Complete nucleotide sequence and coding strategy of rice ... - CiteSeerX

Journal of General Virology (1993), 74, 2463-2468. Printedin Great Britain

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Complete nucleotide sequence and coding strategy of rice hoja blanca virus RNA4 Bertha-Cecilia Ramirez, 1. Ivan L o z a n o , z L u i s - M i g u e l Constantino, 2 Anne-Lise H a e n n i 1 and L e e A. Calvert 2 l Institut Jacques Monod, 2 Place Jussieu-Tour 43, 75251 Paris Cedex 05, France and 2 Centro Internacional de Agricultura Tropical (CIAT), A.A. 6713, Cali, Colombia

The complete sequence office hoja blanca virus (RHBV) RNA4 has been determined, based on the sequence of the corresponding eDNA clones. RNA4 consists of 1991 nucleotides with two open reading frames (ORFs). One putative ORF is located in the 5'-proximal region of the viral RNA4; it encodes a protein of predicted Mr 20076 which corresponds to the major non-structural protein that accumulates in RHBV-infected rice plants, and which bears limited sequence identity with the helper component of tobacco vein mottling potyvirus.

The other ORF is located in the 5'-proximal region of the viral complementary RNA4 and encodes a protein of predicted M r 32469. Between the two ORFs is an intergenic region of 524 nucleotides, part of which can theoretically adopt a stable stem-loop structure; the 5' and 3' ends can potentially base-pair over 16 nucleotides, producing a pan-handle configuration. These characteristics are in favour of an ambisense coding strategy for RHBV RNA4.

Rice hoja blanca virus (RHBV) causes sporadic epidemics in rice throughout most of tropical America (Morales & Niessen, 1985), leading to significant decreases in rice yield (Vargas, 1985). It is a member of the tenuivirus group that includes rice stripe virus (RSV, the type member), maize stripe virus (MStV), European wheat striate mosaic virus and rice grassy stunt virus (Gingery, 1988). It is persistently transmitted by the delphacid planthopper Tagosodes orizicolus Muir [previously known as Sogatodes oryzicola Muir and reclassified by Wilson & Claridge (1991)] in which it replicates (Galvez, 1968). In a population of T. orizicolus, about 10 % of the insects transmit RHBV; the susceptibility of the insect to infection by RHBV is a genetic trait controlled by a single recessive gene (Zeigler & Morales, 1990). Vector colonies with a large percentage of viruliferous planthoppers can be maintained by selective mating of viruliferous individuals. RHBV, like the other tenuiviruses, has thread-like particles 3 to 8 nm in width and of variable length that may adopt a circular configuration (Morales & Niessen, 1985; Espinoza et al., 1992). The protein moiety is a single species of nucleocapsid protein (NC) of M r 34 000

(Morales & Niessen, 1983). The genome of RHBV has been characterized (Ramirez et al., 1992). It consists of four species of ssRNA designated RNA1, -2, -3 and -4 in order of their decreasing size. Three species of dsRNA are also detected in purified ribonucleoprotein particles; they appear to be the double-stranded forms of RNA2, -3 and -4. Hybridization analyses using RNA probes of viral (v) and viral complementary (vc) polarities show that unequal amounts of RNA4 of the two polarities are present; an RNA species of about 800 nucleotides is also present in purified ribonucleoprotein preparations that appears to be derived from vRNA4 and has been designated subgenomic (sg) RNA4. Rice tissues infected with RHBV accumulate a major non-structural viral protein that is antigenetically distinct from the NC (Morales & Niessen, 1983). Translation studies performed in vitro using the individual species of ssRNA as template followed by immunoprecipitation studies using a polyclonal anti-non-structural protein antiserum have revealed that the non-structural protein is produced by vRNA4; it is designated NS4, and has an M r of 21000 as estimated by 0.1% SDS-15% PAGE (Ramirez et al., 1992). To gain some insight into the genome organization of RHBV, we have determined the sequence of RHBV RNA4, and compared certain features of this sequence and of the deduced amino acid sequences with those of RNA4 and corresponding proteins of RSV and MStV.

