Nucleotide sequence of RNA 2 of a ... - Semantic Scholar
Journal of General Virology (1991), 72, 213-216. Printedin Great Britain
213
Nucleotide sequence of RNA 2 of a Czechoslovakian isolate of red clover necrotic mosaic virus T. A. M. Osman, S. J. Miller, A. C. Marriotr~ and K. W. Buck* Department of Biology, Imperial College of Science, Technology and Medicine, London S W 7 2BB, U.K.
The complete nucleotide sequence (1448 nucleotides) of RNA 2 of a Czechoslovakian isolate TpM-34 of red clover necrotic mosaic virus (RCNMV-TpM-34) has been determined. The sequence contained one major open reading frame (ORF) with the potential to encode a protein of 326 amino acids (Mr 35755), designated P2. The nucleotide sequence of RNA 2 of RCNMVTpM-34 and the previously published sequence of RNA 2 of an Australian isolate of the virus (RCNMVAus) were 83 % identical and there was 80% amino acid
sequence identity between the P2 proteins of these isolates. However the N-terminal two-thirds of the P2 proteins shared a higher degree of similarity than the C-terminal regions which were predicted to have a more flexible structure. An ORF in the 3' portion of RNA 2 of RCNMV-Aus, which could encode a protein of Mr 5000, was not present in RNA 2 of RCNMVTpM-34. RNAs 1 and 2 of RCNMV-TpM-34 and RCNMV-Aus are bilaterally compatible.
Red clover necrotic mosaic virus (RCNMV), a member of the Dianthovirus group (Matthews, 1982), has a genome of two positive-strand RNA components, RNA 1 (4.0 kb) and RNA 2 (1.4 kb) (Gould et al., 1981). Both RNA 1 and RNA 2 are required for systemic infection of plants (Gould et al., 1981 ; Okuno et al., 1983; Osman et al., 1986; Paje-Manalo & Lommel, 1989), but RNA 1 can replicate and give rise to virus particles in protoplasts in the absence of RNA 2 (Osman & Buck, 1987; PajeManalo & Lommel, 1989), implying a role for RNA 2 in cell-to-cell movement of the virus. RNA 2 encodes a protein of Mr 34K to 35K (Morris-Krsinich et al., 1983; Lommel et al., 1988), which may be analogous to the movement proteins of other plant viruses (reviewed by Hull, 1989). The complete nucleotide sequences of the RNA components of an Australian isolate of RCNMV (RCNMV-Aus) have been reported (Lommel et al., 1988; Xiong & Lommel, 1989). We now report the nucleotide sequence of RNA 2 of a Czechoslovakian isolate of RCNMV (RCNMV-TpM-34) (Musil, 1969) and compare it with that of RNA 2 of RCNMV-Aus. RCNMV-TpM-34 was propagated, virus particles were extracted and purified, and viral RNA was isolated as described previously (Osman et al., 1986). Doublestranded cDNA was synthesized (Gubler & Hoffman, 1983) either using unfractionated viral RNA as a template with random oligonueleotide primers (Taylor et t Presentaddress: NERC Instituteof Virologyand Environmental Microbiology,MansfieldRoad, OxfordOX1 3SR, U.K.
al., 1976) or with unfractionated, polyadenylated (Ahlquist et al., 1981) viral RNA, using TloG as a primer. Double-stranded cDNA was either tailed with dC and cloned into PstI-linearized dG-tailed pUC8 or pBR322 or cleaved with Sau3A and the fragments were cloned into BamHI-cleaved pUC8. cDNA clones of RNA 1 or RNA 2 were identified by agarose gel electrophoresis of viral RNA, followed by Northern blotting and hybridization with nick-translated or oligolabelled probes prepared from individual clones (Osman & Buck, 1987). The nucleotide sequences of five independently obtained cDNA clones of RNA 2 were determined (Sanger et al., 1977; Chert & Seeburg, 1985) in both directions after subcloning regions into M13 vectors (Messing & Vieira, 1982), by generating unidirectional deletions using exonuclease III (Henikoff, 1984) or by using oligonucleotide primers corresponding to known parts of the sequence. These clones were: GC200 [nucleotide (nt) 1 to 1341], GC4 (nt 1 to 803), TOBA3 (nt 714 to 981), $7 (nt 911 to 982) and SU5 (nt 979 to 1148). The sequences of the 5' end (nt 1 to 74) and also nt 959 to 1432 were determined by primer extension (Ahlquist et al., 1981) on an RNA 2 template. The 3' sequence (nt 1339 to 1448) was determined by 3' end labelling of electrophoretically purified RNA 2 with [3Zp]pCp followed by specific enzymic cleavage and gel electrophoresis (Stahl et al., 1989). The 3'-terminal nucleotide of RNA 2 was determined after complete digestion of 3" end-labelled RNA 2 with T2 ribonuclease, followed by
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Fig. I. Alignment of the nucleotide sequences of RCNMV-TpM-34 R N A 2 (upper line of each pair) and RCNMV-Aus R N A 2 (lower line of each pair). Identical nucleotides are indicated by vertical lines; gaps are denoted as dashes. The initiation and termination codons of P2, and of an ORF with the potential to encode a protein of Mr 5000 in RCNMV-Aus (and a much truncated counterpaa in RCNMV-TpM-34), are underlined.
