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Journal of General Virology(1992), 73, 1309-1312. Printedin Great Britain 1309 Complete nucleotide sequence of RNA 4 of rice stripe virus isolate T, and comparison with another isolate and with maize stripe virus Yafeng Zhu, 1. Takahiko Hayakawa 1 and Shigemitsu Toriyama 2 Plantech Research Institute, c/o M K C Research Center, 1000 Kamoshida-cho, Midori-ku, Yokohama-city 227 and 2National Institute of Agro-Environmental Sciences, 1-1, Kannondai, 3-chome, Tsukuba-city, Ibaraki 305, Japan The complete nucleotide sequence of R N A 4 of rice stripe virus isolate T (RSV-T) was determined and found to consist of 2157 nucleotides, containing two open reading frames (ORFs). One, deduced to be present in the 5'-proximal region of the viral-sense RNA, encodes the stripe disease-specific protein with M, 20541, and the other ORF, in the 5'-proximal region of the viral complementary sense RNA, encodes an unknown protein with Mr 32 474. Between these two ORFs there is an intergenic non-coding region that could form a secondary structure with two base-paired hairpin configurations. These characteristics indicate that RSV-T RNA 4 has an ambisense coding strategy. Comparison of the two ORFs of RSV-T with those of another isolate revealed 97-2 % and 98.0 % identity for the nucleotide sequences, and 98.3% and 98.2% identity for the amino acid sequences. The leader sequences of these two isolates were the same. However, an insertion was found in the intergenic noncoding region of RSV-T. Furthermore, comparison of the nucleotide and amino acid sequences of RSV-T RNA 4 with those of R N A 4 of maize stripe virus, which is another member of the tenuivirus group, revealed greater identity, suggesting a close phylogenetic relationship between these two viruses. Rice stripe virus (RSV) is a type member of the delphacid planthopper-borne tenuivirus group of plant viruses (Francki et al., 1991). Four species of ssRNA and four species of dsRNA are found in the purified virus preparation (Toriyama, 1982; Toriyama & Watanabe, 1989; Ishikawa et al., 1989). Recently, the complete nucleotide sequences of R N A 3 and R N A 4 of RSV have been determined and their ambisense coding strategy identified (Kakutani et al., 1990, 1991 ; Zhu et al., 1991). The virus particles contain a single coat protein with an estimated Mr of 32K (Koganezawa et al., 1975; Toriyama, 1982), which has been shown to be encoded by the viral complementary (vc) sequence of the third longest segment, RNA 3 (Kakutani et al., 1991 ; Zhu et al., 1991). A large amount of a non-structural protein, the stripe disease-specific protein (S protein), is produced in RSV-infected rice plants (Kiso & Yamamoto, 1973; Toriyama, 1986); this protein has been shown to be encoded by the fourth longest segment, R N A 4 (Hayano et al., 1990). Toriyama (1986) observed that the S proteins isolated from RSV-infected rice cultivars, and wheat and maize plants produce peptides of different Mrs. Recently, three isolates of RSV which produce different symptoms in rice plants have been reported (Hayashi et al., 1990); the M,s of their S proteins differ. However, differences between these isolates at the molecular level have not yet been identified. In this paper we report the complete nucleotide sequence of R N A 4 of RSV isolate T (RSV-T), and compare this sequence and the deduced amino acid sequences with those of another isolate (Kakutani et al., 1990), referred to as isolate M (RSV-M) (personal communication), and R N A 4 of maize stripe virus (MStV), which also encodes the non-structural