The nucleotide sequence of the M RNA segment of ... - Semantic Scholar

Journal of General Virology (1992), 73, 2795-2804. Printed in Great Britain

2795

The nucleotide sequence of the M RNA segment of tomato spotted wilt virus, a bunyavirus with two ambisense RNA segments Richard Kormelink,* Peter de Haan, Cor Meurs, Dick Peters and Rob Goldbach Department o f Virology, Binnenhaven 11, 6709 P D Wageningen, The Netherlands

The complete sequence of the tomato spotted wilt virus (TSWV) M R N A segment has been determined. The RNA is 4821 nucleotides long and has an ambisense coding strategy similar to that of the S R N A segment. The M R N A segment contains two open reading frames (ORFs), one in the viral sense which encodes a protein with a predicted size of 33-6K, and one in the viral complementary sense which encodes the precursor to the G 1 and G2 glycoproteins, with a predicted size of 127.4K. Both ORFs are expressed via the synthesis of subgenomic mRNAs that possibly terminate at a stable hairpin structure, located in the

intergenic region. The precursor for the glycoproteins contains a sequence motif (RGD) which is characteristic of cellular attachment domains. Significant sequence homology was found between the G1 glycoproteins of members of the genus Bunyavirus and a corresponding region in the glycoprotein precursor of TSWV, indicating a close evolutionary relationship between these viruses. With the elucidation of the M R N A sequence, the complete nucleotide sequence of T S W V has been determined. TSWV represents the first member of the Bunyaviridae shown to contain two ambisense R N A segments.

Introduction

331.5K which represents the putative viral transcriptase. Expression of this protein has been demonstrated to occur via the synthesis of a full-length m R N A (Kormelink et al., 1992a). The S R N A (2916 nucleotides long) has an ambisense gene arrangement and encodes the N protein of 29K in the viral complementary (vc) sense and a non-structural protein (NSs) of 52-4K in the viral (v) sense (Kormelink et al., 1991). Both proteins are expressed from subgenomic mRNAs, transcribed from complementary strands via a process of cap-snatching (Kormelink et al., 1992b). The m R N A molecules terminate at the central intercistronic region, most probably in a long AU-rich hairpin (de Haan et al., 1990; Ko?melink et al., 1992a). Here we report the complete nucleotide sequence of the genomic M R N A segment of TSWV. It is shown that this R N A segment, like the S R N A segment, has an ambisense gene arrangement, encoding a putative nonstructural protein in the viral sense and a precursor to G 1 and G2 in the viral complementary sense. The complete nucleotide sequence of the TSWV genome is now available, allowing precise comparison with the animalinfecting members of the Bunyaviridae.

Tomato spotted wilt virus (TSWV) is an enveloped plant virus that causes great yield losses in many economically important crops in (sub)tropical and temperate regions. More than 400 species in 50 plant families, both monoand dicotyledons, are susceptible to TSWV (Cho et al., 1987; Peters et al., 1991). The virus is exclusively transmitted by thrips in a persistent manner (Sakimura, 1962; Paliwal, 1974). Based on morphological and molecular data, TSWV has recently been classified into a newly created genus, Tospovirus, within the large family of arthropod-borne Bunyaviridae (Francki et al., 1991 ; Elliott, 1990). Typically for members of the Bunyaviridae, TSWV is a spherical membrane-bound particle, 80 to 110 nm in diameter, covered with surface projections consisting of two glycoproteins denoted G1 (78K) and G2 (58K) (Mohamed et al., 1973; Tas et al., 1977). The virus contains three ssRNA segments, S (2.9 kb), M (5.0 kb) and L (8.9 kb). Each R N A segment is associated with nucleocapsid (N) proteins to form pseudo-circular nucleocapsid structures (van den Hurk et al., 1977; Mohamed, 1981; de Haan et al., 1989). Recently, the nucleotide sequences of the S and L R N A segments have been determined (de Haan et al., 1990, 1991). The L R N A (8897 nucleotides long) is of negative polarity, encoding a single large protein of 0001-1095 © 1992SGM

Methods Virus and plants. The Brazilian isolate BR-01 of TSWV was maintained in Nieotiana rustiea 'America' by thrips transmission and

