Molecular Cloning of Complementary DNA Sequences of Citrus Tristeza Virus RNA

J. gen. Virol. (1983), 64, 1757-1763. Printedin Great Britain 1757 Key words: molecular cloning/citrus tristeza virus/cDNA/dsRNA

By A. ROSNER,* IRITH GINZBURG 1 AND M. BAR-JOSEPH Virus Laboratory, Agricultural Research Organization, The Volcani Center, Bet Dagan 50250 and 1Department of Neurobiology, The Weizmann Institute of Science, Rehovot 76100, Israel (Accepted 20 April 1983)

SUMMARY Complementary DNA (cDNA) of citrus tristeza virus (CTV) RNA, synthesized using calf thymus DNA random primers, was converted to a double-stranded form and inserted into the PstI site of the Escherichia coli pBR322 plasmid by the G-C tailing method. Bacterial clones harbouring virus-specific sequences were detected by colony hybridization with a 32p-labelled viral RNA probe. Hybridization patterns of denatured virus RNA revealed the presence of three types of specific clones: those hybridizing with a distinct narrow band corresponding to the full-length virus RNA, those hybridizing with a broader band of virus RNA sequences, and those hybridizing with several distinct virus-related RNA bands. Similar patterns were obtained when these clones were hybridized to purified double-stranded RNA from CTV-infected plants. None of these cDNA clones hybridized with similarly treated preparations extracted from healthy plants. The origin of variation among the CTV clones is discussed.

INTRODUCTION Citrus tristeza virus (CTV), which causes a serious disease of citrus plants, is a member of the closterovirus group (Bar-Joseph et al., 1979a), the virus particles being 2000 nm long and 10 to 12 nm wide (Kitajima et al., 1964; Bar-Joseph et al., 1970; Garnsey et al., 1977). Recently, an improved procedure for purification of intact non-fixed CTV particles was developed (Gumpfet al., 1981). Using purified CTV preparations, a homogeneous single-stranded RNA with an estimated molecular weight of 6.5 x 106 to 7.0 × 106 was isolated (Gumpf et al., 1981). The products of cell-free translation in a reticulocyte lysate system (Nagel et al., 1982) indicate that CTV RNA has messenger activity and carries information for at least four polypeptides with estimated molecular weights of 26K, 33K, 50K and 65K respectively. The 50K polypeptide is a major product and the 26K polypeptide is the capsid protein. Analysis of the double-stranded (ds) RNA replicative forms from CTV-infected hosts (Dodds & Bar-Joseph, 1983) indicated the presence of a 13.3 x 106 mol. wt. molecule. In addition, several species of dsRNA with molecular weights less than that expected for the replicative form (RF) were consistently found upon electrophoresis. Numerous CTV strains have been isolated which are recognized mainly on the basis of variations in their biological properties, differing considerably in their pathogenic effects. However, little if any serological difference was found among these strains (Gonsalves et al., 1978; Bar-Joseph et al., 1979 b). More detailed studies on strain differentiation at the molecular level have been hampered because of low yields of purified virus particles. This has shown the need to develop molecular probes specific to CTV. In this work we describe the cloning of double-stranded DNA complementary to various regions of the CTV genomic RNA.

METHODS Purification of virus and extraction of viral RNA. VT, a seedling yellows strain of CTV was purified from bark of Ethrog (Citrus medica L.) and Alemow (C. macrophylla W.) seedlings (Bar-Joseph et al., 1970). The virus suspension was loaded (8 ml/tube) on a step gradient (Gumpfet at., 1981) prepared by layering 1 ml each of0-15,

