carrington, 1988). No cleavage products were detected during in vitro translation of a mixture of RRV R N A species (Jones et al., 1985). The Mr of 56859 calculated for the RRV coat protein corresponds well with the estimate of 57000 obtained from gel electrophoresis by Acosta & Mayo (1990b). During electrophoresis, RRV coat protein can adopt a form that migrates with an apparent Mr of 44000 (Acosta & Mayo, 1990b). This change is reversible but can be prevented or reversed by alkylating -SH groups in the protein (Acosta & Mayo, 1990b). However, there is no obvious link between this effect and the cysteine content of RRV coat protein because TBRV coat protein, which does not behave anomalously in electrophoresis, contains nine cysteine residues (Meyer et al., 1986) whereas RRV coat protein contains seven. Comparisons with other nepoviruses

Comparison of the coat protein sequence of RRV with those of TBRV, GCMV, GFLV, T o m R V and ArMV showed 22% to 24% identity and 46% to 49% similarity.

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2770 2790 2~ 10 2830 2~50 2~?0 UGGU~JGGUGUGUCCUGGAAUGCUACUGCGGAUC UCGCCGACAUCGUCACUCGGAAACALrdGGAL~JGUAAAGUCCAAUGAGAL~"JGAC~GGACUI]At)A~GUCCAUAUGGUGAG~ V G V S W N A T A D L A D I V T ~ K H W I V K S N E ~ F E V D L ¥ C P Y G E

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Fig. 1. Nucleotide sequence of RRV RNA-2; the RNA sequence shown is followed by a poly(A) sequence. The amino acid sequence encoded by the single large ORF is shown below the nucleotide sequence; an asterisk indicates a termination codon. The amino acid sequence obtained by Edman degradation of RRV coat protein is underlined.

Very' similar values were obtained for all pairwise comparisons except those between viruses in the same serological subgroup: TBRV and GCMV are 5 7 ~ identical and GFLV and ArMV are 69~. identical. The

comparisons also showed three small clusters of highly conserved and similarly located amino acid sequence in all six coat proteins. These were (F/L)-D-A-(Y/F)-X(R/K), (V/L/I)-C-G-(H/Q) and F-Y-G-X-(S/T) (starting

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V. C. B l o k and others

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Fig. 2. D O T P L O T presentation of comparisons of polyproteins encoded by R R V R N A - 2 (horizontal axes) and T B R V R N A - 2 (a) or G F L V R N A - 2 (b). The horizontal and vertical lines indicate the positions of known cleavage sites in each sequence. The identities of the proteins in the T B R V and G F L V R N A - 2 sequences are shown to the right of each panel. The window length was 30 and the stringency was 17.

from residues 738, 782 and 1167, respectively, in the RRV 123K protein; X represents any amino acid). The extent of the similarity between the coat protein of RRV and that of either TBRV or GFLV is also shown in a DOTPLOT comparison of the sequence of the RRV 123K protein with either the TBRV 150K or the GFLV 131K polyprotein (Fig. 2); the similarities between coat proteins are greatest and most extensive towards the N termini. Although the coat proteins of TBRV and GFLV are almost equally similar to RRV coat protein, this is not the case for the part of the polyproteins on the Nterminal side of the cleavage site of the coat protein. The TBRV 150K protein (Fig. 2a) resembles the RRV 123K protein in this region, as does the 150K protein of GCMV (data not shown), but the GFLV 131K (Fig. 2b) and TomRV proteins (data not shown) do not.

Discussion The sequence of RRV RNA-2 presented here extends the list of sequences known for nepovirus RNA molecules and permits further comparisons to be made. Examination by DOTPLOT and GAP suggests that the coat proteins of nepoviruses, including RRV, from different serogroups (Murant, 1981) are about equally similar in sequence, and that the amino acid sequence similarities detected by Serghini et al. (1990) between TBRV and GFLV coat proteins apply substantially to all nepovirus coat proteins, including that of RRV. In