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mechanical inoculation, Virus was purified according to the method of Tas et al. (1977). Viral nucleocapsids were isolated from infected leaf tissue as described by de A,vila et al. (1990). RNA from virus particles or nucleocapsids was isolated by treatment with 1% SDS, followed by phenol extraction and ethanol precipitation. Molecular cloning and sequence determination. First-strand eDNA to the M RNA segment of TSWV was synthesized using a specific oligonucleotide complementary to the 3' end of the M RNA (M1; de Haan et al., 1989). Second-strand synthesis was performed according to the method of Gubler & Hoffman (1983). Double-stranded eDNA was made blunt-ended using T4 DNA polymerase and subsequently cloned into pUC19 after addition of EcoRI adaptors (Amersham). To verify the sequences at the 5' and 3' ends of the M RNA, an additional cloning experiment was done. To obtain eDNA clones containing the 3' end of the M RNA, 5 l.tgof genomic RNA was polyadenylated at the 3' end using poly(A) polymerase (Bethesda Research Laboratories) according to the method of Devos et al. (1976). First-strand eDNA synthesis was primed with oligo(dT) and subsequently used in a polymerase chain reaction (PCR) to amplify the 3' end sequences of the M RNA. For this purpose, oligo(dT)16 t8 and J 16, identical to nucleotides 4548 to 4562 of the vRNA strand, were used. For cloning the Y-terminal sequence, an oligonucleotide complementary to the M RNA sequence at nucleotide positions 584 to 600 from the 5' end (Z10; dCCCTTCAGGATGACTTG) was used to prime first-strand eDNA, followed by second-strand synthesis (Gubler & Hoffman, 1983). DNA sequencing was performed by the dideoxynucleotide chain termination method (Sanger et al., 1977) on alkali-denatured dsDNA templates (Zhang et al., 1988). Nucleotide and amino acid sequences were compiled and analysed using programs developed by the University of Wisconsin Genetics Computer Group (Devereux et al., 1984). RNA extraction and Northern blot analysis. Total cellular RNA was extracted from TSWV-infected N. rustica plants according to the method of de Vries et al. (1982). Total RNA samples of 7 gg were resolved by electrophoresis through 1% agarose gels after treatment with methylmercuric hydroxide (Bailey & Davidson, 1976). The RNA was blotted onto GeneSereen (New England Nuclear) and hybridized to 32p-labelled riboprobes of 2(SWV M RNA-specific sequences corresponding to the small open reading frame (ORF) (probe M-2; transcribed from a 600 bp EcoRI-HindllI eDNA fragment from clone M 16) or the large ORF (probe M-3; transcribed from a 800 bp EcoRIHindIII eDNA fragment from clone M43) as described previously (Kormelink et al., 1992a).

Results Cloning and sequencing o f the T S W V M R N A

T h e sequence o f 30 n u c l e o t i d e s at the 3' e n d o f the M R N A was d e t e r m i n e d after end-labelling, followed b y p a r t i a l d e g r a d a t i o n w i t h base-specific r i b o n u c l e a s e s (de H a a n et al., 1989). F r o m the d e d u c e d sequence, a synthetic oligonucleotide (M1; dAGAGCAATCAGTG C A A A ) c o m p l e m e n t a r y to the 20 3 ' - t e r m i n a l nucleotides was s y n t h e s i z e d a n d used to p r i m e first-strand synthesis, followed by s e c o n d - s t r a n d synthesis a n d cloning in p U C 1 9 . A set o f o v e r l a p p i n g e D N A clones to M R N A was o b t a i n e d after r e p e a t e d s c r e e n i n g o f the e D N A library, w h i c h y i e l d e d a r e s t r i c t i o n m a p c o v e r i n g a p p r o x i m a t e l y 4800 n u c l e o t i d e s (Fig. 1). T h e n u c l e o t i d e

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Fig. 1. Cloning strategy for the TSWV M RNA segment. M1, J16 and Z10 represent the synthetic oligonucleotides used for cDNA synthesis. The numbers correspond to the cDNA clones used. Restriction enzymes are abbreviated as follows: Bg, BglII; E, EcoRI; H, HindlIl; K, KpnI; S, SstI; X, XbaI.