thin-layer chromatography (Stahl et al., 1989). Computer analysis of sequence data was carried out with HIBIO DNASIS and PROSIS software programs (Pharmacia). The complete nucleotide sequence (1448 nucleotides) of RNA 2 of RCNMV-TpM-34 is shown in Fig. 1. Analysis of open reading frames (ORFs) revealed one major ORF (nt 79 to 1056) with the potential to encode a protein, designated P2, of 326 amino acids (Mr 35755), nine amino acids longer than the P2 protein predicted from the corresponding ORF in R N A 2 of RCNMV-Aus (Lommel et al., 1988). An ORF that could encode a protein of M~ 5K was found in the 3' portion of R N A 2 of RCNMV-Aus (Lommel et al., 1988). Although the start of a corresponding ORF was found in R N A 2 of RCNMV-TpM-34, a termination codon was located after only 18 amino acids (Fig. 2). This result suggests
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Short communication
that R C N M V R N A 2 is monocistronic, in agreement with the conclusion of Lommel et al. (1988) who failed to detect a protein of Mr 5K in the products of in vitro translation of in vitro transcripts containing the 5Kencoding ORF of RCNMV-Aus R N A 2. Comparison of the nucleotide sequence of RNA 2 of RCNMV-TpM-34 with that of RCNMV-Aus (Lommel et al., 1988) revealed 83~ identity (five gaps were introduced to align the sequences) (Fig. 1). Regions with the greatest sequence identity were found at the 3' terminus (nt 1342 to 1448; 89.0~ identity) and in the Nterminal 72~ of the P2-coding region (nt 79 to 786; 87.7~ identity); the lowest sequence identity was found in part of the 3' non-coding region (nt 1057 to 1341; 77.5 ~ identity) and in a region near the C terminus of the P2-coding region (nt 787 to 942; 72.2~o identity). Comparison of the amino acid sequences of P2 of RCNMV-TpM-34 and that of RCNMV-Aus (Fig. 2) revealed 8 0 ~ direct sequence identity. (No gaps were needed to align the sequences.) The sequence of the Nterminal 236 amino acids, showing 9 2 ~ sequence identity, was the most highly conserved region. However the following 52 amino acids (amino acids 237 to 288, corresponding to nt 787 to 942) showed only 4 4 ~ sequence identity. The relatively poor conservation of this region in the P2 proteins of the two isolates suggests that its sequence may not be critical for the putative cellto-cell movement function. Analysis of potential secondary structures (Chou & Fasman, 1978; Garnier et al., 1978) of P2 of RCNMV-TpM-34 and that of RCNMVAus predicted mainly co-helical and fl-sheet structure for the first 220 amino acids, followed by predominantly random coils and//-turns for the remaining C-terminal amino acids of both proteins. This suggests that the Cterminal region has a flexible structure. Xiong & Lommel (1989) reported significant degrees of amino acid sequence homology between the putative RNA polymerase encoded by RNA 1 of RCNMV-Aus and corresponding proteins encoded by viruses in other groups, the carmovirus group (carnation mottle virus, turnip crinkle virus), the luteovirus group (barley yellow dwarf virus) and maize chlorotic mottle virus (unclassified). However we could detect no significant sequence identity between the P2 proteins of RCNMV-TpM-34 or RCNMV-Aus and any proteins corresponding to ORFs of these other four viruses. Nor was any striking identity found when the amino acid sequence of RCNMV-TpM34 P2 was compared with all available protein sequences of plant viruses. The low degree of amino acid sequence identity reported between regions of P2 of RCNMV-Aus and P3a of brome mosaic virus (BMV) (23.6 ~ identity in a 54 amino acid overlap) (Lommel et al., 1988) and P3a of cowpea chlorotic mottle virus (CCCMV) (19.7 ~o identity in a 157 amino acid overlap, including gaps) (Allison et
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al., 1989) was conserved in P2 of RCNMV-TpM-34.