protein (Huiet et al., 1990). Virus particles and viral R N A were prepared as described previously (Zhu et al., 1991). Double-stranded cDNA was synthesized according to Gubler & Hoffman (1983) by using a 20-base oligonucleotide primer (5' GGGCATATCTTTTGAGATTA 3')corresponding to known parts of R N A 4 (Takahashi et al., 1990). The double-stranded cDNA obtained was made blunt using T4 DNA polymerase and inserted into the Sinai site of pUC 19 (Yanisch-Perron et al., 1985). Transformation of Escherichia coli strain DH5 was performed according to Hanahan (1985). Sequencing of cDNA and deletion mutants thereof were carried out as described previously (Zhu et al., 1991). The cDNA covering the full length of the R N A 4 The nucleotide sequencedata reported have been submitted to the DNA Data Bankof Japan and assignedthe accessionnumberD01164. 0001-0664© 1992SGM Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 17 Jun 2017 03:01:36 1310 Short communication IO 20 30 40 50 60 70 80 ACACAAAG~CCA~GGCAUUUGUACAACGAUC~AG~AAUUUAAUCAGAAUCGAA~UGCAAGACGUA~AAAGGA~UAGAAGUUUCUGUU ~ Q D V Q n T I E V S V IO0 110 120 130 140 150 160 170 911 18(1 GGU•CUA•U•UAGGCCUAGA•UA•ACUCUAUUG•JAUGACACU•UGCCUGAGACUGUUAGCGAUAA•AUUACUCUACC•GAUUUGAAAGA•• G P I V G L D Y T L L Y D T L P E T V S D N I T L P D L K D 190 200 210 220 230 240 250 260 270 CCAGA•AGAGUCACGGAAGAUACUAAAAAGCUAAUACUCAAAGGCUGUG•U•1ACAUAGCAUAU•AUCACCCCUUGGAGACUG•CACCCUU P E R V T E D T K K L I L K G C V Y I A Y H H P L E T D T L 280 290 300 310 320 330 340 350 360 ORF 1 UUCAUCAAAGUUCACAAACAUAUACCAGAGUUUUGUCACUCAUUCC~AUCACACC~UCUAGGAGG~GAAGAUGAUGACAAUGCUCUUA~ F I K V II g 1t I P E F C It S F L S II L L G G E D b D N A L 1 370 380 390 400 410 420 430 440 GACAUUGGUCUGU•UUUCAACAUGUUGCAAC•UU•UUUGGGCGGUUGGAUAA•C•AGAAUU•UCUU•GACACCC••AAUAGGAUGUCUAAG D I G L F F N b I L Q P S L G G W I T K N F L R H P N I I H S g 450 460 470 480 490 500 510 520 830 GACCAAAU•AAAAUGCUCCUGGAUCAGAUCAU•AAGAUGGCUAAGGCUGAGAG•UCAGA••CAG•AGAG••AUGAA•AAGUG••GGAAGA•G D Q I K N L L D Q I I K H A K A E S S D T E E Y E K V W K K 540 550 560 570 580 590 | 600 610 620 630 AUGCCAACUUAUUUUGAAUCAAUUAUCCAACCUCUUCUUCAUAAAACAUAGI UUAUUUCAUAUCACAUUUUCCACCCACGAGUGCUAGCA H P T Y F E S I I Q P L L I I K T * 540 650 660 670 680 690 700 710 720 UUAUAGAAGUGGAGCU~GU~UGGGU~GAC~UUCUUGUAUGAAAAUACGAGUAU~Uh~AGUCU~AA~A~GAUG~UUUUCCAC~ 730 740 750 760 770 780 790 600 810 UA•GCU•GACAGUUUGUAAAUAAUUGUGUGUGAUUGUGCGUGUGUGUAUGUAUG•}UAAUGUAUAACUGUGCAUAGGUGUGUAGAUAUAAA 820 830 840 850 860 870 880 890 •A•UAGAAGCAAUUAAAUUGAUAUAUUCCUAU•AACU•AUAUACAUACUUGUGAGUCUUAGC••C•CUA•AUAC•CACAUAGAAACA••GA 900 910 920 930 940 950 960 970 980 GA•C•UUAUA•AAGAC•CAUUGCAAAAAUACUAAUAA•UUAG••UUUUACAAUGUCAAAA•UAAAAACUGAAAUA•CAAAAACAUGAGAAA 990 1000 1010 11120 1030 1040 1050 1060 1070 10811 AUAGA~AAUC~AAAACAAUGAAUG~UGCUAAGCAC~A~AUCCGGAUGUGGUGCGUAGC~CCAUUUUCA~A~CAUUA~UCUAUA~CCUACU 1090 1100 lll0 1120 1130 11,10 1150 1160 U••AGCAA•AACACCCAUAUGCAUGAAGUGCAUAGCA•UCU•UUA•GGGUUGACUACUAUGA•UACGUGAA•GCAU•GCAUUCUCC••GGA 1170 1180 1190 1200 1210 1220 1230 1240 |1260 1260 AAAAAGCAGAGCAGAACAAACCACAUCAAGUGCAUAGCACAAGAUGUUAGCAUACUCCGGAACUGGUUAUCUCA( ~'UACAUGAUGACAGA N l V S 1270 1280 1290 1300 1310 1320 1330 1340 1350 AACUUCAGAUUUUGAUU•CUUUG•GAUGGGUAUCUUCUUU•GCUGCUUCACCACACC•AACUCCUUCUCAG•1G•]C1•AGCUCAGGGAAGC• V E S K S K K T I P I K K F Q K V V G F I ~ X E T D L E F F S 1360 1370 1380 1390 14OO 1,110 1420 1430 CUUGCUGGUGAAAGCACCUCCAACAGCUU•CU•AACAACAUUAGUCGUAAA•UUACUAAAGCUGGACAAAUGG••UUAUAGCC•••AUC••UC K S T F A G G V A K Q V V N T T F N S F S S L t l N I A K D E 1.140 1450 1460 1470 1480 1490 1500 1510 1620 1530 