sequence o f the T S W V M R N A s e g m e n t was d e t e r m i n e d from e D N A clones M201, M10, M l l , M43, M I 2 , M13, M14, M16, M17, M18, M24, M28 a n d M31 (Fig. 1). T h e 3 ' - t e r m i n a l s e q u e n c e o f the M R N A was verified in a n i n d e p e n d e n t e x p e r i m e n t using R N A f r o m a s e p a r a t e virus purification. G e n o m i c R N A was polya d e n y l a t e d at the 3' e n d a n d first-strand e D N A was synthesized by p r i m i n g with oligo(dT). S i n g l e - s t r a n d e d c D N A c o n t a i n i n g the 3 ' - t e r m i n a l sequence o f the M R N A was a m p l i f i e d by P C R using oligo(dT) a n d o l i g o n u c l e o t i d e J16 ( d G T T G A A T C G A T G C A G ) as p r i m e r s (Fig. 1). T h e D N A was s u b s e q u e n t l y cloned into p U C 1 9 a n d clones were selected using a 250 b p K p n I restriction f r a g m e n t o f clone M l l as a p r o b e in a hybridization experiment. The conserved 5'-terminal sequence o f the M R N A was p r e s e n t in clones M24, M28 a n d M31 as they c o n t a i n e d the sequence 5' A G A G C A A T C A G T G C A . . . . c o m p l e m e n t a r y to the sequence at the Y e n d o f the T S W V M R N A . T h e 5 ' - t e r m i n a l sequence was also verified in a n i n d e p e n d e n t e D N A synthesis e x p e r i m e n t . F o r this purpose, p r i m e r Z10 (Fig. 1) was used to p r i m e firsts t r a n d e D N A synthesis, followed by s e c o n d - s t r a n d synthesis a c c o r d i n g to the m e t h o d o f G u b l e r & H o f f m a n (1983). T h e e D N A was m a d e b l u n t - e n d e d by using T4 D N A p o l y m e r a s e a n d s u b s e q u e n t l y c l o n e d into the S i n a i site o f p U C 1 9 . A 400 b p E c o R I - B g l I I r e s t r i c t i o n f r a g m e n t f r o m clone M28 was used as a p r o b e to select positive clones in a colony h y b r i d i z a t i o n e x p e r i m e n t . T h e i r n u c l e o t i d e sequences were i d e n t i c a l to those o f clones M24, M28 a n d M31, e x c e p t for t h e 16 5 ' - t e r m i n a l nucleotides, w h i c h were p r e s e n t only in M24, M28 a n d M31. Characteristics o f the T S W V M R N A sequence

T h e c o m p l e t e n u e l e o t i d e sequence o f t h e T S W V M R N A (Fig. 2) is 4821 nucleotides long, a n d has a base

Nucleotide sequence of T S W V M RNA

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Fig. 2. The complete nucleotide sequence of TSWV M R N A (numbered ~ o m the 5' end of the v R N A strand) and the predicted gene products. The deduced amino acid sequence of the protein encoded by the v R N A is shown above the R N A sequence. The sequence of the protein e n c ~ e d by the v c R N A strand is shown below the R N A sequence. Potential ~glycosylation sites are underlined. The asterisks indicate termination codons. The cell attachment site is boxed.

composition of 31-8~o A, 32-5~ U, 18.0~ C and 17.6~ G. The termini of the M R N A show some complementarity for about 64 nucleotides, and can be folded into a stable panhandle structure with a free energy of - 187-3 kJ/mol (Fig. 3 a). The length of the panhandle is similar to those found for the L and S RNA segments, which also involve basepairing of approximately 65 to 70 nucleotides (de Haan et al., 1990, 1991). An internal inverted sequence of A-rich stretches followed by U-rich stretches is located between positions 1075 and 1245 (numbered from the 5' end of the v R N A strand), and can be folded into a hairpin structure with a free energy of -151-2 kJ/mol. The stem of the hairpin structure is slightly shorter (85 nucleotides) than that found in TSWV S R N A (126 nucleotides). A sequence located at the top of the hairpin ( C A A A C U U U G G ; Fig. 3 b) is conserved in the S R N A segments of TSWV and a second tospovirus, Impatiens necrotic spot virus (INSV) ( C A A U U U G G ) (de Haan et al., 1992). This nucleotide sequence motif may therefore have a possible role as a signal for transcription termination of the ORFs within the ambisense R N A segments of tospoviruses. Predicted gene products of T S W V M R N A

