Nucleotide Sequence of the 3' Ends of the Double ... - CiteSeerX

J. gen. Virol. (1988), 69, 423-428.

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423

Key words: nepovirus/GCMV/RNA sequencing

Nucleotide Sequence of the 3' Ends of the Double-stranded RNAs of Grapevine Chrome Mosaic Nepovirus B y O L I V I E R L E G A L L , T H I E R R Y C A N D R E S S E * AND J E A N D U N E Z Station de Pathologie VOg~tale, INRA, BP 131, 33140 Pont de la Maye, France (Accepted 6 November 1987) SUMMARY

Attempts were made to label the termini of dsRNAs corresponding to the two genomic RNAs of grapevine chrome mosaic nepovirus (GCMV). It was not possible to label the 5' ends of the dsRNAs with [y-32p]ATP, which suggests that a genome-linked protein blocks their 5' ends. Both dsRNA species were labelled at their 3' ends with pCp. The T-terminal sequences were determined by 'wandering spot' or by partial enzymic cleavage analysis. One strand (presumably positive) ended in a poly(A) 30 to 50 nucleotides long whereas the other (presumably negative) ended in 3'-ACCUUUUAAAAAG (RNA1) or Y-ACCUUUUAAUAAAG (RNA2). The sequences resemble closely those complementary to the 5' ends of the R N A s of tomato black ring virus (strain S), which is distantly related to GCMV. Grapevine chrome mosaic nepovirus (GCMV) (Martelli & Quacquarelli, 1972) is distantly related to tomato black ring virus (TBRV) and resembles it in having a genome divided between two positive-sense ssRNAs. TBRV RNAs are polyadenylated at their 3' ends (Mayo et al., 1979) and have a polypeptide (VPg) covalently linked to their 5' ends (Mayo et al., 1982). Pseudorecombinants have been obtained between GCMV and TBRV (Dozet al., 1980). The viruses are related serologically (Kerlan & Dunez, 1983) and by nucleic acid hybridization (Dodd & Robinson, 1984). TBRV is probably the nepovirus about which most molecular detail is known; the complete nucleotide sequences of satellite and genome RNAs of strain S are known (Meyer et al., 1984, 1986; C. Fritsch, personal communication). However, dsRNAs associated with TBRV multiplication have never been studied. We report here the isolation of GCMV dsRNAs and the nucleotide sequences at their 3' ends. dsRNA was purified from infected Chenopodium quinoa tissue by an essentially standard method (Dodds et al., 1984), involving cellulose (CF11, Whatman) column chromatography. GCMV-infected C. quinoa plants contained two virus-specific dsRNAs of about 7200 and 4800 base pairs [as determined by comparison with a Bethesda Research Laboratories (BRL) 1 kb DNA Ladder upon electrophoresis in a 6% polyacrylamide gel; Fig. 1] that corresponded in size to the genome RNAs of the virus. No dsRNAs were observed in preparations obtained from healthy plants. The dsRNAs were most abundant in infected C. quinoa about 7 days after inoculation, which is when symptoms first appear and when the concentrations of both coat protein and viral RNAs in the plant are greatest (Bretout, 1987). About 10 ~tg dsRNA2 and 3 ~tg dsRNA1 were obtained from 400 g of infected plant material (as determined by fluorescence after ethidium bromide staining) and their dsRNA nature was confirmed by their resistance to digestion by RNase A in 2 × SSC but not in 0.2 x SSC (1 × SSC is 0.15 M-NaC1, 0.015 M-sodium citrate pH 7.0), The dsRNAs were further purified and partially separated by electrophoresis in 1% agarose (Low Melting Point Agarose, BRL), phenol extraction and ethanol precipitation according to Maniatis et al. (1982). In an attempt to label 5' ends, the purified dsRNAs were treated with alkaline phosphatase and then with [y-32p]ATP (Amersham, 1000 to 3000 Ci/mmol) and T4 polynucleotide kinase (BRL) as described by Lomonossoff et al. (1985). The 5' labelling was at 0000-7960 O 1988 SGM

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Fig. 2

Fig. 2. 'Wandering spot' analysis of labelled dsRNA1 (a) and d s R N A 2 (b). First dimension electrophoresis (1D) was for 2 h at 2000 V in a 10~ polyacrylamide denaturing gel (200 × 400 × 0.4 mm) containing 7 M-urea in 0.03 M-sodium citrate pH 3.5. Second dimension (2D) electrophoresis was overnight at 300 V in a 2 0 ~ polyacrylamide gel (350 x 450 x 0.4 mm) in 0.04 M-Tris-citrate pH 8.0.