However, a search of the NBRF protein database using the Lipman & Pearson (1985) algorithm revealed similar, or greater, degrees of identity with regions in over 100 unrelated proteins. Hence the functional significance of the identity between regions in P2 of R C N M V and P3a of BMV and CCMV remains to be determined. When RNAs of RCNMV-TpM-34 and RCNMV-Aus were separated (Osman et al., 1986; Osman & Buck, 1987) and inoculated in homologous or heterologous mixtures to cowpea leaves, lesions were formed in numbers similar to those induced by inocula of the parent isolates. Hence these two isolates can form pseudorecombinants in both directions. Previous studies have shown that isolates of R C N M V and other dianthoviruses can form pseudorecombinants either bilaterally (Okuno et al., 1983; Osman et al., 1986) or unilaterally (Rao & Hiruki, 1987). The ability to form viable pseudorecombinants probably depends, amongst other things, on the ability of the viral R N A replicase to recognize sequences at the 5' and 3' ends of the heterologous RNA. It is noteworthy that 22 out of 23 nucleotides at the 3' end of RNA 2 of RCNMV-TpM-34 and RCNMV-Aus were identical, allowing formation of a stem-loop structure (Lommel et al., 1988) which may be an R N A polymerase recognition site. Eight nucleotides at the 5' termini of the RNAs were identical. We thank Dr S. M. Dodd for preliminary experiments, Dr S. A. Lommel for supplying the Australian isolate of RCNMV, the Agricultural and Food Research Council for a grant and the Science and Engineering Research Council for research studentships for S.J.M. and A.C.M.
References AHLQUIST, P., DASGUPTA,R. & KAESBERG,P. (1981). Near identity of 3' secondary structure in bromoviruses and cucumber mosaic virus. Cell 23, 183-189. ALLISON, R. F., JANDA,M. & AHLQUIST, P. (1989). Sequence of cowpea chlorotic mottle virus RNAs 2 and 3 and evidence of a recombination event during bromovirus evolution. Virology 172, 321-330. CHEN, E. Y. & SEEBURO, P. H. (1985). Supercoil sequencing: a fast and simple method for sequencing plasmid DNA. DNA 4, 165-170. Crlou, P. Y. & FASMAN,G. D. (1978). Empirical predictions of protein conformation. Annual Review of Biochemistry 47, 251-276. GARNIER, J., OSGUTHORPE, D. J. 8,; ROBSON, B. (1978). Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. JournalofMolecular Biology 120, 97-120. GOULD, A. R., FRANCKI, R. I. B., HATTA, T. & HOLLINGS, M. (1981). The bipartite genome of red clover necrotic mosaic virus. Virology 108, 499-506. GUBLER, U. & HOFFMAN, B. J. (1983). A simple and very efficient method for generating cDNA libraries. Gene 25, 263-269. HENIKOFF, S. (1984). Unidirectional digestion with exonuclease III creates target break point for DNA sequencing. Gene 28, 351-359. HULL, R. (1989). The movement of viruses in plants. Annual Review of Phytopathology 27, 213-240. LIPMAN, D. J. & PEARSON, W. R. (1985). Rapid and sensitive protein similarity searches. Science 227, 1435-1439.
