Nucleotide Sequence of the 3'-Terminal Region of RNA1 of Satsuma Dwarf Virus*

日植病報 62: 4-10 (1996)

Ann. Phytopathol. Soc. Jpn. 62: 4-10 (1996)

Toru IWANAMI**,Fumiaki YAMAO***, Takeshi SENO*** and Hiroyuki IEKI**

Abstract The sequenceof the 3'-terminal3116 nucleotides of satsuma dwarf virus (SDV)RNA1 was determined.The sequencecontains a part of a singleopen reading frameof 2868nucleotides and a non-coding regionof 248nucleotides upstream of the poly(A) tail. The C-terminalregion of the ORFis apparently homologousto the RNA-dependentRNA polymerase of viruses.The aminoacid sequenceof this region showshomology with that of the genus Comovirus(28%, cowpea mosaic virus) followedby the genus Nepovirus(25%, grapevine fanleaf virus). There is no significanthomology with the virusesof other genera,suggesting that SDV is comparativelycloser to the como-and nepoviruses.However, the low homologyof RNA-dependentRNA polymerasegene among SDV,the genus Comovirusand the genus Nepovirusin contrast to highconservation among viruses of the genusComovirus (51-61%) and the genus Nepovirus(35-70%) suggests that SDVis distinctfrom those so far sequencedcomo- and nepoviruses. (ReceivedJanuary 19, 1995;Accepted May 7, 1995)

Key words: satsuma dwarf virus, comovirus,nepovirus, RNA-dependent RNA polymerase.

genome structure of SDV in order to elucidate the

INTRODUCTION relationship between SDV and the como- and nepoviruses. In this paper, we report cDNA cloning and Satsuma dwarf virus (SDV), which is the causal agent sequencing of the 3'-terminal 3116 nucleotides of RNA1. of satsuma dwarf of satsuma mandarin (Citrus unshiu In addition, sequence comparison was made between Marc.)9,20) is an unclassified virus7,18,19). Recently, SDV RNAs of comoviruses {cowpea mosaic virus (CPMV)13), was tentatively placed in the genus Nepovirus in the cowpea severe mosaic virus (CPSMV)1) and red clover Family Comoviridae2). Field observation suggests that mottle virus (RCMV)17)}, nepoviruses {grapevine SDV is transmitted through soil but no vector has been chrome mosaic virus (GCMV)12), grapevine fanleaf virus indentified11). It had been reported that SDV shows many (GFLV)16) and tomato black ring virus (TBRV)3)}, and common features with the comoviruses and the nepovi- polio picornavirus (human poliovirus, HPV, Sabin ruses. The virus has small isometric particles about 30 strain10). nm in diameter. The particles sediment as three compo- nents with identical coat proteins but vary in their RNA MATERIALS AND METHODS content18). They encapsidate two species of RNAs of about 1.9•~106 (RNA1) and 1.7•~106 (RNA2), which Virus and viral RNA preparation. SDV strain together constitute the viral genome19). The virus S-58 was propagated in Physalis floridana and purified as induces the formation of characteristic tubules contain- described previously18) with slight modifications. Viral ing single rows of virions in the infected cells6). All of RNA was extracted from purified virions by the conven- these properties are common to the como- and nepovi- tional SDS-phenol method and concentrated by ethanol ruses. In spite of these similarities, Usugi et al. could not precipitation. RNA was purified by oligo (dT)-latex classify SDV in the Comovirus or nepovirus group treatment which is a modification of oligo (dT)-cellulose because serological relationship was not confirmed18). affinity chromatography14). We therefore carried out a detailed investigation of the cDNA synthesis and cloning. The first strand