Analysis of all possible reading frames of the v- and vcRNA revealed the presence of two ORFs located on opposite strands (Fig. 4). One smaller ORF is located in the vRNA strand, with the first A U G codon being at

position 101, and terminating with a U A G stop codon at position 1007. This ORF encodes a protein of 302 amino acids (Fig. 2) with a predicted Mr of 33"6K. Analysis of this amino acid sequence did not reveal any hydrophobic domains that could function as signal sequences or transmembrane domains, according to the hydropathy algorithms of Hopp & Woods (1981) and Kyte & Doolittle (1982) (data not shown). Instead, a rather acidic C terminus was found, as can be seen by the number of aspartic (D) and glutamic (E) acid residues (Fig. 2). An acidic C terminus is also present in the large protein encoded by the L R N A segment (de Haan et al., 1991). The amino acid sequence encoded by the small ORF contains two potential N-glycosylation sites, but it is not known whether these are used in vivo. A search of the EMBL protein and nucleotide sequence database did not reveal significant homology to any other published sequence. Since a protein of 33.6K is not found in purified virus particles on SDS-polyacrylamide gels, the predicted protein is probably non-structural and therefore is tentatively named NSM. A second, larger ORF is located in the vcRNA strand, with the first A U G codon being at position 4737 (numbered from the 5' end of the vRNA) and terminating with a U G A codon at position 1332. This ORF has the capacity to encode a protein of 1136 amino acids with an Mr of 127"4K. Previous studies on morphologically defective isolates of TSWV have linked the genetic information for the glycoproteins to the M R N A

2799

Nucleotide sequence of T S W V M RNA

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Fig. 3. (a) The complementary sequences at the 5' and 3' ends, and (b) the secondary structure in the intergenic region of TSWV M RNA. The nucleotide positions are numbered from the 5' end of the viral RNA. Asterisks represent gaps corresponding to unpaired nucleotides in the sequence. The conserved sequence motif, a putative transcription termination signal, is indicated with a bar,

(Verkleij & Peters, 1983). Analysis of the amino acid sequence of the predicted translation product of the vcORF demonstrated the presence of eight potential N-glycosylation sites (Fig. 2) and several hydrophobic regions (Fig. 5), as expected for a precursor to the glycoproteins. Furthermore, a search of the EMBL protein database revealed sequence similarity to the glycoprotein precursor encoded by the M RNA of Bunyamwera and snowshoe hare (SSH) viruses, members of the genus Bunyavirus. This was mainly restricted to G 1 with 45 ~ similarity and 22 % identity in a 485 amino acid overlap. However, no identity was found with members from other genera of the Bunyaviridae (Fig. 6a; Lees et al., 1986; Collett et al., 1985; Ihara et al., 1985; Ronnholm & Pettersson, 1987; Schmaljohn et al., 1987). Alignment of the amino acid sequences of GI of SSH, Bunyamwera, La Crosse and Germiston viruses (Eshita & Bishop, 1984; Lees et al., 1986; Grady et al., 1987; Pardigon et al., 1988) with the C-terminal part of the protein encoded by the vcORF of TSWV M R N A demonstrated the presence of a homologous stretch of amino acids, indicating motifs in glycoprotein G1 conserved among members of the genera Bunyavirus and Tospovirus (Fig. 6b). Based on these data, and because the two envelope glycoproteins of TSWV have previously been found not to be encoded by either S or L R N A (de Haan et al., 1990, 1991), it is concluded that the vcORF in the M R N A segment,