The nucleotidesequencedata have been submittedto the GenBank databank and assignedaccessionnumber L14952. 0001-1932 © 1993 SGM

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Plants and insect vectors were maintained and ribonucleoproteins purified as described (Morales & Niessen, 1983; Ramirez et al., 1992). RHBV RNA was purified as indicated (Ramirez et al., 1992). Total nucleic acids were extracted from non-infected and from RHBV-infected rice tissue. After grinding the tissue (0.5 g) in liquid nitrogen, 2-5 ml of an extraction buffer containing 0.1 M-Tris-HC1 pH 7.4, 0.02 M-EDTA, 2 % SDS and 0-2 ml of vanadyl-ribonucleoside complex (BRL) was added. A similar procedure was used to extract nucleic acids from uninfected and RHBV-infected T. orizicolus: after grinding 20 or 100 mg of leafhoppers in liquid nitrogen, 12"5volumes of extraction buffer were added. In all cases, the preparations were subjected to one phenol and four phenol-chlorofom extractions, followed by ethanol precipitation. The pellets were resuspended in 10 mu-Tris-HC1 pH 8.0 and 1 mMEDTA. Rabbit polyclonal anti-RHBV antiserum and antiNS4 antiserum were kindly provided by Dr F. Morales and used in the ELISA as described by Zeigler & Morales (1990). cDNA clones specific for RHBV RNA were prepared by standard procedures using random primers or the primer 5' ACACAAAGTC 3' that corresponds to the conserved sequences at the 5' end of the four RSV RNA species (Takahashi et al., 1990). The cDNA products were synthesized following the protocol of Gubler & Hoffman (1983); they were fractionated on a Sephacryl 300 column, incubated with the Klenow enzyme to fill in the termini, and ligated into the SmaI-digested vector Bluescript pKS + (Stratagene). The resulting constructs were purified on PlasmidQuick columns (Stratagene). Whenever necessary, subcloning was performed using appropriate restriction enzymes. Clone RHB4C-341 from a previously described cDNA library was also used to confirm the exact sequence of the 3' end of RNA4; it was obtained by polyadenylation of total RHBV RNA before cDNA synthesis (Ramirez et al., 1992). Sequencing of the double-stranded cDNA inserts was carried out using the dideoxynucleotide method (Sanger et al., 1977). The entire sequence of RHBV RNA4 was determined on both strands, and most of the sequence was derived from more than one cDNA clone. The sequence data were analysed using DNASIS (Pharmacia). The pKS + vector containing the viral cDNA fragments was linearized with the appropriate restriction enzyme and transcription in vitro was performed using T7 RNA polymerase (Stratagene) in the presence of [~-32P]ATP. The labelled transcripts served as probes to detect RHBV RNA4. The RHBV RNAs to be analysed were separated by 1% agarose gel electrophoresis, denatured with NaOH,

ORF NS4

IR 3'

vRNA4 (1991

r]llllllllllllllA~"

3"

IR

Sg

nt)

vcRNA4

ORF NSvc4

R4-26B

I

R4-26

Bglll

R4-18 -

-

R4-1 23

RHB4C-341 Fig. 1. Schematic representation of the positions of the ORFs on RHBV vRNA4 and vcRNA4 deduced from the sequence data, and of the cDNA clones used for sequencing and hybridization experiments. On v- and vcRNA4, ORFs are boxed, the intergenic region (IR) is represented as a thick line and non-coding regions as thin lines. R426B, R4-26, R4-18, R4-123 and RHB4C-341 are cDNAs. The position of the BglII site on R4-26 is indicated.