Fig. 1. Polyacrylamide gel electrophoresis of G C M V dsRNAs. Lanes 1 to 4, ethidium bromide-stained gel; lanes 5 to 6, autoradiography of pCp-labelled dsRNAs. Lane 1, BRL 1 kb d s D N A Ladder (positions are indicated in base pairs); lane 2, dsRNAs after cellulose chromatography; lanes 3 and 5, agarose-purified d s R N A I ; lanes 4 and 6, agarose-purified dsRNA2.

3054-----~

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7126----~ 6108----.~ 5090----~

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425

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(b)

(a) 1

2

3

4

5

(b) 1

2

3

4

5

G A A A A A

G A A A U A A

O U U U C C Fig. 3

U U U U C C Fig. 4

Fig. 3. Complete RNase T1 digests of pCp-labelled dsRNA1 (a) and dsRNA2 (b) analysed in a 15~ polyacrylamide denaturing gel containing 7 M-urea in TBE buffer. The positions of bromophenol blue (B), xylene cyanol (X) dyes, the poly(A) tract (brackets) and the bands excised for further sequencing (arrows) are indicated. Fig. 4. Enzymic sequencing patterns of the RNase TI oligonucleotides obtained from dsRNA l (a) and dsRNA2 (b). Lanes 1, RNase TI (G-specific); lanes 2, RNase U2 (A-specific); lanes 3, alkaline lysis (non-specific); lanes 4, RNase Phy M (A + U-specific); lanes 5, B. cereus RNase (C + U-specific). Electrophoresis was in a 20~ polyacrylamide denaturing gel containing 7 M-urea in TBE buffer.

best extremely inefficient and further sequencing revealed a complex pattern (results not shown). We therefore assume that the weak labelling had occurred either at hidden breaks or at the 5' end of contaminating nucleic acids and that the 5' ends of G C M V d s R N A s are always blocked. This is in contrast with the results of Lomonossoff et al. (1985) who found that some cowpea mosaic virus d s R N A molecules were free of VPg and could therefore be 5' labelled. The 3' ends of both d s R N A s were efficiently labelled, as shown in Fig. 1, by treating about 1 ~tg of purified d s R N A with [5'-32P]pCp (Amersham, 1500 to 3000 Ci/mmol) and T4 R N A ligase (BRL) as described by Lomonossoff et al. (1985). This showed that 3' ends were not phosphorylated. After electrophoresis, the labelled d s R N A s were eluted from the polyacrylamide gel according to Maxam & Gilbert (1980), by crushing the excised bands in elution buffer (containing no magnesium), gently shaking the mixture overnight at 4 °C, centrifuging it and ethanol precipitating R N A from the solution. After partial alkaline hydrolysis (20 min in 0-5 ~NaHCO3 pH 9-5 at 90°C), the 3" termini were sequenced by 'wandering spot' analysis according to De Wachter & Fiefs (1982). The resulting patterns are shown in Fig. 2. In both

426

Short communication (a)

(b) 1

3

4

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2

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4

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Fig. 5. Determination of the Y-terminal nucleotide of dsRNA t (lanes 2) and dsRNA2 (lanes 3) derived Tl-oligonucleotides by thin-layer chromatography on PEI-cellulose developed in 1 M-LiCI (a) and cellulose developed in isobutyric acid/NH4OH/H20 pH 4.3 (577/38/385 v/v/v) (b) after complete digestion with RNase T2. Controls are pCp-labelled poly(A) (lanes 4) eluted from the bracketed region of the gel shown in Fig. 2 and unlabelled oligo(rA) (Pharmacia) (lanes L)RNase T2 digests. An RNase T2 digest of 5 lag of turnip yellow mosaic virus RNA was added to lanes 2, 3 and 4 in order to localize the four mononucleotide positions by u.v. absorbance at 260 nm (circles). The origin of migration is arrowed at the bottom of the plates.