contrast, the 5'-terminal non-coding sequence of RRV RNA-2 (Fig. 1) does not conform to the consensus sequence of U-(U/G)GAAAA(U/A)(U/A)(U/A) proposed for nepovirus RNAs by Fuchs et al. (1989). The results of amino acid sequencing of RRV coat protein suggest that it is cleaved from the 123K polyprotein by proteolysis of a C-A bond. Cleavage sites for the release of other nepovirus coat proteins from polyprotein precursors are R-A (GCMV; Brault et al., 1989), R-G (GFLV, ArMV; Serghini et al., 1990; Bertioli et al., 1991) and K-A (TBRV; Demangeat et al., 1991). A cleavage site of C-A is unusual in that it has not been reported for any other plant virus or for picornaviruses (Palmenberg, 1987). However, proteases of some alphaviruses are known to cleave this bond (Strauss & Strauss, 1990). Rott et al. (1991) noted that the regions of the RNA-2 polyprotein sequences of TomRV and GFLV which are most similar are those immediately to the N-terminal side of the coat proteins, and suggested that these may be related to a possible transport function. An alignment of these parts of the RRV 123K polyprotein and the TBRV 150K polyprotein is shown in Fig. 3. There are patches of identical or nearly identical sequence in the two polyproteins, just as there are in the sequences of the GFLV and TomRV polyproteins (Rott et al., 1991), but none of the sequences common to either pair of viruses is common to all four. Thus comparisons in this region of the polyprotein distinguish two groups of viruses: RRV

Raspberry ringspot nepovirus R N A - 2

(a) (b)

SENSARKLAEVGWNNADICGVDLAVKSH]AVG'FPVBVIISLVDGACSDMPTATMCAFEVNLASQN , ,i,t tl ....... : : [ [ '' ,, I i( ,, ~: LYNELNKLAESOWKEAKSVCLNLHIRSYVPV~LYAFCVIMWGHSSDAETASLCGAGVYLGDQE

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NRSLNLPLLSLPFSRLLADLHDFQNRVKIACQFRDPEGFNVG~MLSFSSLEFSELKQTAFERNS

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KQPLLNKSRSMRI QSFVSFRGSNIPVGRRIDNTAEAINYELGRASTSSVSSRLDESKCNLK

Fig. 3. Alignmentsbetween sequencesto the N-terminal side of the coat protein in RNA-2-encodedpo]yproteins. The sequences were aligned by GAP. The sequencesare from residues351 (RRV, a) or 593 (TBRV, b), and are contiguousuntil the C-A (RRV) or K-A (TBRV) cleavage sites for the release of coat protein from the polyprotein. Matching amino acids are shown by a vertical line.

and TBRV (29~ identical by GAP), and GFLV and TomRV (39~ identical by GAP); comparisons between viruses in different groups gave estimates of about 1 9 ~ identity and no similarities were detectable by using the program COMPARE. This contrasts with the results of pairwise comparisons among the coat proteins of the four viruses, which suggest no marked grouping among them. A feature which is shared by the viruses in either pair is the genus of the nematode vector: RRV and TBRV are

transmitted by species of Longidorus whereas TomRV a n d G F L V a r e t r a n s m i t t e d by s p e c i e s o f Xiphinema. T h e s p e c i f i c i t y o f n e m a t o d e t r a n s m i s s i o n is d e t e r m i n e d by R N A - 2 ( H a r r i s o n et al., 1974) a n d c o r r e l a t e s w i t h t h e serological specificity of the coat protein (Harrison, 1964). It m a y b e t h a t a s e c o n d R N A - 2 - e n c o d e d p r o t e i n influences nematode transmission and that the sequence s i m i l a r i t i e s w e h a v e d e t e c t e d a r e in s o m e w a y r e l a t e d to t h i s f u n c t i o n . W h e t h e r o r n o t t h i s s p e c u l a t i o n p r o v e s to be c o r r e c t , it is i n t e r e s t i n g t h a t c o m p a r i s o n s b e t w e e n sequences of nepovirus RNA-encoded non-structural proteins have suggested relationships between viruses that correlate with their biological properties rather than w i t h t h e p h y s i c a l p r o p e r t i e s u s e d to d e f i n e s u b g r o u p s o f nepoviruses. We thank Mr A. Willis, MRC Immunochemistry Unit, Oxford, for the protein sequencing. This work was supported in part by flexibility funding from the Scottish Office Agriculture and Fisheries Department.

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(Received 29 January 1992; Accepted 11 May 1992)