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LOMMEL, S. A., WESTON-FINA, M., XIONG, Z. & LOMONOSSOFF, G. P. (1988). The nucleotide sequence and gene organization of red clover necrotic mosaic virus RNA-2. NucleicAcids Research 16, 8587-8602. MATITIEWS, R. E. F, (1982). Classification and nomenclature of viruses. Intervirology 17, 1-199. MESSING,J. & VIEIRA, J. (1982). A new pair of M 13 vectors for selecting either DNA strand of double-digest restriction fragments. Gene 19, 269-276. MORRIS-KRSINICH, B. A. M., FORSTER, L. S. & MossoP, D. W. (1983). Translation of red clover necrotic mosaic virus RNA in rabbit reticulocyte lysate: identification of the virus coat protein cistron on the larger RNA strand of the bipartite genome. Virology 124, 349356. MUSIL, M. (1969). Serological properties of certain isolates of red clover necrotic mosaic virus. Acta virologica 13, 226-234. OI~UNO, T., HIRUKI, C., RAO, D. V. & FIGUEIREDO, G. C. (1983). Genetic determinants distributed in two genomic RNAs of sweet clover necrotic mosaic, red clover necrotic mosaic and clover primary leaf necrosis viruses. Journal of General Virology 64, 19071914. OSMAN, T. A. M. & BUCK, K. W. (1987). Replication of red clover necrotic mosaic virus RNA in cowpea protoplasts: RNA I replicates independently of RNA 2. Journal of General Virology 68, 289-296.
OSMAN, T. A. M., DODD, S. M. & BUCK, K. W. (1986). RNA 2 of red clover necrotic mosaic virus determines lesion morphology and systemic invasion in cowpea. Journalof General Virology67, 203-207. PAJE-MANALO, L. & LOMMEL, S. A. (1989). Independent replication of red clover necrotic mosaic virus RNA-I in electroporated host and non-host Nicotiana species protoplasts. Phytopathology 79, 457461. RAO, A. L. N. & HIRUKI, C. (1987). Unilateral compatibility ofgenome segments from two distinct strains of red clover necrotic mosaic virus. Journal of General Virology 68, 191-194. SANGER, F., NICKLEN, S. & COULSON, A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proceedings of the National Academy of Sciences, U.S.A. 74, 5463-5467. STAHL, D. A., KRUPP, G. & STACKEBRANDT, E. (1989). RNA sequencing. In Nucleic Acids Sequencing: A PracticalApproach, pp. 137-183. Edited by C. J. Have & E. S. Ward. Oxford: IRL Press. TAYLOR, J. M., ILLMENSEE, R. & SUMMERS, J. 0976). Efficient transcription of RNA into DNA by avian sarcoma virus polymerase. Biochimica et biophysica acta 442, 325-330. XIONG, Z. & LOMMEL, S. A. (1989). The complete nucleotide sequence and genome organization of red clover necrotic mosaic virus RNA-1. Virology 171, 543-554.
(Received 16 July 1990; Accepted 12 October 1990)
213
Nucleotide sequence of RNA 2 of a Czechoslovakian isolate of red clover necrotic mosaic virus T. A. M. Osman, S. J. Miller, A. C. Marriotr~ and K. W. Buck* Department of Biology, Imperial College of Science, Technology and Medicine, London S W 7 2BB, U.K.
The complete nucleotide sequence (1448 nucleotides) of RNA 2 of a Czechoslovakian isolate TpM-34 of red clover necrotic mosaic virus (RCNMV-TpM-34) has been determined. The sequence contained one major open reading frame (ORF) with the potential to encode a protein of 326 amino acids (Mr 35755), designated P2. The nucleotide sequence of RNA 2 of RCNMVTpM-34 and the previously published sequence of RNA 2 of an Australian isolate of the virus (RCNMVAus) were 83 % identical and there was 80% amino acid
sequence identity between the P2 proteins of these isolates. However the N-terminal two-thirds of the P2 proteins shared a higher degree of similarity than the C-terminal regions which were predicted to have a more flexible structure. An ORF in the 3' portion of RNA 2 of RCNMV-Aus, which could encode a protein of Mr 5000, was not present in RNA 2 of RCNMVTpM-34. RNAs 1 and 2 of RCNMV-TpM-34 and RCNMV-Aus are bilaterally compatible.