d s R N A species, a homopolymeric poly(A) sequence about 40 nucleotides long is clearly visible as well as a heteropolymeric sequence that presumably corresponds to the 3' end of the negative strand. These heteropolymeric sequences can be interpreted as Y - U U U U A A A A A for d s R N A 1 and Y - U U U U A A U A A A for dsRNA2. They differ by one deletion/insertion. Since the first spots deriving from both strands migrate in the same region of the gel, interpretation of the terminal sequences is difficult. In order to separate the 3' ends of the negative and positive strands the d s R N A s were heatdenatured, digested to completion with ribonuclease T1 (Pharmacia) and the mixture was electrophoresed on a 1 5 ~ polyacrylamide denaturing gel containing 7 M-urea in TBE buffer (Maniatis et al., 1982). For each d s R N A the- label migrated to two regions of the gel (Fig. 3). These corresponded to a 30 to 50 nucleotide long ladder, which is the heterogeneous length of the poly(A), at the 3' end of the positive strand (brackets) and a major band of about 13 to 15 nucleotides corresponding to the oligonucleotide terminating at the first G of the negative strand (arrows). The dsRNAl-derived oligonucleotide (lane a) is one base shorter than the one derived from d s R N A 2 (lane b), confirming the deletion previously noted. The additional band in the dsRNA1 track which comigrated with the dsRNA2-derived oligonucleotide is probably due to contamination of d s R N A 1 with d s R N A 2 since the sequence of this oligonucleotide was found to be the same as that of d s R N A 2 (result not shown). The labelled T1 oligonucleotides were then eluted from the gel as described above.

427

Short communication GCMV

RNA1 B-UGGAAAAU

RNA2

~oI~A C C U U U U A A U +

TBRV-S

RNA1, RNA2

÷

Satellite RNA +

~ G G A A A & U U A

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- - / / - -

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. . . . . .

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/ - -

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Fig. 6. Comparison between terminal sequences of GCMV and TBRV-S RNA positive strands. For GCMV, the sequence is derived from the 3'-terminal sequence of the negative strands; for TBRV-S, see Meyer et al. (1984, 1986) and Hemmer et al. (1987). The regions identical to GCMV RNA2 are framed. B is a blocking structure, probably a VPg molecule.

The 3'-terminal oligonucleotides of the negative strands were sequenced enzymically using the base-specific RNases T1, U2, Phy M and Bacillus cereus RNase (RNA sequencing kit, Pharmacia). The resulting pattern shown in Fig. 4 reveals the same sequence as the 'wandering spot' analysis and shows that two additional C residues are present at the 3' end of both oligonucleotides. Since the T-terminal nucleotide could not be determined by either of the methods described above, each oligonucleotide was digested to completion with ribonuclease T2 (BRL) and chromatographed on plates of either polyethyleneimine (PEI)-cellulose (Collmer & Kaper, 1985) or cellulose (Kikuchi et al., 1982) together with markers. The autoradiograms in Fig. 5 show that, in both systems, the label comigrated with authentic Ap, demonstrating that the labelled phosphate of pCp had been transferred to a T-terminal Aon in both dsRNA species. Assuming that dsRNAs end in a perfect duplex, we propose the structure given in Fig. 6 for GCMV dsRNA ends. Sequencing of DNA complementary to the GCMV genomic RNAs will allow us to verify this hypothesis. There is evidence in this report for the presence of a poly(A) tract at the 3' end of each GCMV RNA positive strand. This has been confirmed in our laboratory by sequencing DNA complementary to the viral RNAs (Bretout, 1987). It is quite interesting to note that, as in almost all VPg-containing RNA viruses, the 5'terminal nucleotide, which we suppose is covalently linked to a VPg, is a U. Except for an extra U at position 10 in RNA2, the 5' ends of GCMV RNAI and 2 are identical (Fig. 6). They are also very similar to those of TBRV-S genomic RNAs (Hemmer et al., 1987). Along with homologies in the 3' non-coding regions of GCMV and TBRV-S genomic RNAs (Meyer et al., 1986; Bretout, 1987), our results strengthen the case for GCMV being a strain of TBRV. These terminal sequence homologies may explain, at least in part, why it is possible to obtain pseudorecombinants between TBRV-S and GCMV (Doz et al., 1980). Nevertheless such homologies are probably not the complete explanation because although GCMV and TBRV-S genomic RNAs show about the same level of homology in the 5' ends with the TBRV-S satellite, GCMV cannot act as a helper for this molecule (Doz et al., 1980). We wish to thank C. Fritsch for making information available to us concerning the 5'-terminal sequence of TBRV-S RNA1 before publication, and K. D. Mayo for help with the English of the manuscript. REFERENCES BRETOUT, C. ([ 987). Contribution ~ l'~tude du phknombne de la prOmunition entre deux souches du virus des anneaux noirs de la tomate ( T B R V ) sur Chenopodium quinoa Wild. Ph.D. thesis, Universit~ de Bordeaux II. COLLMER,C. W. &KAVER,J. M. (1985). Double-stranded RNAs of cucumber mosaic virus and its satellite contain an unpaired guanosine: implications for replication. Virology 145, 249-259.

