日 植 病 報 62: 4-10 (1996) Ann. Phytopathol. Soc. Jpn. 62: 4-10 (1996) Nucleotide Sequence of the 3'-Terminal Region of RNA1 of Satsuma Dwarf Virus* Toru IWANAMI**,Fumiaki YAMAO***, Takeshi SENO*** and Hiroyuki IEKI** Abstract The sequenceof the 3'-terminal3116 nucleotides of satsuma dwarf virus (SDV)RNA1 was deter- mined.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 nepovi- ruses. 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 nu- cleotides 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).