Red clover necrotic mosaic virus (RCNMV), a member of the Dianthovirus group (Matthews, 1982), has a genome of two positive-strand RNA components, RNA 1 (4.0 kb) and RNA 2 (1.4 kb) (Gould et al., 1981). Both RNA 1 and RNA 2 are required for systemic infection of plants (Gould et al., 1981 ; Okuno et al., 1983; Osman et al., 1986; Paje-Manalo & Lommel, 1989), but RNA 1 can replicate and give rise to virus particles in protoplasts in the absence of RNA 2 (Osman & Buck, 1987; PajeManalo & Lommel, 1989), implying a role for RNA 2 in cell-to-cell movement of the virus. RNA 2 encodes a protein of Mr 34K to 35K (Morris-Krsinich et al., 1983; Lommel et al., 1988), which may be analogous to the movement proteins of other plant viruses (reviewed by Hull, 1989). The complete nucleotide sequences of the RNA components of an Australian isolate of RCNMV (RCNMV-Aus) have been reported (Lommel et al., 1988; Xiong & Lommel, 1989). We now report the nucleotide sequence of RNA 2 of a Czechoslovakian isolate of RCNMV (RCNMV-TpM-34) (Musil, 1969) and compare it with that of RNA 2 of RCNMV-Aus. RCNMV-TpM-34 was propagated, virus particles were extracted and purified, and viral RNA was isolated as described previously (Osman et al., 1986). Doublestranded cDNA was synthesized (Gubler & Hoffman, 1983) either using unfractionated viral RNA as a template with random oligonueleotide primers (Taylor et t Presentaddress: NERC Instituteof Virologyand Environmental Microbiology,MansfieldRoad, OxfordOX1 3SR, U.K.
al., 1976) or with unfractionated, polyadenylated (Ahlquist et al., 1981) viral RNA, using TloG as a primer. Double-stranded cDNA was either tailed with dC and cloned into PstI-linearized dG-tailed pUC8 or pBR322 or cleaved with Sau3A and the fragments were cloned into BamHI-cleaved pUC8. cDNA clones of RNA 1 or RNA 2 were identified by agarose gel electrophoresis of viral RNA, followed by Northern blotting and hybridization with nick-translated or oligolabelled probes prepared from individual clones (Osman & Buck, 1987). The nucleotide sequences of five independently obtained cDNA clones of RNA 2 were determined (Sanger et al., 1977; Chert & Seeburg, 1985) in both directions after subcloning regions into M13 vectors (Messing & Vieira, 1982), by generating unidirectional deletions using exonuclease III (Henikoff, 1984) or by using oligonucleotide primers corresponding to known parts of the sequence. These clones were: GC200 [nucleotide (nt) 1 to 1341], GC4 (nt 1 to 803), TOBA3 (nt 714 to 981), $7 (nt 911 to 982) and SU5 (nt 979 to 1148). The sequences of the 5' end (nt 1 to 74) and also nt 959 to 1432 were determined by primer extension (Ahlquist et al., 1981) on an RNA 2 template. The 3' sequence (nt 1339 to 1448) was determined by 3' end labelling of electrophoretically purified RNA 2 with [3Zp]pCp followed by specific enzymic cleavage and gel electrophoresis (Stahl et al., 1989). The 3'-terminal nucleotide of RNA 2 was determined after complete digestion of 3" end-labelled RNA 2 with T2 ribonuclease, followed by
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Short communication
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239
CAACUGCCAAAGGUAAUAGUGUCGCGCUUAA•JUACACCCACGUAAUCCUUUCACUAGCACCAACAAUUGGUGUGGCAAUCCCAGGACACGUCACCGUAGAACUGGUAAACCCAAACGUGG
358