* Contribution No .B-215 of the Fruit Tree Research Station. The nucleotide sequence data reported in this paper will appear in the GSDB, DDBJ, EMBL and NCBI nucleotide sequence databases with the following accession number D45026. ** Okitsu Branch , Fruit Tree Research Station, Okitsu, Shimizu 424-02, Japan 果樹試験場興津支場 *** National Institute of Genetics , Yata 1111, Mishima 411, Japan 国立遺伝学研究所 Ann. Phytopathol. Soc. Jpn. 62 (1). February, 1996 5

cDNA was synthesized using a mixture of RNA1 and RNA2 as templates and oligo (dT) as a primer, and the second strand cDNA was synthesized using RNase H, Escherichia coli DNA polymerase I and T4 DNA polymerase as described by Gubler and Hoffman4). The double-stranded cDNA was ligated into the SmaI site of pUC19. The DNA was used to transform competent E. coli JM 109 cells, and the colonies were screened on LB medium with ampicillin, IPTG and X-gal. Screening and analysis of cDNA clones. The selected white colonies were grown on a small scale, and plasmid DNAs were prepared by the simple single-step procedure5). The size of the DNA insert was estimated by agarose gel electrophoresis after digestion with PstI and EcoRI. Recombinant plasmids with cDNA inserts larger than 500bp were selected, eluted electropho- retically from the gel and used as probes for Northern blot analysis with viral RNAs, employing enhanced chemiluminescence (ECL) system (Amersham). DNA sequencing. The recombinant clone S58CD5, which contained the largest cDNA insert was selected for sequence analysis. Series of unidirectionally deleted cDNAs were prepared after digestion with PstI and SalI using exonuclease III and S1 nuclease (Phar- macia). The dideoxynucleotide chain termination reac- Fig. 1. Northern blot hybridization of SDV RNAs with tion on a DNA thermal cycler was conducted with Taq cDNA probes using enhanced chemilumines- DNA polymerase and M13 reverse dye primers (Applied cence (ECL) system. The RNAs electrophoresed Biosystems). For sequencing of another orientation of in a 1.0% agarose gel containing formaldehyde S58CD5, series of cDNA were prepared from S58CD5 by were transferred to a nylon membrane and generation of blunt ends and self-ligation using T4 DNA hybridized with inserts of S58CD5 after diges- polymerase (Takara) after digestion with EcoRI and one tion with PstI and EcoRI (lane 1) or BamHI and of the following enzymes; AflII, AxyI, BstEII, BstXI, EcoRI (lane 2). EspI, HpaI, MluI, NcoI, SacII, SpeI, StuI. These cDNAs

Fig. 2. Nucleotide sequencing strategy. The relationship between SDV RNA1 and the cDNA insert of S58CD5 is

presented. Arrows below the map indicate the directions of the nucleotide sequence determined from subclones. The abbreviations for the restriction enzymes are as follows. Af, AflII; Ax, AxyI; BE , BstEII; BX, BstXI; Es, EspI; Hp, HpaI; Ml, MluI; Nc, NcoI; S1, SacI; S2, SacII; Sp , SpeI; St, StuI. The arrows (•¨, •©) indicate the nucleotide sequence determined using -21M13 dye primers and M13 reverse dye primers , respectively (See text). 6 日本植物病理学会報第62巻第1号平成8年2月 as well as SacI-digested and self-ligated cDNA were Computer analysis. Nucleotide and amino acid used for the dideoxynucleotide chain termination reac- sequence data were analyzed and compared with those tion on a DNA thermal cycler with Taq DNA polymer- in the EMBL-GDB, GenBank, NBRF-PDB and SWISS- ase and -21M13 dye primers (Applied Biosystems). The PROT, using a GENETYX-MAC/CD software (Soft- DNA sequencing was analyzed in an automated DNA ware Development Co., Japan). A phylogenetic tree was sequencer (373A, Applied Biosystems). generated using Unweighted Pair Group Means Analysis Ann. Phytopathol. Soc. Jpn. 62 (1). February, 1996 7