UAGUGGUUCAAAUUCAAACAUCACAGUGU•AUUGGUCUUCAUGGACACUGGCAGAUCAGUCCUCU•AAUCCCCCAGAACCAAAG•GUUCU L P E F E F H V T D N T K H S v P L D T R E | G W F W L T R 1540 1550 1560 1570 1580 1500 1600 1610 1620 AGAGAUGACACAGUUCUGCACU•AGCU•UCAUCAACAGAUACGGACACUUGAAGAUUAUGCUUAUCCUCCAAAGCC•GA•AAUUCGCCAG S I V C N Q V S S D D V S V S V Q L N I I K D E L A L F N A L 1630 1640 1650 1660 1670 1680 1690 1700 1710 AGAACCCAA•ACAG•AAAAUUCUUGCUAAUUGGGUAGCGAACCUCUA•CUCAACUUGA1•CUGAGGGGUUCACAUAGGACUUG••CUAUUAU O R F 2 S G L V A F N K S I P Y R V E V E V Q D S P N V Y S K D I I 1720 1730 1740 1750 1760 1770 1780 1790 1800 GCGGAGGGUAGUUAUU~CA~UAGCUCUACC~UUAA~UCCAAUC~AGACCAUAGCAAAAGUUU~CACUCU~AGAAUGGGUAAUGGGUGAG R L T ~ I G S A R G K I G I W V H A F T E V R F F P Y I I T L 1610 1820 1830 1840 1850 1860 1870 1880 1890 AGGUUGAUGAAACCAAUAUGGAUCAACA~GUAUGU~GAAAGUUGCUUUCCUAUGGGCC~UCACAG~AUAG1j~CUGUUUGGCUAUGAACAU P Q H F W Y P D V L I N F T A K R H A K V S Y D Q K A I F H 1900 1910 1920 1930 1940 1950 1960 1970 1980 A•CAUACUUGUUCAC•••GACAU•UGAGAAG••GAAGGGUU•CAAU•CAAG•AUUGUGG•AGC••U•GUCAAUUGUAACCC•A••CUGGUU D Y K N V K V D S F S F P E L G L H T A A Q D I T V R G Q N 1990 2000 2010 2020 2030 2040 2050 2060 2070 •A•AGGC•UUUUGGAGAGAGCUAGAGAUUU••UAUU••UAUUAUCAACUCU•UUUUGGGACUCCUCACUAAGGUCA••CAUAGAGUACC•1U L P R K S L A L S K R N K N D V R K Q S E E S L D D Y L V K 2080 2090 2100 2110 2120 2130 2140 2150 ACUUUUUAAAGUGGACAAAAGUCGAGACAAAGCCAIAA•UAAAGUAUAUAUUAGCUUAAU•UCAAA•GAUAUG•CCUGACUUUGUGU 8 K L T S L L R S L A H , J Fig. 1. CompletenucleotidesequenceofRSV-TRNA 4andtwodeducedORFs(ORFl and ORF2). The numbers indicate the distance ~om the 5' terminus of vRNA. ORF1 (nucleotides 55 to 591) is predicted to be the S protein encoding region. The protein encoded by the ORF within nucleotides 1246 to 2106 was deduced ~om the vcRNA sequence and contains 287 amino acids. The underlined sequence indicates the 20-base inse~ion relative to this region of RSV-M. segment was obtained by primer extension. The nucleotide sequence o f the c D N A was determined in both directions. Parts o f the genomic c D N A were sequenced from two or three independent clones. For confirmation, the c D N A s obtained by polymerase chain r e a c t i o n (PCR) amplification (Saiki et al., 1988) were sequenced directly (Brow, 1990). Computer analysis was performed using Genetyx (Software Development). Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 17 Jun 2017 03:01:36 Short communication The complete nucleotide sequence of RSV-T R N A 4 is given in Fig. 1. RSV-T R N A 4 consists of 2157 nucleotides, and two open reading frames (ORFs) were predicted from the nucleotide sequence. One putative O R F , located in the Y-proximal region of the viral-sense R N A (vRNA), encoded a protein of 178 amino acids with an Mr of 20541 (20K protein) (Fig. 1, ORF1). Its amino acid composition was more than 98 % identical to that of purified S protein (data not shown), confirming that the 20K protein is the S protein. The other O R F , in the Y-proximal region of the v c R N A , encoded a protein of 286 amino acids with an Mr of 32474 (32K protein) (Fig. 1, ORF2). No O R F of significant length was deduced to exist in the Y-proximal regions of either v R N A or v c R N A . These observations confirmed the previous report that RSV-M R N A 4 shows an ambisense coding strategy (Kakutani et al., 1990). Between the two O R F s there is an intergenic noncoding region of 634 nucleotides. Computer analysis showed that this intergenic region can form two basepaired hairpin configurations. One involves 36 nucleotides (918 to 953) with a calculated