22.5 and 30% (w/w) Cs2SO, dissolved in 0.04 M-sodium phosphate pH 8.2, containing 10% (w/w) sucrose. Tubes were centrifuged in a Beckman SW41 rotor for 2-5 h at 38500 rev/min at 8 °C. The virus band, located immediately below a green zone, was collected using a peristaltic pump, diluted 1:5 in 50 mM-Tris HC1 pH 7.5, and the virus particles were precipitated by centrifugation at 42000 rev/min for 2 h in a Beckman 50Ti rotor. The virus pellet was resuspended in 200 ~tl 50 mM-Tri~HC1 pH 7.5, and SDS was added to a final concentration of 0.50/0. The solution was heated to 50 °C for 5 min and RNA was extracted twice with an equal volume of phenol/chloroform (1:1) and precipitated with 2.5 vol. ethanol at -20 °C. Isolation ofdsRNA. Frozen bark tissue from graft-inoculated Egyptian lime (C. aurantifolia S.) seedlings was ground to powder in liquid N2 with a mortar and pestle. The nucleic acids were extracted from the powdered tissue with phenol and the dsRNA was isolated by chromatography on CF-I 1 (Whatman) columns (Morris & Dodds, 1979; Dodds & Bar-Joseph, t983; Bar-Joseph et al., 1983). Electrophoresis of RNA and blotting. Fractionation of RNA in a denaturing agarose gel by electrophoresis and the transfer of RNA bands onto nitrocellulose were carried out as detailed by Fellous et al. (1982). Preparation of recombinant plasmids containing CTV DNA sequences. Single-stranded DNA complementary to CTV RNA was synthesized using calf thymus DNA random primers (Taylor et al., 1976) with avian myeloblastosis virus (AMV) reverse transcriptase (obtained from Dr J. Beard, Life Sciences, St Petersburg, Fla., U.S.A.). Double-stranded cDNA was prepared by a procedure in which single-stranded cDNA was not purified prior to second strand synthesis (Ginzburg et al., 1980). The second DNA strand was synthesized with DNA polymerase I followed by $1 nuclease treatment (Wickens et al., 1978), The dsDNA and PstI-cut pBR322 plasmid were tailed by terminal transferase homopolymer addition of poly(dC) and poly(dG) respectively, and annealed. E. coli MM294 cells were transformed with the DNA mixture (Ginzburg et al., 1980), and cells harbouring plasmids were selected on tetracycline-containing plates. Selection of clones containing inserts complementary to CTV RNA. Bacterial clones resistant to tetracycline but sensitive to ampicillin were grown in microtitre plates in LB medium. Replicas were transferred to nitrocellulose and their DNA was denatured as described by Ginzburg et al. (1980). Colonies harbouring recombinant plasmids containing CTV sequences were detected by hybridizing the filters to each of the following probes: (i) 32p-labelled cDNA prepared using virus RNA as template (Ginzburg et al., 1980); (ii) virus RNA, 32p-labelled with [y- 32p]ATP using polynucleotide kinase (Maizels, 1976; Rosner et at., 1983); and (iii) virus RNA further purified by electrophoresis in 1% agarose gel and the part of the gel containing the high molecular weight distinct virus band, as visualized under u.v, illumination of ethidium bromide-stained RNA, was cut out. The RNA was electroeluted from the gel and labelled with [y-3zp]ATP (as in ii). Hybridization conditions were as described by Ginzburg et aL (1980). Large scale preparation of recombinant plasmid from the E. coli cells was carried out as described previously (Ginzburg et aL, 1980). Restriction endonucleases PstI, EcoRI and HindIII were obtained from New England Biolabs and reaction conditions were according to the manufacturers" instructions.

RESULTS

Construction and identification of recombinant plasmid clones containing CTV cDNA sequences As CTV RNA is of a large size, i.e. molecular weight of 6.5 × 106 to 7 × 106, a library of clones representing the whole genome was prepared. The concentration of calf thymus DNA required for optimal priming ofcDNA synthesis was measured by adding increasing amounts of calf thymus DNA to a standard reaction mixture containing labelled [32p]dATP and the other three unlabelled nucleotides. Maximal incorporation was observed at a calf thymus DNA concentration of 360 ~tg/ml; this concentration was used in further cloning experiments. Above 400 ~tg/ml there was inhibition of the reaction which was attributed to masking of the template RNA. cDNA was synthesized with CTV RNA as template, converted into the double-stranded form, and treated with $1 nuclease, resulting in dsDNA molecules of about 600 nucleotides long. Some larger molecules of up to 1000 base pairs (bp) were also observed. The double-stranded molecules were tailed with poly(dC) and inserted into the PstI site of pBR322 plasmid tailed with poly(dG). The recombinant plasmids were introduced into E. coli cells by transformation, as described in Methods. Over 600 tetracycline-resistant, ampicillin-sensitive bacterial clones were isolated. Colonies harbouring viral cDNA sequences were detected by hybridization with the three 32p-labelled probes described in Methods. Hybridization with all three probes gave similar results: most of the clones that positively hybridized with the purified virus probe also hybridized with the two other probes. Several colonies which strongly hybridized with all three probes were chosen for further analysis. Cloning of citrus tristeza virus cDNA 1759 Plasmid