Fig. 3. Nucleotide sequence of the 3'-terminal 3116 nucleotides of SDV RNA1. The predicted amino acid sequence

of the single large ORF is presented below the nucleotide sequence. The restriction site of BamHI is shown

by •b at the nucleotide position 2188. The region which contains four blocks of conserved motifs of RNA-

dependent RNA polymerase shown in Fig. 4 is indicated by •¥, •¥. The stop colon at the end of the large ORF

is indicated by an asterisk. The nucleotide sequence identical with a putative polyadenylation signal is

underlined. 8 日本植物病理学会報第62巻第1号平成8年2月

(UPGMA) after the amino acid sequences were aligned, reading frames of the (+) and (-) strands contained which was performed by the program MAlign in the many stop colons and few extended ORFs. GENETYX-MAC software. The 3' non-coding region of RNA1 of SDV contained a putative polyadenylation signal AAUAAA at 116 RESULTS nucleotides upstream from the poly (A) tail. Comparison of the putative proteins of the ORF Homology of the 3' region of RNA1 and RNA2 with other viral proteins Both SDV RNA1 and RNA2 were successfully To assess the function of the putative ORF product of purified by oligo (dT)-latex treatment, suggesting that SDV, we searched for amino acid sequence homologies both RNAs contain poly (A) tails at the 3' termini. Northern blot analysis showed that all the eight cDNA inserts including S58CD5 hybridized with both RNA1 and RNA2. The fragment of the S58CD5 which had been prepared by digestion with BamHI and EcoRI hybridized only with RNA1 (Fig. 1). Sequence analysis revealed that BamHI site of S58CD5 corresponds to the positions 924-928 nucleotides upstream of the poly (A) tail (positions 2188-2192 in Fig. 3). These results shows that S58CD5 is complementary to RNA1 and that there is a region of strong sequence homology in the 3'- terminal region of RNA1 and RNA2. This 3'-conserved region of RNA1 and RNA2 was shown to be 242 nucleotides by sequencing the clone S58CD5 and S58CD21 which is specific to RNA2 (data not shown). Sequence determination of 3' region of RNA1 of SDV S58CD5 was used for sequence analysis of RNA1 of SDV. The nucleotide sequencing strategy and orienta- Fig. 4. Alignments of the putative RNA-dependent RNA polymerase domains of the protein of tion of S58CD5 are shown in Fig. 2. The sequence of the SDV (indicated by •¥, •¥ in Fig. 3) with the four 3'-terminal 3116 nucleotides upstream of the poly (A) tail motifs of the RNA-dependent RNA polymer- is shown in Fig. 3. ases of CPMV (amino acid positions 1427 to Gene organization of the 3'-terminal region 1583), CPSMV (1417 to 1573), RCMV (1424 to Computer analysis revealed a part of one large open 1580), GFLV (1726 to 1876), TBRV (1712 to reading frame (ORF) consisting of 2868 nucleotides in 1866), GCMV (1695 to 1849) and HPV (1970 to one of the reading frames of the (+) strand (virion 1989). The lengths separating the motifs are polarity). The deduced amino acid sequence of the indicated. Highly conserved amino acids are protein encoded by this ORF is presented in Fig. 3. Other boxed.