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similar to the M RNAs of animal-infecting bunyaviruses, encodes the precursor to the glycoproteins (GI and G2). Taking into account the fact that the sizes of glycoproteins G 1 (78K) and G2 (58K) are overestimated owing to glycosylation, the size of the predicted protein of the large ORF fits with this conclusion. From the similarity between G1 of the bunyaviruses and the glycoprotein precursor of TSWV it is assumed that the C-terminal portion of the glycoprotein precursor encodes the G1 glycoprotein. The hydropathy plot of the precursor is in agreement with this, indicating that the larger glycoprotein (G1; 78K) is located at the C terminus and the smaller glycoprotein (G2; 58K) at the N terminus (Fig. 5, Fig. 6b). Furthermore, analysis of this plot shows a hydrophobic N-terminal stretch of 30 residues that is probably a signal sequence for translocation across the endoplasmic reticulum (ER) membrane. This sequence would be cleaved from the glycoprotein precursor at amino acid residue 35 (VLLAFLIFRATDA^KV), as deduced using the algorithms of Von Heijne (1986). Similarly, the hydrophobic domain between amino acid residues 400 and 500 probably functions as a signal sequence for the G1 protein. Hydrophobic domains found between amino acid residues 300 and 400, and 1000 and 1100 may represent domains for anchoring the two glycoproteins in the ER membrane (Fig. 5). Based on these assumptions, the Mrs

1000

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Fig. 6. (a) Dot plot comparison of the glycoprotein precursors of TSWV, Bunyamwera, Punta Toro and Hantaan viruses using the Compare (window 30, stringency 16) and Dotplot programs of the GCG package. Sequence data were obtained from Lees et al. (1986) (Bunyamwera virus), Ibara et al_ (1985) (Punta Toro virus) and Schmaljohn et al. (1987) (Hantaan virus). Homologous sequences are indicated by diagonal lines. (b) The conserved amino acid sequence within the G 1 glycoprotein of TSWV compared with that of members of the genus Bunyavirus. Hydrophobic domains are indicated by hatched areas. X and hyphens refer to non-identical amino acids•

of G2 and G1 can be estimated at about 46K and 75K, respectively. The latter is in good agreement with the size of G1 estimated previously (78K). However, the calculated size of G2 is considerably smaller than the size estimated previously from SDS-polyacrylamide gels (58K), possibly due to glycosylation. Analysis of the amino acid sequence of the glycoprotein precursor also revealed the presence of an RGD motif (Fig. 2; Ruoslahti & Pierschbacher, 1986, 1987) at the N terminus, immediately downstream of the hydrophobic signal sequence. As this latter sequence is proposed to be cleaved from the precursor protein, it is anticipated that the RGD motif is not found close to a membrane anchor site, but rather is exposed on the viral envelope. Transcriptional expression o f the M R N A segment

Analysis of M RNA-derived RNA species in infected N. rustica plants recently demonstrated the presence of a possible (4 kb) subgenomic mRNA transcribed from the 3' part of the vRNA (Kormelink et al., 1992a). This result was the first indication of the presence of two distinct cistrons in M RNA. The nucleotide sequence of this RNA also demonstrates the presence of two cistrons in M RNA, arranged in an ambisense manner. To demonstrate that these cistrons in M RNA are expressed

Nucleotide sequence o f T S W V M R N A

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2

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nucleocapsid R N A fractions demonstrated the presence o f M R N A strands of both polarities (Fig. 7, lanes 1, 3, 5 and 7), the M v c R N A species being present in smaller amounts (Fig. 7, lanes 3 and 7; Kormelink et al., 1992a). In addition to genome-length M R N A species, a viralsense subgenomic R N A species of approximately 1.1 kb (Fig. 7, lanes 1 and 2) was detected with riboprobe M2-vc, corresponding to the small ORF. A riboprobe (probe M3-v) corresponding to the glycoprotein precursor gene also hybridized with genome-length M R N A species and revealed a second subgenomic R N A species of approximately 3-5 kb (Fig. 7, lanes 7 and 8). A fast migrating R N A species (Fig. 7, lane 8) was detected with probe M3-v, but appeared to be non-specific as it was also detected in a total R N A extract from healthy plants. No subgenomic M-specific R N A species were found when probes M2-v and M3-vc were used in a hybridization experiment (Fig. 7, lanes 3 to 6). In view of the location and polarity of both probes on the physical map of the M RNA, and in view of the sizes of the NS M gene and glycoprotein precursor gene encoded by this R N A segment, it is concluded that the M RNA-derived species probably represent subgenomic m R N A s corresponding to. coach of the two cistrons. As these m R N A s are transcribed from opposite strands, the ambisense coding strategy of the TSWV M R N A is confirmed.