neutralized and transferred to a nitrocellulose membrane. Hybridization with the 32P-labelled riboprobes was carried out at 65 °C in a solution containing 50% formamide, 6×SSPE (1 × : 0.9M-NaC1, 0"05MNa2HPO 4 and 0.006 M-EDTA, pH 7.4), 7 × Denhardt's solution [1 ×: 0.1% Ficoll 400 (Pharmacia), 0.1% polyvinylpyrrolidone (Sigma) and 0"1% BSA fraction V (Sigma)], and denatured herring sperm DNA. The initial wash was at 37 °C for 30min in 1 × SSC (1 x : 0.15 MNaC1 and 0-015 M-sodium citrate) containing 0.2 % SDS, and the subsequent three washes were at 65 °C for 30 min in 0"2 x SSC containing 0.01% SDS. The membranes were autoradiographed for the times indicated. Of the cDNAs produced from total RHBV RNA, a subset specifically hybridized with RNA4. Four clones, R4-26, R4-18, R4-123 and RHB4C-341, corresponded to the entire RNA4 except for four nucleotides (nt) at the 5' end (Fig. 1). Other clones (not shown) corresponded to RNA4 beginning at nt 2 from the 5' end as deduced from the complementarity with the 3' end. Sequencing of these clones yielded the complete primary structure of RHBV RNA4 as presented in Fig. 2. The viral RNA is composed of 1991 nt and two ORFs can be predicted from the sequence. One putative ORF, located in the 5'-proximal region of vRNA4, encodes a protein of 174 amino acids whose predicted M r of 20076 is in agreement with the estimated size of NS4 produced upon in vitro translation of RHBV RNA4. The other putative ORF, located in the Y-proximal region of vcRNA4, can encode a protein of 283 amino acids with an M r of 32469; it is designated NSvc4. Between the two ORFs is a non-coding region of 524 nt. Computer analyses showed that part of this region can adopt a hairpin structure, involving nt 918 to

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M D F L K T D V B V O P Z E Q L N Y R R L Y 10

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D Z L P N E V B D N Z T L P R ' L K N P D K V ~ E E N K K L Z L ~ G F I Y V A y H ~ O ~ C D ~ C ~ c c A G A U ~-U~(X~~AU~IOC0~CCAC 130

140

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H P Z E ~ D P D F T B V H K H M P G Z B H B F L E H L L G T D ~ B N N T Z D L G 250

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K L P D Z L Q B R L G D W I T M N F L K H N N R M B K D Q Z K T L C E ~ I V D L 370

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A K A R G G D T E ~ Y E A V W K K M p A y y S Z L L Q Q Z L H K t 490

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~I~rC~JOACAI~ 500

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P,AAD 720

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UU~J~00~UG~nO~ 950 960

* A ~ I M V R K K Q V L P V G P Q E V V O P ~ Z G p K D ~ L A E

R T T F ~ T T V A R Q V V D T T F N K F K B L H N V T R D P L P E F E F M V T K U 1330

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Q E 2 N P V P F D ~ R E I O W F W L B R B I I C N Q V G ~ D K I V Z ~ V Q M K D A D E Y B M F N P L B O L V A F T E ~ N P T C Z R V E V Q R R P D T Y B R D Z I R F ~ V T G N A R G K I G L W I V A F T A Z R F F P F T Q L P K R P F L P B B D I T F H T R R N A C V B T D Q K ~ V P M D T G N A R V D A P B F P H L G L V T A A A D I T N R O K T L P R N K L A L K R K N K K E F K E A A R R B L D D T L I V 1991

BKKFPNYRSZSN Fig. 2. Completesequenceof RHBV RNA4 and predictedgeneproducts. The amino acid sequencesencodedby the vRNA and the vcRNA are written above and belowthe RNA sequencerespectively.Asterisksrepresentterminationcodons. 967, with a calculated free energy of -40.1 kJ (Fig. 3 a). In the case of the Punto Toro phlebovirus S segment, the terminal loop of the potential hairpin structure located in the intergenic region is the site of transcription termination (Emery & Bishop, 1987). In addition, the Yand 3'-terminal sequences of RHBV RNA4 are composed of 16 nt potentially capable of base-pairing (Fig. 3b); this feature presumably accounts for the circular configuration observed by electron microscopy (Morales & Niessen, 1985; Espinoza et al., 1992). The amino acid sequences deduced from the nucleotide sequences of the ORFs in RHBV RNA4 were compared (Fig. 4) with those of RSV (Hayano et aI., 1990; Kakutani et al., 1990) and MStV (Huiet et al., 1990, 1992). The RHBV NS4 protein has a 59 % amino acid

identity with the corresponding protein of RSV and of MStV (Fig. 4a). In RSV and in MStV as in RHBV, this protein corresponds to the major non-structural protein that accumulates in infected plant tissue. Between RSV and MStV, these proteins present 74% identity. The RHBV NSvc4 protein has 57 % and 56 % amino acid identity respectively with the corresponding proteins of RSV and MStV (Fig. 4b); the amino acid identity is 76 % between the corresponding proteins of RSV and MStV. Furthermore, a computer-aided search (Fig. 5) identified a region of limited but probably significant sequence identity (35%) between a 67 amino acid stretch of RHBV NS4 and a 64 amino acid segment of the helper component (Domier et al., 1987) of tobacco vein mottling potyvirus (TVMV). The corresponding proteins in RSV