Fig. 5. Phylogenetic tree of the RNA-dependent RNA polymerase gene of satsuma dwarf virus (SDV), the comoviruses {cowpea mosaic virus (CPMV), cowpea severe mosaic virus (CPSMV), red clover mottle virus (RCMV)}, and the nepoviruses {grapevine chrome mosaic virus (GCMV), grapevine fanleaf virus (GFLV), tomato black ring virus (TBRV). The values of the horizontal lines show the calculated evolutionary distance. Ann. Phytopathol. Soc. Jpn. 62 (1). February, 1996 9 between the protein and other proteins in databases. of cowpea severe mosaic virus genomic RNAs: Infec- Homology was scored with the comoviruses followed by tious transcripts and complete nucleotide sequence of the nepoviruses. Homologies with other viruses were RNA1. Virology 191: 607-618. low. The putative amino acid sequence of the C-terminal 2. Goldbach, R., Martelli, G.P. and Milne, R.G. (1995). In region of the ORF was apparently homologous to the Virus Taxonomy: Sixth Report of the International RNA-dependent RNA polymerase (RdRp) of viruses8). Committee on Taxonomy of Viruses (Murphy, F.A. et Four blocks of conserved motifs of RdRp15)were found al. eds.), Springer-Verlag, Wien, pp. 341-347. 3. Grief, C., Hemmer, O. and Fritsch, C. (1988). Nu- between nucleotide positions 1654 and 2121 (Figs. 3, 4). cleotide sequence of tomato black ring virus RNA1. J. The sequence homologies between the predicted RdRp Gen. Virol. 69: 1517-1529. of SDV and the corresponding regions of the other 4. Gubler, U. and Hoffman, B.J. (1983). A simple and very viruses shown in Fig. 4 were 28% (CPMV), 27 efficient method for generating cDNA libraries. Gene (RCMV), 26% (CPSMV), 25% (GFLV), 24% (TBRV), 25: 263-269. 24% (GCMV), 20% (HPV), while homologies within 5. He, M., Wilde, A. and Kaderbhai, M.A. (1990). A sim- comoviruses and nepoviruses ranged from 51% (CPMV ple single-step procedure for small-scale preparation of vs. CPSMV) to 61% (CPMV vs. RCMV), 35% (GFLV vs. Escherichia coli plasmids. Nucl. Acids Res. 18: 1660. TBRV) to 70% (GCMV vs. TBRV), respectively. The 6. Hibino, H., Tsuchizaki, T., Usugi, T. and Saito, Y. homologies between the genus Comovirus and the genus (1977). Fine structures and developmental process of Nepovirus were 30% (RCMB vs. TBRV) to 32% (CPMV tubules induced by mulberry ringspot virus and satsuma vs. GCMV). The phylogenetic tree of the RdRp polymer- dwarf virus infections. Ann. Phytopathol. Soc. Jpn. 43: ase gene revealed that SDV was clustered into neither 255-264. the comoviruses nor the nepoviruses (Fig. 5). 7. Iwanami, T., Koizumi, M. and Ieki, H. (1993). Diversity The function of the N-terminal region of the part of of properties among satsuma dwarf virus and related the ORF could not be elucidated because the homology viruses. Ann. Phytopathol. Soc. Jpn. 59: 642-650. 8. Kamer, G. and Argos, P. (1984). Primary structural with other proteins in the database was very low. comparison of RNA-dependent polymerases from plant, animal and bacterial viruses. Nucl. Acids Res. 12: 7269- DISCUSSION 7282. 9. Kishi, K. and Tanaka, S. (1964). Studies on the indica- The affinity of both RNA1 and RNA2 to oligo (dT)- tor plants for citrus viruses. 2. Mechanical transmission latex suggested that both RNAs contain poly (A) tails at of the virus causing satsuma dwarf to sesame (Sesamum the 3' termini. The poly (A) tail of RNA1 was confirmed indicum L.). Ann. Phytopathol. Soc. Jpn. 29: 142-148. by sequencing. The Northern blot analysis revealed the 10. Kitamura, N., Semler, B.L., Rothberg, P.G., Larsen, presence of a region with strong homology in the 3'- G.R., Adlere, C.J., Dorner, A.J., Emini, E.A., Hanecak, terminal region of RNA1 and RNA2. These features are R., Lee, J.L., Van der Werf, S., Anderson, C.W. and similar to those of the como- and nepoviruses. Wimmer, E. (1981). Primary structure, gene organiza- The computer search indicated that SDV RNA1 en- tion and polypeptide expression of poliovirus RNA. codes RdRp in the 3' region. RdRp is also encoded in the Nature 291: 547-553. 3' region of RNA1 of the como- and nepoviruses. 11. Koizumi, M., Kano, T., Ieki, H. and Mae, H. (1988). In The homology in the amino acid sequence of the Proc. 10th Conf. IOCV (Timmer, L.W. et al. eds.), IOCV, Riverside, Calif., pp. 348-352. putative RdRp between SDV and the como- and nepoviruses suggests that these viruses are comparatively 12. Le Gall, O., Candresse, T., Brault, V. and Dunez, J. (1989). Nucleotide sequence of Hungarian grapevine similar than other plant viruses. However, the low chrome mosaic nepovirus RNA1. Nucl. Acids Res. 17: homology of RdRp gene among SDV, the genus 7795-7807. Comovirus and the genus Nepovirus (24-28%) in contrast 13. Lomonossoff, G.P. and Shanks, M. (1983). The nu- to high conservation among viruses of the genus cleotide sequence of cowpea mosaic virus B RNA. Comovirus (51-60%) and the genus Nepovirus (35-70%) EMBO J. 2: 2253-2258. suggests that SDV is distinct from these definite 14. Nakazato, H. and Edmonds, M. (1974). Purification of comoviruses and nepoviruses which have been se- messenger RNA and heterogeneous nuclear RNA quenced. Therefore, we consider that a new genus for containing poly (A) sequences. Methods Enzymol. 29: SDV should be established in the Family Comoviridae 431-443. following the nomenclature described in the sixth report 15. Poch, O., Sauvaget, I., Delarue, M. and Tordo, N. (1989). of the International Committee on Taxonomy of Identification of four conserved motifs among the RNA- Viruses2). Further investigations for the determination dependent polymerase encoding elements. EMBO J. 8: of the complete sequence of RNA1 and RNA2 of SDV 3867-3874. are under way to confirm this hypothesis. 16. Ritzenthaler, C., Viry, M., Pinck, M., Margis, R., Fuchs, M. and Pinck, L. (1991). Complete nucleotide sequence and genetic organization of grapevine fanleaf nepovirus Literature cited RNA1. J. Gen. Virol. 72: 2357-2365. 1. Chen, X. and Bruening, G. (1992). Cloned DNA copies 17. Shanks, M. and Lomonossoff, G.P. (1992). The nu- 10 日本植物病理学会報第62巻第1号平成8年2月