4

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Fig. 7. Identification of M-specific subgenomic RNA species in TSWV-infected N. rustica plants. Northern blots were prepared and analysed accordingto proceduresgiven in Methods. Total RNA was hybridized with strand-specificriboprobescorrespondingto the small (lanes 1 to 4) or large (lanes 5 to 8) ORF to detect M-specificvRNA species (lanes 1, 2, 5 and 6) and M-specificvcRNA species(lanes 3, 4, 7 and 8). The subgenomicRNA speciesare indicated. Lanes 1, 3, 5 and 7 contain 0.5 gg of purified nucleocapsid RNA, and lanes 2, 4, 6 and 8 contain 7 gg of total RNA from TSWV-infected N. rustica plants purified 8 days post-inoculation. The notation M2-v and M2-vc indicates that riboprobe M2, detecting vcRNA and vRNA strands respectively,was used in the hybridizationexperiment. Northern blot M3-v was exposed three times as long as blot M3-vc.

via subgenomic m R N A s transcribed from opposite strands, R N A extracts of TSWV-infected N. rustica plants were analysed by Northern blotting using strandspecific riboprobes. Previous analyses of cytoplasmic

Discussion With the determination of the M R N A sequence the genetic organization of the complete genome of TSWV has been unravelled, the features of which are shown in Fig. 8. The sequence data show that the M R N A is 4821 nucleotides long. Like the S and L R N A segments, the M R N A has termini complementary for about 64 nucleotides which are involved in the formation of a stable panhandle structure, causing the R N A segments to appear as pseudo-circular structures in virus particles (Peters et al., 1991). The terminal sequences of the M R N A are identical for the first eight nucleotides to the 5' and 3' termini of the S and L RNAs, and probably have an important function in genome transcription and replication. From the nucleotide sequence it can be deduced that the M R N A has an ambisense coding strategy. One small ORF encoding a putative protein of 33-6K occurs in the viral sense. Based on its absence from purified virus particles, this protein is tentatively called NSM. A second, but larger, O R F is located in the vc sense and encodes a protein with a predicted Mr of 127.4K. From its similarity to glycoprotein G 1 of the bunyaviruses and the hydropathy plot of the predicted protein, it is concluded that this O R F encodes the precursor to both

2802

R. Kormelink and others

L RNA L (331-5K) I Translation I Transcription vRNA 5'

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glycoproteins, in the order NHz-G2-G1-COOH. The ambisense gene arrangement in the M RNA has been confirmed by Northern blot analyses, revealing the presence of a 1-1 kb (v sense) and a 3.5 kb (vc sense) subgenomic RNA species in infected plant cells. Analysis of the nucleotide sequence of the intergenic region showed the presence of internal inverted A- and U-rich stretches capable of forming a stable hairpin structure. As previously discussed for TSWV S RNA,