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(a)

**

RRBV NS4 TVMVBC

(85-121) (106-140)

* *

* *

* *

* *

FLEBI~LGT-D FL-~DLFT~

*

ESNNTIDL~ R~'~'~4T-AA

**

**

**

*

**

**

**

***

LFDII~GDWIT-M~PFL I~IEII,--RLI GDRNEA-PFA

A/U\G

l 940K yI G:C

~m~v xs4

(b)

A:u-._ G:C/u C:G C:G 950

He

C:O G:C G:C G:C ~/A:U ~C:G U:A O:C

A:U

A:U

U:A

A:U

C:G

A:U

G:C

C:G

920

U:A

A:U

918

G:C O:C

1 A:U

**

*

*

****

*

*

****

(122-1sl)

~mma~m~

(141-169)

-mma--L~ :55~aP

~m'uz--~I

* *

*

wLm~zc-~

D S ~ S D S r . LZ

Table 1. Values of the ELISA test for non-infected and

1991

(a) RI~BV RSV MStV

--M~)FLKTDV SVGPIEGLNY MQDVQRTVE ...... V..D. ---MQRSAD. . I . . . T ....

RHBV RSV MStV

KKL~LCGFIY ..... K.CV. ..... K . C V E

VAYHHPIETD PDFTS%q4KNM PGISESFLEH LLGT-DESNN I ..... L . . . T L . I K .... I T E F C H . . . S . . . . G E . D D . A T ..... L . . . . L . A ..... L . D F C H . . . . . . . . G E Q D E . S

97 i00 97

RHBV RSV MStV

TIDLGKLFDI L..I.LF.NM L..I.EF.KL

LQERLGDWIT ..PS..G... ..PS ......

MNFLKHNNRM SKDQIKTLCE TIVDLAKAEG K . . . R , P . . . . . . . . . L . L D Q . I K M .... S KYY...P.K. .GI ..... L N Q . I N M .... S

147 150 147

RRBV RSV

GDTEIYEAVW K~AYY-SI S...E..K ...... T.FE.. S...T..K ...... S.F-..

LL~ILHK IQ-PD...T V.TPL...W

175 178 176

RRLYDILPNK TL...T..ET TD...S..SS

VSDNITLPRL . . . . . . . . D. . . . . . . . LD.

~NPDK~2TEEN .D.ER...DT .E.ER...AT

48 50 47

(b) RHBV RSV

**

C:O

967

Fig. 3. Putative secondary structures in RHBV vRNA4. (a) Hairpin structure predicted for nt 918 to 967 in the intergenic region. (b) Potential base-pairing between the 5' and 3' extremities. Colons and dots indicate potential base-pairing in the stem regions. Numbers refer to distances from the 5' end of vRNA4.

MStV

**

*

Fig. 5. Comparison of a 67 amino acid segment of the N S 4 protein of R H B V with a 64 amino acid stretch from the helper component of TVMV. The positions of the amino acid regions considered for the NS4 protein (Fig. 2) and the helper component (HC; Domier et al., 1987) are in parentheses. Two asterisks indicate that residues are identical, and one asterisk indicates that residues are members of one of the following groups (Kamer & Argos, 1984; Domier et al., 1987): acidic and polar (D, E, N, Q), basic (K, R), hydrophobic (A, C, F, H, I, L, M, V, W, Y), polar (T, S) or strong-turn formers (D, G, N, P). A dash indicates that the sequence has been shifted by a single amino acid residue to obtain an optimal alignment.

• :

A:U C:S AG=-40.1kJ U.G 930 C:G C:G A:U C:G U C U U 960

*

MStV

MSISRYN?FK ,AL..LL~5 .ALMKLFSRS

KSVILTDDLS ERAAEKFEK~ ..I~V.Y . . . . . E S O K R V D N . NGKV.V ..... EGQKRLDLA

PalBV RSV MStV

AAAT%rLGLEP FSFADV~S Q...M ........ S..KV.K Q .............. IKV.K

RHBV RSV MStV

KPLQTPPFFR IATFAVIWLG IKGRANGTVT Q . . T H Y .... V .... M V . I . . . . . . S . I T . • . . T Y Y . . . . . . . . . M V . I . V .... A . . T .