cleotide sequence of red clover mottle virus bottom 温州萎縮ウイルス(SDV)の2成分のウイルス核酸(RNA1, component RNA. J. Gen. Virol. 73: 2473-2477. RNA2)のうち,RNA1について3'末端のポリA配列の上流の 18. Usugi, T. and Saito, Y. (1977). Some properties of 3116塩基の塩基配列を決定した。その結果,上流から続くと考 satsuma dwarf virus. Ann. Phytopathol. Soc. Jpn. 43: えられる一つの長い読み取り枠(ORF)と248塩基からなる3' 137-144. 末端非翻訳領域が認められ,このORFのC末端領域にはRNA 19. Usugi, T. and Saito, Y. (1979). Satsuma dwarf virus. 依存性RNAポリメラーゼがコードされていると考えられた。 CMI/AAB Description of Plant Viruses. No.208. RNAポリメラーゼのアミノ酸配列はコモウイルス属の 20. Yamada, S. and Sawamura, K. (1952). Studies on the cowpea mosaic virus (CPMV)およびネポウイルス属のgrape- dwarf disease of satsuma orange. Citrus unshiu Mar- vine fanleaf virus (GFLV)にそれぞれ28%と25%の相同性を covitch (preliminary report). Bull. Hort. Div. Tokai- 示し,その他の属のウイルスとはほとんど相同性がなく,SDV Kinki Agric. Exp. Stn. 1: 61-71. が比較的コモウイルス属およびネポウイルス属のウイルスに近縁であることが示唆された。しかし,SDVとこれらのウイルス和文摘要とのRNAポリメラーゼの相同性は,コモウイルス属あるいはネポウイルス属内のウイルス間の相同性(51～61%, 35～70%) 岩波徹・山尾文明・瀬野桿二・家城洋之:温州萎縮ウイルスより低いので,SDVはこれまでに塩基配列の解読されたコモウ RNA1の3'末端領域の塩基配列イルス属およびネポウイルス属のウイルスとはやや異なるウイルスと考えられた。