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CB~C~GCU~kGG~.CAGBGBBGCGC~BJkABBACACCCACGBAGU~CBUUCGC~GC~CCG~C.~kADDGG~GBGGCj~kD~C~GGACACGDC~CCGDAG~kB~GAB~kJkBC~.~G~GG 359 AUGGACCUUUUCAAGU~AUGAGUGGUCAGACCUUGUCAUGGUCACCAGGAGCAGGCAAACCCUGCC~CAUGAUAUUUUCAGUCCACCACCAACUCAACUCGGAUCACGAGCCUUUUAGGG
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AAGGACCGUUUCAAGUUAUGUCAGGUCAGACCUUGUCGUGGUCGCCAGGAGCUGGAAAGCCCUGCCUGAUGAUAUUCUCAGUCCACCACCAACUCAACUCGGAUCACGAGCCUUUUAGGG
479
UGCGUAUCACUAAUUCCGGAAUACCUACCAAGAAGUCGUAUGCAAGAUGUCAUGCGUACUGGGGGUACGACGUGGGUACGAGACACCGCUAUUACAAGUCAGAACCAGCGCGUCUGAUUG
598
IIII IIIII I I IIIIII I II llill IIIIIIIIIIIIII 11111111 llillll 11111111 II lllllIll lllllllillll IIII II 111111111 UGCGAAUCACGAAUACCGGAAUCCCAACGAAGAAAUCGUAUGCAA~AUGCCAUGCGU~UUGGGGGUUUG~CGUGGGA/kCUCGACACCGCUAUUACA/k~UCAGAACCAGCGCGUCUG~UUG
599
AGUUGGAAGUAGGUUACCAACGGACGUUACUGUCGAGCAUCAAGGCAGUGGAAGCAUACGUGCAAUUUACCUUUGACACAAGCAGGAUGGAGAGCAAUCCUCAAUUGUGCACCAAAUCUA
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GCAACGAAACCACAAAGUUGCCAGCGUCGGGACCCACUCUCAGGCCCGGUUCCGCUUUGAGUAUGAAGCGGgGAUCUCGCAGGAAUAUUGCGUACUCAUCUAAUCCGAUUAAACGUAACC
958
III I IIIIII I I llll III II III II 11
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1078
I IIII 111 i II IIIIII IIIII III I IIIIIIIIII II IIIIIIIIII I III lllllllllllillll I III I III i IT-IIIIIIIII II 11111 AAGAUGGCAAUUGGUUAGGUGACCACCUAUCUGACAAGGGCCGGGUUA-CCGACCCAAAUCCGGAAAGAC~CUAGACGAGCCGGGGAAGUCAGAUGCCUG~CCACUGAAAAAGUGGAAUC
1078
CUAUUCGAAGUACAGAAGGAGACAAGAAUUGCGUUGGCUUCGGCGAGCGACGAAAAGGUUUAAAUACAGGC~ACGAGUUCUCCUCGUAGGGUC~CUU~CCUCCGGCCUUAAGUGGGCUUU
1198
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1198
UAAUAGGGUUUCUCAUCUACAGCGUAUGGACCAUGGAUGUUCUACGGCUAGGCACGGUGUUUGCGAUAGCAUCUUGUCCUAGGUUGAUUUCUUUGCGAUUGUGGAGGGGUUGGACCCUCC
1318
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1438
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Fig. I. Alignment of the nucleotide sequences of RCNMV-TpM-34 R N A 2 (upper line of each pair) and RCNMV-Aus R N A 2 (lower line of each pair). Identical nucleotides are indicated by vertical lines; gaps are denoted as dashes. The initiation and termination codons of P2, and of an ORF with the potential to encode a protein of Mr 5000 in RCNMV-Aus (and a much truncated counterpaa in RCNMV-TpM-34), are underlined.
thin-layer chromatography (Stahl et al., 1989). Computer analysis of sequence data was carried out with HIBIO DNASIS and PROSIS software programs (Pharmacia). The complete nucleotide sequence (1448 nucleotides) of RNA 2 of RCNMV-TpM-34 is shown in Fig. 1. Analysis of open reading frames (ORFs) revealed one major ORF (nt 79 to 1056) with the potential to encode a protein, designated P2, of 326 amino acids (Mr 35755), nine amino acids longer than the P2 protein predicted from the corresponding ORF in R N A 2 of RCNMV-Aus (Lommel et al., 1988). An ORF that could encode a protein of M~ 5K was found in the 3' portion of R N A 2 of RCNMV-Aus (Lommel et al., 1988). Although the start of a corresponding ORF was found in R N A 2 of RCNMV-TpM-34, a termination codon was located after only 18 amino acids (Fig. 2). This result suggests
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Fig. 2. Comparison of the amino acid sequences of P2 of RCNMVTpM-34 (upper line) and P2 of RCNMV-Aus (lower line). A line represents identical amino acids.