this structure could be involved in termination of transcription of the subgenomic mRNAs. A conserved sequence found at the top of the hairpins of TSWV and INSV S RNA was also found at the top of the TSWV M RNA hairpin, indicating a possible function in transcription termination. Whether both structural features are a requirement for termination of transcription remains to be investigated. Ambisense coding strategies have recently been demonstrated for the S RNA segments of two different tospoviruses, TSWV and INSV (de Haan et al., 1990, 1992), and are also found for the S RNA segments of viruses of the genus Phlebovirus (Ihara et al., 1984; Marriot et al., 1989; Simons et al., 1990; Giorgi et al., 1991). However, TSWV is the first member of the Bunyaviridae with an ambisense M RNA, thus having a genome with two ambisense RNA segments. Computer alignments of L and S RNA-encoded gene products of TSWV with those of their animal-infecting Bunyaviridae counterparts reveal significant homology only between the L RNA-encoded RNA polymerases of TSWV and Bunyamwera virus, the type species' of the genus Bunyavirus (de Haan et al., 1991). No significant homology is found between the other proteins of TSWV and those of other members of the Bunyaviridae (de Haan et al., 1990). The data presented now reveal significant homology between the G1 glycoproteins of TSWV and those of members of the genus Bunyavirus. This finding seems to confirm that TSWV is most closely related to members of the genus Bunyavirus, although they do not have an ambisense S RNA. This could suggest that the creation of ambisense RNA molecules is a relatively late event in bunyavirus evolution. No significant homology was found between the G2 glycoprotein of TSWV and those of members of the genus Bunyavirus, but analysis of the putative G2 glycoprotein of TSWV revealed the presence of a putative cell attachment site (RGD) in the N-terminal region. This motif has only been found in glycoproteins that are found in the extracellular matrix of animal and plant cells, and are thought to be involved in adhesion of cells to the extracellular matrix (Pierschbacher & Ruoslahti, 1984; Ruoslahti & Pierschbacher, 1986, 1987; D'Souza et al., 1988; Schindler et al., 1989; Sanders et al., 1991). The RGD sequence of such adhesive proteins is crucial for the recognition of cell surface receptors (referred to as integrins). For TSWV it is tempting to assume that this sequence is only involved in the insect part of the virus multiplication cycle, possibly in binding of virus particles to cell receptors in the midgut of the thrips vectors. Indeed, morphologically defective isolates, i. e. virus mutants lacking a lipid membrane, are still able to infect plants but are deficient in transmission by thrips (Ie, 1982; Verkleij & Peters, 1983; Resende et

Nucleotide sequence of T S W V M RNA al., 1991). The relevance of the RGD motif in the glycoprotein precursor of TSWV remains to be determined because analysis of the glycoprotein precursor proteins of other members of the Bunyaviridae has revealed this motif only in Germiston and SSH bunyaviruses, and Punta Toro phlebovirus. The TSWV M RNA encodes, in addition to the glycoproteins, a putative non-structural protein (NSu). A search in the EMBL protein database did not reveal significant homology with other known proteins. Alignment with the glycoprotein precursors (containing potential NSM sequences) encoded by the M RNAs of different members of the Bunyaviridae also did not reveal any homology. The protein has not yet been detected in TSWV-infected plant cells and appears to be absent from virus preparations. With the determination of the M RNA sequence the genetic organization of the complete genome of TSWV has been elucidated. This will be a good starting point for future research aimed at studying the adaptation of this bunyavirus to plants. Comparison of the genome of TSWV with those of other members of the Bunyaviridae indicates that no additional genes are present in the TSWV genome. Therefore, it is likely that the adaptation of TSWV to plants has been achieved by sequence divergence of one or more ancestral bunyaviral genes. After submission of this article, the nucleotide sequence of the M RNA of another tospovirus, INSV, was published (Law et al., 1992), indicating an ambisense organization similar to that reported here. The authors wish to thank Tony de Rover and Mart van Grinsven of Zaadunie Biotechnology Department, Enkhuizen, The Netherlands, for providing the synthetic oligonucleotides and for helpful discussions. This work was supported in part by the Netherlands Foundation for Chemical Research (S.O.N.) with financial aid from the Netherlands Organization for Scientific Research (N.W.O.).