P.HBV RSV MStV

YPMAKTFAVLGSLP~MSYED~DKMQVEIV .,IS.N ....... A;.LAL. .KHNL..SVS ..ICKN .......... LAL. .KTNLR.SVS

NKRKLALKNR PLTKGRMTID .RKS..,SK. ..NQ..V... . N K . K L . S A . . . . . . . . $..

YDMFVAKQDY SVCANRRTHF .... I . . . . . . . K . H . . A T . .... T . . . . . . I K . . . V A T .

50 50 50

TIDSSPLFFR M.LVD.YW.H C.AVD.FW.H

100 I00 i00

FRIIDRSYTD L .... K . . V N LK...K..V.

PERQVEVEIC S S D ..... V R .QD ..... V T

150 150 150

IKDDSVQNCI V D . S ..... V . Q G A T .... V

ISRSLWFWGI ...T ...... ...A ......

200 200 200

RHBV RSV MStV

ERTDFPVPME SQKT~EFE PLPDRTV~tHL SKF~TTDV .... L . . S . K T N D . . . . . . . . . E . K A I . . . . S . S .... N. .... L . . S . K T T D . . . . . . . . . E . F N . . . . . S . S K . . . N .

VQRAVTTAFT ..K..GG.., ..K..GG..V

250 250 250

PJIBV RSV MStV

TREALEDKPG SKSFP,LDTE .KSFP.LDSQ

IEFGVVKQPG VPLVQK~VM K ........ K KIPIT..SKS K; . . . . . . . K K I P I M . P K R S

IEA EVSVIM .FD

283 286 283

Fig. 4. Comparison of the two non-structural proteins encoded by RHBV RNA4 with the corresponding proteins of RSV and MStV. (a) Comparison of the amino acid sequences between the NS4-type proteins. (b) Comparison of the amino acid sequences between the NSvc4-type proteins. Dots represent amino acid identity between the RHBV protein and the corresponding RSV or MStV protein. A dash indicates that the sequence has been shifted by a single amino acid residue to obtain an optimal alignment.

RHB V-infected rice plants and planthopper vectors using polyclonal anti-RHBV and anti-NS4 antisera* Sample

Sample buffer Non-infected rice RHBV-infected rice Non-infected plauthoppers 1st to 4th instars; adults RHBV-infected planthoppers 1st to 4th instars Adults

Anti-RHBV

Anti-NS4

0-04] 0.06 0.65

0"04t 0"06 0.38

0-04

0.06

0.21-0.43 0.55

0-03~).07 0.04

* Non-infected insects were from a virus-free vector colony. RHBVinfected insects were from a vector colony with a 70% level of viruliferous planthoppers. t Values are mean A405 for four replicates.

and MStV share 30% and 27% identity respectively with this segment of the TVMV helper component (not shown). This same stretch in TVMV also shares similarity with the gene II product or aphid transmission factor of the cauliflower mosaic caulimovirus (Domier et al., 1987). Both the helper component and the gene II product appear to be intimately associated with transmission of the virus by the insect vector. The similarity observed between the RHBV NS4 protein and the TVMV helper component suggests that the NS4 protein of tenuiviruses may be involved in a similar function, although the mode of vector transmission is different for the viruses of these three groups. As it is known that tenuiviruses multiply in their insect vector, we examined whether NS4 accumulates in viruliferous planthoppers. Using ELISA and the appropriate antibodies, the NC but not the NS4 protein was detected in T. orizicolus, whereas both proteins accumulated in rice plants (Table 1). A similar result has been obtained with MStV, i.e. the NC but not the NS4 protein can be detected in MStV-infected planthoppers

Short communication 1

2

3

4

5

dsRNA4 I ~

ssRNA4 I ~ sgRNA4 II~

Fig. 6. Northern blot analysis of RHBV R N A and of total nucleic acids extracted from rice and planthopper tissues. Clone R4-26 was used to obtain an R N A transcript detecting vRNA4. RHBV R N A (lane 1); total nucleic acids extracted from rice tissues (lanes 2 and 3) or from planthoppers (lanes 4 and 5); extracts from non-infected (lanes 2 and 4) or from RHBV-infected (lanes 3 and 5) tissues. The positions of the RHBV RNAs are indicated to the left of the panel. All lanes are from the same gel. Exposure times were 2 h (lane 1), 8 h (lane 3), and 10 days (lanes 2, 4 and 5).