Short communication
that R C N M V R N A 2 is monocistronic, in agreement with the conclusion of Lommel et al. (1988) who failed to detect a protein of Mr 5K in the products of in vitro translation of in vitro transcripts containing the 5Kencoding ORF of RCNMV-Aus R N A 2. Comparison of the nucleotide sequence of RNA 2 of RCNMV-TpM-34 with that of RCNMV-Aus (Lommel et al., 1988) revealed 83~ identity (five gaps were introduced to align the sequences) (Fig. 1). Regions with the greatest sequence identity were found at the 3' terminus (nt 1342 to 1448; 89.0~ identity) and in the Nterminal 72~ of the P2-coding region (nt 79 to 786; 87.7~ identity); the lowest sequence identity was found in part of the 3' non-coding region (nt 1057 to 1341; 77.5 ~ identity) and in a region near the C terminus of the P2-coding region (nt 787 to 942; 72.2~o identity). Comparison of the amino acid sequences of P2 of RCNMV-TpM-34 and that of RCNMV-Aus (Fig. 2) revealed 8 0 ~ direct sequence identity. (No gaps were needed to align the sequences.) The sequence of the Nterminal 236 amino acids, showing 9 2 ~ sequence identity, was the most highly conserved region. However the following 52 amino acids (amino acids 237 to 288, corresponding to nt 787 to 942) showed only 4 4 ~ sequence identity. The relatively poor conservation of this region in the P2 proteins of the two isolates suggests that its sequence may not be critical for the putative cellto-cell movement function. Analysis of potential secondary structures (Chou & Fasman, 1978; Garnier et al., 1978) of P2 of RCNMV-TpM-34 and that of RCNMVAus predicted mainly co-helical and fl-sheet structure for the first 220 amino acids, followed by predominantly random coils and//-turns for the remaining C-terminal amino acids of both proteins. This suggests that the Cterminal region has a flexible structure. Xiong & Lommel (1989) reported significant degrees of amino acid sequence homology between the putative RNA polymerase encoded by RNA 1 of RCNMV-Aus and corresponding proteins encoded by viruses in other groups, the carmovirus group (carnation mottle virus, turnip crinkle virus), the luteovirus group (barley yellow dwarf virus) and maize chlorotic mottle virus (unclassified). However we could detect no significant sequence identity between the P2 proteins of RCNMV-TpM-34 or RCNMV-Aus and any proteins corresponding to ORFs of these other four viruses. Nor was any striking identity found when the amino acid sequence of RCNMV-TpM34 P2 was compared with all available protein sequences of plant viruses. The low degree of amino acid sequence identity reported between regions of P2 of RCNMV-Aus and P3a of brome mosaic virus (BMV) (23.6 ~ identity in a 54 amino acid overlap) (Lommel et al., 1988) and P3a of cowpea chlorotic mottle virus (CCCMV) (19.7 ~o identity in a 157 amino acid overlap, including gaps) (Allison et
215
al., 1989) was conserved in P2 of RCNMV-TpM-34.
However, a search of the NBRF protein database using the Lipman & Pearson (1985) algorithm revealed similar, or greater, degrees of identity with regions in over 100 unrelated proteins. Hence the functional significance of the identity between regions in P2 of R C N M V and P3a of BMV and CCMV remains to be determined. When RNAs of RCNMV-TpM-34 and RCNMV-Aus were separated (Osman et al., 1986; Osman & Buck, 1987) and inoculated in homologous or heterologous mixtures to cowpea leaves, lesions were formed in numbers similar to those induced by inocula of the parent isolates. Hence these two isolates can form pseudorecombinants in both directions. Previous studies have shown that isolates of R C N M V and other dianthoviruses can form pseudorecombinants either bilaterally (Okuno et al., 1983; Osman et al., 1986) or unilaterally (Rao & Hiruki, 1987). The ability to form viable pseudorecombinants probably depends, amongst other things, on the ability of the viral R N A replicase to recognize sequences at the 5' and 3' ends of the heterologous RNA. It is noteworthy that 22 out of 23 nucleotides at the 3' end of RNA 2 of RCNMV-TpM-34 and RCNMV-Aus were identical, allowing formation of a stem-loop structure (Lommel et al., 1988) which may be an R N A polymerase recognition site. Eight nucleotides at the 5' termini of the RNAs were identical. We thank Dr S. M. Dodd for preliminary experiments, Dr S. A. Lommel for supplying the Australian isolate of RCNMV, the Agricultural and Food Research Council for a grant and the Science and Engineering Research Council for research studentships for S.J.M. and A.C.M.