I X B1 A10 D1 E8 B12 D2 A3 D7

bp kbp

~23 ~9-4 ~6.6 ~4.4

~2-3 ~2-0

1350~. 1078~ 872~ 603 310 281~ 271~'-" 234 t 194/'~

Fig. 1. Size of the CTV cDNA inserts in the recombinant plasmids. The plasmid DNAs of seven selected clones harbouring CTV cDNA sequences in pBR322 and a control plasrnid not containing an insert (A10) were digested with PstI restriction endonuclease and fractionated in 1.1 ~ agarose gel by electrophoresis. 2 DNA digested by HindlII, and ~bX174DNA cut by HaelII were used as size markers.

Sizing of DNA inserts in the bacterial plasmids In order to determine the size of the inserted viral cDNA sequences, purified plasmid DNA preparations from CTV-VT clones were cut with restriction endonuclease PstI and subjected to electrophoresis on a 1.1 ~ agarose gel (Fig. 1). The inserts were estimated to be 250 to 900 bp long (see Fig. 1, plasmids B12 and E8), in comparison with size markers. An unusually large insert of about 2.3 kbp was also observed in clone DI. Three selected clones, A3, D7 and B 1 were also analysed by restriction cleavage with EcoRI and HindlII endonuclease, and found to be different in their partial restriction maps (not shown).

Hybridization of labelled plasmid DNA to a blot of fractionated viral RNA The cloned viral cDNA sequences were further hybridized with the genomic RNA of CTV. Viral RNA fractionated on a 1.1 ~ agarose formamide/formaldehyde denaturing gel (Fig. 2) was transferred onto nitrocellulose filters and hybridized with nick-translated plasmids as described by Fellous et al. (1982). Three typical patterns of hybridization were observed: clones A3 and D1 hybridized only with a high molecular weight narrow virus band (see arrow); clone BI hybridized with a somewhat broader band, including the high molecular weight one; clones D7 and E8 hybridized with the high molecular weight RNA band as well as with several distinct faster migrating bands. None of the clones hybridized with preparations from healthy plants which were purified similarly (Fig. 2, lanes H). 1760 A. ROSNER, I. GINZBURG AND M. BAR'JOSEPH

Clone

I D7 B1 Dl E8 A3 I IV .IIv IIv HIIv .llv .I 4

kbp .~--V 9.4 6.6 4.4

2-3 2.0

0"5 '

Fig. 2. Formamide/formaldehydeRNA blot ofCTV RNA hybridized with various recombinant CTV clones. CTV RNA (V) and RNA from uninfected plants (H) were fractionated in a 1-1~ formamide/formaldehyde denaturing gel and transferred to nitrocellulose paper. DNA of six different plasmid clones were 32p-labelled by nick translation and each of them was hybridized to a strip of nitrocellulose containing viral RNA and a similar preparation from healthy plants. H/ndIII fragments of 2DNA were used as size markers.