free energy of - 36 kJ (Tinoco et al., 1973), and the other involves 46 nucleotides (1010 to 1055) with a calculated free energy of - 160 kJ (Fig. 2). These configurations might function in termination of the subgenomic R N A , as suggested previously (Auperin et al., 1984; Zhu et al., 1991). We compared the nucleotide and deduced amino acid sequences of RSV-T R N A 4 with those of RSV-M R N A 4 (Kakutani et al., 1990). A schematic summary of the comparison is shown in Fig. 3. The nucleotide sequences of ORF1 and O R F 2 were 97.2% and 98.0% identical, respectively; at the amino acid level, 93.3 % and 98.2 % of residues were identical. The intergenic non-coding region of RSV-T, however, showed 86.9% identity with that of RSV-M, this region of RSV-T containing a 20-base insertion (Fig. 1, underline). This was confirmed by direct sequencing of the c D N A synthesized by PCR. The insertion does not alter the stable hairpin structure described above. This kind of insertion or deletion has been observed in the intergenic non-coding region of the S R N A of two isolates of tomato spotted wilt virus, which also has an ambisense coding strategy (de H a a n et al., 1990; Maiss et al., 1991). These observations suggested that the intergenic non-coding region, which is predicted to have a complex higher structure, is susceptible to mutation, and that the secondary structure rather than the nucleotide sequence is important for viral activity. It remains to be elucidated whether the sequence divergence between the two isolates of RSV relates to their biological characteristics, including disease symptoms. In addition to the O R F s and the intergenic non-coding region, the leader sequences from each viral strand were also compared and shown to be 1311 AG = -160 kJ AG = -36 kJ C~G A~:U C~G C~Gi: C~;6 ;G:~IC: U:,::G A:~ U A:~U: A A:--U C:~:G; .A~ U A::~: U, C A G:~:6 ;U::~:A:: G~::C G~::C ::U:~A G-:C U-:A - U-A A-U C:~G - G A~:U: ::U;~:A :A:~:U A~:U G.:::U - - Nucleotides 918 to 953 1010 to 1055 Fig. 2. The two predicted hairpin structures in the intergenic noncoding region. Dashes and asterisks show base-pairing in the stem configuration. The numbers indicate the distance from the 5' terminus of the viral ssRNA 4 (see Fig. 1). Nucleotides Intergenic Leader ORF1 non-coding sequence region (a) I I I I 1 54 591 1245 I100 ] 97"2 [ 1 54 1 -~-~----[ (b) [ 179 - . ~ _ ~ 1 86.9 591 ] O R F 2 Leader sequence I 98.0 1225 21062157RSV_ T 1100 I RSV-M 20862137 .............. . . . . . . . . . . . . . . . . 179 ....................... ............... 287 1 1 98.2 ] RSV-T i RSV-M RSV-T 111111! .sv_M 287 1 Fig. 3. Schematic representation of the identities between the nucleotide and deduced amino acid sequences of RSV-T and RSV-M. The percentage identity in each region (leader sequence, ORF and intergenic non-coding region) is shown in the corresponding box. The upper bar (a) shows a comparison of the nucleotide sequences. The middle and lower bars (b and c) represent comparisons of the amino acid sequences encoded by v- and vcRNAs, respectively.The numbers in (a) indicate the distance from the 5' terminus of the viral ssRNA. In (b) and (c), the numbers indicate the amino acid positions relative to the first methionine of each ORF encoded by the v- and vcRNA 4, respectively. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 17 Jun 2017 03:01:36 1312 Short communication MQDVQRTIEVSVGPIVGLDYTLLYDTLPETVSDNITLPDL II II III II II Ill II lllllIl 40 II MQRSADVSIGPITGLNYTDLYDSLPSSVSDNITLLDL KDPERVTEDTKKLILKGCVYIAYHHPLETDTLFIKVHKHI I lllill I I l l i l i i l l l i l l l i l l l l II IIII KEPERVTEATKKLILKGCVETAYHHPLETDPLFASVHKHL PEFCHSFLSHLLGGEDDDNALIDIGLFFNMLQPSLGGWIT I IIIIII llllll I I IIIII II llllll Ill PDFCHSFLEHLLGGEQDENSLIDIGEFFKLLQPSLGDWIT KNFLRHPNRMSKDQIKMLLDQIIKMAKAESSDTEEYEKVW I I III II lit II III l l l l l l l l l l lilll KYYLKHPNKMSGIQIKTLLNQIINMAKAESSDTETYEKVW KKMPTYFESI IQPLLHKT [III II IIIII K K M P SYF S I V L T P L L H K V V 37 80 77 120 117 160 157 179 176 Fig. 4. Comparison of the amino acid sequence of the S protein deduced from the RSV-T RNA 4 nucleotide sequence with that of NCP encoded by MStV RNA 4. Identical amino acids are shown by dashed lines. The upper line is the RSV-T-eneoded sequence and the lower line is that of MStV. Maximum matching analysis was performed using Genetyx. identical. This observation suggests that the leader sequence could be an important factor in R N A replication and/or protein translation. Huiet et al. (1990) partially sequenced the RNA 4 segment of MStV, another member of the tenuivirus group, and reported that it encodes a major non-capsid protein (NCP). Comparison of the ORFs encoding NCP and the S protein of RSV-T revealed 69.6~o identity at the nucleotide sequence level and 74.1 ~o identity for the deduced amino acid sequence (Fig. 4). Recently, the complete nucleotide sequence of MStV RNA 3 has been reported (Huiet et al., 1991), and comparison of R N A 3 of RSV-T and MStV revealed more than 65 ~o identity in the two ORFs, and about 53~o identity in the intergenic non-coding region. These results demonstrate the close phylogenetic relationships between RSV and MStV. The function of the S protein is not known; however, the high degree of identity between the S protein of RSV and the NCP of MStV indicates that they should have similar functions. References AUPERIN, D. D., ROMANOWSKI,V., GALINSKI, M. & BISHOP, D. H. L. (1984). Sequencing studies of Pichinde arenavirus S RNA indicate a novel coding strategy, an ambisense viral S RNA. Journalof Virology 52, 897-904. BRow, M. A. D. (1990). Sequencing with Taq DNA polymerase. In PCR Protocols. A Guide to Methods and Applications, pp. 189-196. Edited by M. A. Innis, D. H. Gelfand, J. J. Sninsky & T. J. White. New York & London: Academic Press. 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Science 239, 487-491. TAKAHASHI, M., TORIYAMA, S., KIKUCHI, Y., HAYAKAWA, T. & ISHIHAMA, A. (1990). Complementarity between the 5'- and 3"-terminal sequences of rice stripe virus RNAs. Journal of General Virology 71, 2817-2821. TINOCO, J. I., BORER, P. N., DENGLER, B., LEVINE, M. D., UHLENBECK, O. C., CROTHERS, n . W. & GRALLA, J. (1973). Improved estimation of secondary structure in ribonucleic acids. Nature New Biology 246, 40-41. TORIYAMA, S. (1982). Characterization of rice stripe virus: a heavy component carrying infectivity. Journal of General Virology 61, 187-195. TORIYAMA, S. (1986). Rice stripe virus: prototype of a new group of viruses that replicate in plants and insects. MicrobiologicalSciences 3, 347-351. TORIYAMA, S. & WATANABE,Y. (1989). Characterization of single- and double-stranded RNAs in particles of rice stripe virus. Journal of General Virology 70, 505-511. YANISCH-PERRON, C., VIEIRA, J. & MESSING, J. (1985). Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mpl8 and pUC19 vectors. Gene 33, 103-119. ZHU, Y., HAYAKAWA, T., TORIYAMA, T. & TAKAHASHI, M. (1991). Complete nucleotide sequence of RNA 3 of rice stripe virus: an ambisense coding strategy. Journalof General Virology 72, 763-767. (Received 3 October 1991; Accepted 2 January 1992) Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 17 Jun 2017 03:01:36