References AHLQUIST, P., DASGUPTA,R. & KAESBERG,P. (1981). Near identity of 3' secondary structure in bromoviruses and cucumber mosaic virus. Cell 23, 183-189. ALLISON, R. F., JANDA,M. & AHLQUIST, P. (1989). Sequence of cowpea chlorotic mottle virus RNAs 2 and 3 and evidence of a recombination event during bromovirus evolution. Virology 172, 321-330. CHEN, E. Y. & SEEBURO, P. H. (1985). Supercoil sequencing: a fast and simple method for sequencing plasmid DNA. DNA 4, 165-170. Crlou, P. Y. & FASMAN,G. D. (1978). Empirical predictions of protein conformation. Annual Review of Biochemistry 47, 251-276. GARNIER, J., OSGUTHORPE, D. J. 8,; ROBSON, B. (1978). Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. JournalofMolecular Biology 120, 97-120. GOULD, A. R., FRANCKI, R. I. B., HATTA, T. & HOLLINGS, M. (1981). The bipartite genome of red clover necrotic mosaic virus. Virology 108, 499-506. GUBLER, U. & HOFFMAN, B. J. (1983). A simple and very efficient method for generating cDNA libraries. Gene 25, 263-269. HENIKOFF, S. (1984). Unidirectional digestion with exonuclease III creates target break point for DNA sequencing. Gene 28, 351-359. HULL, R. (1989). The movement of viruses in plants. Annual Review of Phytopathology 27, 213-240. LIPMAN, D. J. & PEARSON, W. R. (1985). Rapid and sensitive protein similarity searches. Science 227, 1435-1439.
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LOMMEL, S. A., WESTON-FINA, M., XIONG, Z. & LOMONOSSOFF, G. P. (1988). The nucleotide sequence and gene organization of red clover necrotic mosaic virus RNA-2. NucleicAcids Research 16, 8587-8602. MATITIEWS, R. E. F, (1982). Classification and nomenclature of viruses. Intervirology 17, 1-199. MESSING,J. & VIEIRA, J. (1982). A new pair of M 13 vectors for selecting either DNA strand of double-digest restriction fragments. Gene 19, 269-276. MORRIS-KRSINICH, B. A. M., FORSTER, L. S. & MossoP, D. W. (1983). Translation of red clover necrotic mosaic virus RNA in rabbit reticulocyte lysate: identification of the virus coat protein cistron on the larger RNA strand of the bipartite genome. Virology 124, 349356. MUSIL, M. (1969). Serological properties of certain isolates of red clover necrotic mosaic virus. Acta virologica 13, 226-234. OI~UNO, T., HIRUKI, C., RAO, D. V. & FIGUEIREDO, G. C. (1983). Genetic determinants distributed in two genomic RNAs of sweet clover necrotic mosaic, red clover necrotic mosaic and clover primary leaf necrosis viruses. Journal of General Virology 64, 19071914. OSMAN, T. A. M. & BUCK, K. W. (1987). Replication of red clover necrotic mosaic virus RNA in cowpea protoplasts: RNA I replicates independently of RNA 2. Journal of General Virology 68, 289-296.
OSMAN, T. A. M., DODD, S. M. & BUCK, K. W. (1986). RNA 2 of red clover necrotic mosaic virus determines lesion morphology and systemic invasion in cowpea. Journalof General Virology67, 203-207. PAJE-MANALO, L. & LOMMEL, S. A. (1989). Independent replication of red clover necrotic mosaic virus RNA-I in electroporated host and non-host Nicotiana species protoplasts. Phytopathology 79, 457461. RAO, A. L. N. & HIRUKI, C. (1987). Unilateral compatibility ofgenome segments from two distinct strains of red clover necrotic mosaic virus. Journal of General Virology 68, 191-194. SANGER, F., NICKLEN, S. & COULSON, A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proceedings of the National Academy of Sciences, U.S.A. 74, 5463-5467. STAHL, D. A., KRUPP, G. & STACKEBRANDT, E. (1989). RNA sequencing. In Nucleic Acids Sequencing: A PracticalApproach, pp. 137-183. Edited by C. J. Have & E. S. Ward. Oxford: IRL Press. TAYLOR, J. M., ILLMENSEE, R. & SUMMERS, J. 0976). Efficient transcription of RNA into DNA by avian sarcoma virus polymerase. Biochimica et biophysica acta 442, 325-330. XIONG, Z. & LOMMEL, S. A. (1989). The complete nucleotide sequence and genome organization of red clover necrotic mosaic virus RNA-1. Virology 171, 543-554.
(Received 16 July 1990; Accepted 12 October 1990)