Analysis of dsRNA in CTV-infected plants The three types of virus clone were used to analyse dsRNA species extracted from CTV- infected Egyptian lime seedlings. The dsRNA was purified from plants and fractionated on both denaturing (Fig. 3a) and non-denaturing (Fig. 3b) agarose gels. The fractionated RNA preparations were transferred to nitrocellulose sheets and hybridized with the above labelled plasmids. The A3 clone that was shown to be unique for the high molecular weight viral band (Fig. 3a, lane A3-V), hybridized with the dsRNA molecule equal in size to the denatured viral RNA (Fig. 3a, lane A3-ds). The D7 clone which was found to hybridize with a heterogeneous family of virus-related RNA molecules (Fig. 3a, lane D7-V) also hybridized with a heterogeneous population of dsRNA species (Fig. 3a, lane D7-ds). It should be noted, however, that not all the distinct bands observed in the dsRNA lane (Fig. 3 a, lane D7-ds) coincide with those observed in the viral single-strand RNA preparation (Fig. 3a, lane D7-V). The BI clone hybridized with a broad band in both the viral (Fig. 3a, lane BI-V) and dsRNA (Fig. 3a, lane Bl-ds) preparations (better seen upon longer autoradiographic exposures). None of the clones hybridized with dsRNA prepared from uninfected plants as a control (Fig. 3a, lanes H). The hybridization patterns of the A3 and D7 probes with blots of non-denaturing dsRNA gels (Fig. 3 b) were similar to the denaturing one. The A3 clone hybridized only with the high molecular weight RF ofCTV (mol. wt. 13.3 x 106) (Fig. 3b, lane A3-ds), whereas the D7 clone hybridized with the RF as well as with several smaller segments (Fig. 3 b, lane D7-ds) which were similar to Cloning of citrus tristeza virus cDNA 1761 (a) Clone (b) Clone < I A3 D7 B1 I I A3 D7 I :c z IH ds vl[. ds VII. ds vl Iv ds HIIV ds H I

kbp

9"4~ v. "W 6.6-- 4.4-- --28S Ilv-

2-3-- 2-0-- --18S

0.5--~

Fig. 3. Analysis ofdsRNA from infected plants by blot hybridization with CTV clones. (a) CTV RNA (V), dsRNA from CTV-infected plants (ds) and dsRNA from healthy uninfected plants (H) were subjected to electrophoresis in parallel in a 1.1 ~ formamide/formaldehyde denaturing gel, transferred to nitrocellulose, and hybridized with three of the isolated clones A3, B1 and D7, labelled with 32p by nick translation. Ribosomal RNA (28S and 18S) and 2DNA fragments generated by a HindlII digestion were used as size markers. (b) RNA samples as in (a) were fractionated in duplicate in a non- denaturing 1.1 ~ agarose gel containing E buffer, transferred to nitrocellulose, and hybridized with clones D7 and A3 as in (a). the dsRNA fractions obtained from CTV-infected plants by Dodds & Bar-Joseph (1983). The difference in mobility of the major dsRNA band, relative to that of the viral band in denaturing and non-denaturing gels, is due to strand separation leading to reduction in the apparent molecular weight of the dsRNA molecules under denaturing conditions.

DISCUSSION The aim of the present work was to obtain eDNA probes containing sequences homologous to various regions of the CTV genome. The synthesis of DNA complementary to several plant RNA viruses has already been reported (e.g. Gonda & Symons, 1978). cDNA has been used to study the homology between virus strains (Palukaitis & Symons, 1980; Van de Valle & Siegel, 1982), between satellites (Gould et al., 1978; Mossop & Francki, 1979; Kiefer et al., 1982), between RNA species of multi-component viruses (Talianski et al., 1979), for the elucidation of genome structure and replication modes (Goelet & Karn, 1982; Bar-Joseph et al., 1983), for the diagnosis of virus infection (Kummert, 1980; Maule et al., 1983), and for sequencing (Goelet et al., 1982; Meshi et al., 1982). CTV is a single, plus-stranded RNA virus of unusually large size (Bar-Joseph et al., 1979a; Dodds & Bar-Joseph, 1983; Gumpf et al., 1981; Nagel et al., 1982). The recently developed procedure (Gumpf et al., 1981) for purifying virus particles enabled us to obtain virus RNA suitable for cloning and we have obtained a selection of eDNA clones. Two main groups of cloned virus sequences were obtained: one category hybridized exclusively to the full-length virus RNA while the other hybridized to full-length virus RNA and also to several distinct RNA species of smaller sizes. It is possible that the smaller hybridizing RNAs are subgenomic species, although their possible origin by specific breakage cannot be excluded; the particles of beet yellows virus, another closterovirus, appear to break at specific places (Bar-Joseph & Hull, 1974). A distinct high molecular weight band and several smaller distinct dsRNA molecules have 1762 A. ROSNER, I. GINZBURG AND M. BAR-JOSEPH been detected in extracts of CTV-infected plants (Dodds & Bar-Joseph, 1983). It is possible that some CTV dsRNAs correspond to virus RNA bands seen upon hybridization with the D7 cDNA clone (Fig. 3). The presence of several smaller RNAs within the CTV viral particles, some corresponding to dsRNAs found in infected plants, may indicate that only a part of the subgenomic molecules are packaged in virus particles smaller than normal size, as has been reported for tobacco mosaic virus (Goelet & Karn, 1982; Zelcer et al., 1981 ; Dawson & Dodds, 1982; Bar-Joseph et al., 1983). Strains of CTV are numerous and vary considerably in their effects on citrus trees. Strain typing on the basis of biological tests is difficult and time-consuming (Bar-Joseph et al., 1979a). The availability of a molecular probe for CTV is expected to have practical application in diagnosing CTV strains by rapid biochemical tests, and it will enable elucidation of the molecular structure and diversity of the virus.