(Falk et al., 1987), but it is in contradiction with what has been reported for RSV (Toriyama, 1986). This may be due to the different insect/virus combinations and/or experimental designs used. In another series of experiments, s2p-labelled riboprobes were used to search for the presence of RNA4related species among the total nucleic acids extracted from RHBV-infected rice and planthoppers (Fig. 6). To this end, a vcRNA4 probe hybridizing with vRNA4 was prepared from clone R4-26 (Fig. 1). Using this probe, ssRNA4 and dsRNA4 were detected in RHBV (Fig. 6 a, lane 1), and ssRNA4 and sgRNA4 were detected in RHBV-infected rice tissues (lane 3). In contrast, only ssRNA4 could be detected in RHBV-infected planthoppers (lane 5) after about 10-fold longer exposures of the autoradiograms than required for the RHBV-infected rice tissues. Upon long exposures, hybridization signals were obtained with three and two populations of nucleic acids extracted respectively from non-RHBV-infected rice tissues (lane 2) and planthoppers (lane 4). The two species detected in the nucleic acids extracted from nonRHBV-infected planthoppers migrated in the same position as the two larger species extracted from nonRHBV-infected rice tissues. To examine in more detail the region of the riboprobe responsible for hybridization

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with nucleic acids extracted from non RHBV-infected rice and insect tissues, clone R4-26B was used. This clone was obtained by BgIII (in insert)-BamHI (in polylinker) deletion of clone R4-26; the corresponding transcript had retained the 5' leader sequence and most of the NS4 ORF (Fig. 1). No hybridization was detected between this riboprobe and nucleic acids extracted from nonRHBV-infected rice or insect tissues (not shown), suggesting that the intergenic region of RNA4 was responsible for the hybridization observed with R4-26. In a parallel analysis (not shown) with the same samples as those in Fig. 6, and a vcRNA4 probe from clone R4123 (Fig. 1) complementary to the 3' end of vRNA4, ssRNA4 and dsRNA4 but not sgRNA4 were detected, indicating that the sgRNA4 contained the gene for NS4. The fact that RNA4 was detected in planthoppers but not the NS4 protein indicates either that the protein was present in amounts too small to be detected by the ELISA used, or that it was unstable, or that it was present transiently during virus multiplication and did not accumulate, or that it was not synthesized. The presence of ORFs in the vRNA4 and vcRNA4 is strong evidence that the ambisense coding strategy is used by RHBV, and that this strategy is probably a common feature of tenuiviruses. The genome organization and the degree of similarity between RNA4 of RHBV, RSV and MStV reflect the evolutionary relatedness that exists between these viruses. The amino acid similarity between the proteins corresponding to the NS4 proteins of RSV and MStV is greater than that with the NS4 protein of RHBV. This is also the case for the NSvc4 protein. The amino acid similarities of the predicted proteins encoded by RNA3 of RHBV (unpublished data), RSV and MStV show a similar trend. It thus appears that RSV and MStV are evolutionarily more closely related to each other than to RHBV. In addition, tenuiviruses share several characteristics with tospoviruses, and phleboviruses, suggesting a possible evolutionary relationship between these viruses. We are grateful to F. Morales and F. Chapeville for their interest and encouragement. B.C.R. is the holder of a fellowship from the Ministbre des Affaires Etrang~res of France. This study was supported in part by the Rockefeller Foundation and the Appel d'Offre :' Virologie Fondamentale' o f the Ministbre de l'Education Nationale de la Jeunesse et des Sports. The Institut Jacques Monod is an Institut Mixte, CNRS-Universit6 Paris 7.

References DOM]~R, L. L., SHAW,J. G. & RHOADS, R. E. (1987). Potyviral proteins share amino acid sequence homology with picorna-, como-, and caulimoviral proteins. Virology 158, 20-27. EMERY, V.C. & BrSHOP, D. H. L. (1987). Characterization of Punta Toro S m R N A species and identification o f an inverted complementary sequence in the intergenic region of Punta Toro phlebovirus ambisense S R N A that is involved in m R N A transcription termination. Virology 156, 1-11.

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(Received 2 July 1993; Accepted 9 July 1993)