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DE WACHTER,R. & FIERS, W. (1982). Two-dimensional gel electrophoresis of nucleic acids. In Gel Electrophoresisof Nucleic Acids." A Practical Approach, pp. 77-116. Edited by D. Rickwood & B. D. Haines. Oxford: I R L Press. DODD, S. M. & ROBINSON, D. J. (1984). Nucleotide sequence homologies among R N A species of strains of tomato black ring virus and other nepoviruses. Journal of General Virology 65, 1731-1740. DODDS, J. A., MORRIS, T. J. & JORDAN, R. L. (1984). Plant viral double-stranded R N A . Annual Review of Phytopathology 22, 151 168. DOZ, B., MACQUAIRE,G., DELBOS,R. & DUNEZ, J. (1980). Caract+ristiques et r61e du R N A 3 , R N A satellite du virus des anneaux noirs de la tomate. Annales de Virologie 131E, 489-499. HEMMER, O., MEYER, M., GREIF, C. & FRITSCH, C. (1987). Comparison of the nucleotide sequences of five tomato black ring virus satellite R N A s . Journal of General Virology 68, 1823 1833. KERLAN, C. & DUNEZ, J. (1983). Applications de l'immuno61ectromicroscopie ~ la d~tection de deux souches d'un m6me virus. Annales de Virologie 134E, 417-428. KIKUCHI, Y., KAZIMIERZ,T., FILIPOWICZ, w., SANGER, H. L. & GROSS, H. J. (1982). Circularization of linear viroid R N A via 2'-phosphomonoester, 3',5'-phosphodiester bonds by a novel type of R N A ligase from wheat germ and Chlamydomonas. Nucleic Acids Research 10, 7521-7529. LOMONOSSOFF, G. P., SHANKS, M. & EVANS, D. (1985). The structure of cowpea mosaic virus replicative form R N A . Virology 144, 351-362. MANIATIS, T., FRITSCH, E. F. & SAMBROOK,J. (1982). Molecular Cloning: A Laboratory Manual. New York: Cold Spring Harbor Laboratory. MARTELLI, G. P. & QUACQUARELLI,A. (1972). Grapevine chrome mosaic virus. CMI/AAB Descriptions of Plant Viruses, no. 103. MAXAM,A. M. & GILBERT, W. (1980). Sequencing end-labeled D N A with base-specific chemical cleavages. Methods in Enzymology 65, 499-560. MAYO,M. A., BARKER,H. & HARRISON,B. D. (1979). Polyadenylate in the R N A of five nepoviruses. JournalofGeneral Virology 43, 603 610. MAYO, M. A., BARKER, H. & HARRISON, B. D. (1982). Specificity and properties of the genome-linked proteins of nepoviruses. Journal of General Virology 59, 149-162. MEYER,M, HEMMER,O. & FRITSCH,C. (1984). Complete nucleotide sequence of a satellite R N A of tomato black ring virus. Journal of General Virology 65, 1575-1583. MEYER, M., HEMMER,O., MAYO,M. A. & FRITSCH,C. (1986). The nucleotide sequence of tomato black ring virus R N A 2. Journal of General Virology 67, 1257-1271.

(Received 20 July 1987)