We gratefully acknowledge the technical support of Mrs Mira Moscovitz. This work was supported by the United States-Israel (Binational) Agricultural Research and Development Fund (BARD).

REFERENCES BAR-JOSEPH, M. & HULL, R. (1974). Purification and partial characterization of sugar beet yellows virus. Virology 62, 552-562. BAR-JOSEPH, M., LOEBENSTEIN, G. & COHEN, J. (1970). Partial purifications of virus-like particles associated with citrus tristeza disease. Phytopathology 60, 75-78. BAR-JOSEPH, M., GARNSEY, S. M. & GONSALVES, D. (1979a). The closteroviruses: a distinct group of elongated plant viruses. Advances in Virus Research 25, 93-168. BAR-JOSEPH, M., GARNSEY, S. M., GONSALVES, D., MOSCOVITZ, M., PURCIFULL, D. E., CLARK, M. F. & LOEBENSTEIN, G. (1979b). The use of enzyme-linked immunosorbent assay for detection of citrus tristeza virus. Phytopathology 69, 190-194. BAR-JOSEPH, M., ROSNER, A., MOSCOVITZ, M. & HULL, R. (1983). A simple procedure for the extraction of double- stranded RNA from virus-infected plants. Journal of Virological Methods 6, 1-8. DAWSON, W. O. & DODDS, J. A. (1982). Characterization of subgenomic double stranded RNAs from virus-infected plants. Biochemical and Biophysical Research Communications 107, 1230-1235. DODDS, J. A. & BAR-JOSEPH, M. (1983). Double-stranded RNA from plants infected with closteroviruses. ,Phyto- pathology. 73, 419-423. FELLOUS, A., GINZBURG, I. & LITrAUER, U. Z. (1982). Modulation of tubulin mRNA levels by interferon in human lymphoblastoid cells. EMBO Journal 1, 835-839. GARNSEY, S. M., GONSALVES,D. & PURCIFULL, D. E. (1977). Mechanical transmission of citrus tristeza virus. Phyto- pathology 67, 956-968. GINZBURG, I., DE BAETSELIER,A., WALKER, M. D., BEHAR, L., FRISCHANT,A. M. & LITTAUER, U. Z. (1980). Brain tubulin and actin cDNA sequences : isolation of recombinant plasmids. Nucleic Acids Research 8, 3553-3564. GOELET, P. & KARN, J. (1982). Tobacco mosaic virus induces the synthesis of a family of 3' coterminal messenger RNAs and their complements. Journal of Molecular Biology 154, 541 550. GOELET, P., LOMONOSSOFF, E. P., BUTLER, P. J. G., AKAM, M. E., GAIT, M. J. & KARN, J. (1982). Nucleotide sequence of tobacco mosaic virus RNA. Proceedings of the National Academy of Sciences, U.S.A. 79, 5818-5822. GONDA, T. J. & SYMONS,R. H. (1978). The use of hybridization analysis with complementary DNA to determine the RNA sequence homology between strains of plant viruses: its application to several strains of cucumoviruses. Virology 88, 361-370. GONSALVES, D., PURCIFULL, D. E. & GARNSEY, S. M. (1978). Purification and serology of citrus tristeza virus. Phyto- pathology 68, 553-559. GOULD, A. R., PALUKAITIS, P., SYMONS, R. H. & MOSSOP, D. (1978). Characterization of satellite RNA associated with cucumber mosaic virus. Virology 84, 443-455. GUMPF, J. D., BAR-JOSEPH, M. & DODDS, J. A. (1981). Purification of citrus tristeza virus on sucrose-cesium sulphate cushion gradients and estimation of its RNA size. Phytopathology 71, 878. KIEFER, M. C., DAUBERT, S. D., SCHNEIDER,I. R. & BRUENING, G. (1982). Multimeric forms of satellite of tobacco ring- spot virus RNA. Virology 121, 262-273. KITAJIMA, E. W., SILVA, D. M., OLIVEIRA, A. R., MULLER, G. W. & COSTA, A. S. (1964). Electron microscopical investigations of tristeza. In Proceedings of the 3rd Conference of the International Organization of Citrus Virologists, pp. 1-9. Edited by W. E. Price. Gainesville: University of Florida Press. KUMMERT, J. (1980). Synthesis and characterization of DNA complementary to carnation mottle virus RNA. Virology 105, 35-40. MAIZELS, N. (1976). Dictyostelium 17S, 25S and 5s-r DNAs lie within a 38,000 base pair repeated unit. CeU9, 431- 438. MAULE, A. J., HULL, R. & DONSON, J. (1983). The application of spot hybridization to the detection of DNA and RNA viruses in plant tissues. Journal of Virological Methods 6 (in press). Cloning of citrus tristeza virus cDNA 1763