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DE HAAN, P., WAGEMAKERS, L., PETERS, D. & GOLDBACH, R. (1990). The S RNA segment of tomato spotted wilt virus has an ambisense character. Journal of General Virology 71, 1001-1007. DE HAAN, P., KORMELINK, R., RESENDE, R. DE O., VAN POELWIJK, F., PETERS, D. & GOLDBACH, R. (1991). Tomato spotted wilt virus L RNA encodes a putative RNA polymerase. Journal of General Virology 72, 2207-2216. DE HAAN, P., DE ,~VILA, A. C., KORMELINK, R., WESTERBROEK, A., GIELEN, J. J. L., PETERS, n . & GOLDBACH, R. (1992). The nucleotide sequence of the S RNA of Impatiens necrotic spot virus, a novel tospovirus. FEBS Letters (in press). DEVEREUX, J,, HAEBERLI, P. & SMITHIES, O. (1984). A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Research 12, 387-395. DEVOS, R. G. E., GILLIS, E. & FIERS, W. (1976). The enzymatic addition of poly(A) to the 3' end of RNA using bacteriophage MS2 RNA as a model system. European Journal of Biochemistry 62, 401-410. DE VRIES, S. C., SPRINGER, J. & WESSELS, J. G. H. (1982). Diversity of abundant mRNA sequences and patterns of protein synthesis in etiolated and greened pea seedlings. Plants 156, 129-135. D'SOUZA, S. E., GINSBERG, M. H., BURKE, T. A., LAM, S. C.-T. & PLOW, E. F. (1988). Localization of an Arg-Gly-Asp recognition site within an integrin adhesion receptor. Science 242, 91 93. ELLIOTT, R. M. (I 990). Molecular biology of the Bunyaviridae. Journal of General Virology 71, 501 522. ESHITA, Y. & BISHOP, D. H. L. (1984). The complete sequence of the M RNA of snowshoe hare bunyavirus reveals the presence of internal hydrophobic domains in the viral glycoprotein. Virology 137, 227-240. FRANCKI, R. I. B., FAUQUET, C. M., KNUDSON, D. L. & BROWN, F. (1991). Classification and nomenclature of viruses: fifth report of the International Committee on Taxonomy of Viruses. Archives of Virology, supplementum 2. GIORGI, C., ACCARDI, L., NICOLETTI, L., GRO, M. C., TAKEHARA, K , HILDITCH, C., MORIKAWA,S. & BISHOP, D. H. L. (1991). Sequences and coding strategies of the S RNAs of Toscana and Rift Valley fever viruses compared to those of Punta Toro, Sicilian sandfly fever, and Uukuniemi viruses. Virology 180, 738-753. GRADY, L. J., SANDERS,M. L. & CAMPBELL,W. P. (1987). The sequence of the M RNA of an isolate of La Crosse virus. Journalof General Virology 68, 3057-3071. GUBLER, U. & HOPI:MAN, B. J. (1983). A simple and very efficient method for generating cDNA libraries. Gene 25, 263 269. HoPp, T. P. & WOODS, K. R. (1981). Prediction of protein antigenic determinants from amino acid sequences. Proceedingsof the National Academy of Sciences, U.S.A. 78, 3824-3828. IV, T. S. (1982). A sap-transmissible, defective form of tomato spotted wilt virus in plant cells. Virology 59, 387-391. 1HARA,T., AKASHI, H. & BISHOP,D. H. L. (1984). Novel coding strategy (ambisense genomic RNA) revealed by sequence analyses of Punta Toro phlebovirus S RNA. Virology 136, 293-306. IHARA, T., SCHMIDT, J., DALRYMPLE,J. M. & BISHOP, D. H. L. (1985). Complete sequences of the glycoproteins and M RNA of Punta Toro phlebovirus compared to those of Rift Valley fever virus. Virology 144, 246-259. KORMELINK, R., KITAJIMA,E. W., DE HAAN, P., ZUIDEMA, D., PETERS, D. & GOLOBACH, R. (1991). The nonstructural protein (NSs) encoded by the ambisense S RNA segment of tomato spotted wilt virus is associated with fibrous structures in infected plant cells. Virology 181, 459-468. KORMELINK, R., DE HAAN, P., PETERS, D. & GOLDBACH, R. (1992a). Viral RNA synthesis in tomato spotted wilt virus-infected Nicotiana rustica plants. Journal of General Virology 73, 687-693. KORMELINK, R., VAN POELWHK, F., PETERS, O. & GOLDBACH, R. (1992b). Non-viral heterogeneous sequences at the 5' ends of tomato spotted wilt virus mRNAs. Journal of General Virology 73, 2125-2128. KYTE, J. & DOOLITTLE, R. F. (1982). A simple method for displaying the hydropathic character of a protein. Journal of Molecular Biology 157, 105-132.

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(Received 12 May 1992; Accepted 21 July 1992)