MES1-/I, I., TAKAMATSU, N., OHNO, T. & OKADA, Y. (1982). Molecular cloning of the complementary DNA copies of the common and cowpea strains of tobacco mosaic virus RNA. Virology 118, 64 75. MORRIS, X. J. & DODDS, J. A. (1979). Isolation and analysis of double-stranded RNA from virus-infected plants and fungal tissue. Phytopathology 69, 854-858. MOSSOP, D. W. & FRANCKI, R. L B. (1979). Comparative studies on two satellite RNAs of cucumber mosaic virus. Virology 95, 395-404. NAGEL, J., HIEBERT, E. & LEE, R. F. (1982). Citrus tristeza virus RNA translated with a rabbit reticulocyte lysate: capsid protein identified as one of the products. Phytopathology 72, 953. PALUKAITIS, P. & SYMONS, R. H. (1980). Nucleotide sequence homology of thirteen tobacco virus RNAs as determined by hybridization analysis with complementary DNA. Virology 107, 354-361. ROSNER, A., BAR-JOSEPH, M., MOS¢OVlTZ, M. & MEVARECH, M. (1983). Diagnosis of specific viral RNA sequences in plant extracts by hybridization with a polynucleotide kinase mediated 3~-P-labeled double-stranded RNA probe. Phytopathology 73 (in press). TALIANSKI, M. E., BOYKOV, S. V., MOLISHENKO, S. I., KASVAN, V. M. & ATABEKOV, J. G. (1979). A study of barley stripe mosaic virus (BSMV) genome. I. Determination of sequence homology between BSMV RNA species. Molecular and General Genetics 175, 89 92. TAYLOR, J. M., ILLM~NSEE, a. & SUMMERS, J. (1976). Efficient transcription of RNA into DNA by avian sarcoma virus polymerase. Biochirnica et biophysica acta 442, 324-330. VAN DE VALLE, M. J. & SIEGEL, A. (1982). Relationships between strains of tobacco mosaic virus and other selected plant viruses. Phytopathology 72, 390-395. WICKENS, M. P., BUELL, G. N. & SCrI1MKE, R. T. (1978). Synthesis of double-stranded DNA complementary to lysozyme, ovomucoid and ovalbumin mRNA. Journal of Biological Chemistry 253, 2483-2495. ZELCER, A., WEABER,K. F., BALAZS,E. & ZAITLIN, M. (1981). The detection and characteristics of viral related double- stranded RNAs in tobacco mosaic virus infected plants. Virology 113, 417-427.

(Received 18 February 1983)