Journal of General Virology (1992), 73, 2517-2523.

2517

Printed in Great Britain

Replication of grapevine fanleaf virus satellite RNA transcripts in Chenopodiumquinoaprotoplasts F. Hans, 1 M.

Fuchs2~ and

L. Pinck ~*

l [nstitut de Biologie Moldculaire des Plantes du CNRS, Laboratoire de Virologie, 12 rue du G~ngral Zimmer, 67084 Strasbourg Cedex and 2Station de Recherches Vigne et Vin, INRA, 68021 Colmar Cedex, France

A set of full-length cDNA clones of the satellite R N A of grapevine fanleaf nepovirus isolate F l 3 (GFLVF13) was constructed with a variable number of additional, non-viral nucleotides at the 5' and 3' ends. The biological activity of the RNAs transcribed from these constructs was tested in Chenopodium quinoa protoplasts using a helper virus. When inoculated with arabis mosaic virus S (ArMV-S) R N A as helper, transcripts with 33 non-viral nucleotides at the 5' end (tr45p4) did not replicate, whereas transcripts with

only one non-viral nucleotide at the 5' end (tr3S and tr3M) did replicate. Capping of the transcripts enhanced their replication. On the other hand, the presence of extra nucleotides at the 3' end had little influence on the biological activity of the in vitro transcripts. In contrast with ArMV-S, G F L V isolate 24 was not a helper for tr3M transcripts, indicating a specific interaction between the helper strain and the satellite RNA.

Introduction

as the satellites of chicory yellow mottle virus (CYMV; Rubino et al., 1990), tomato black ring virus (TBRV; Hemmer et al., 1987) and a lilac isolate of arabis mosaic virus (ArMV-L; Liu et al., 1990). GFLV RNA3 and the satellite R N A of ArMV-L share 83% nucleotide sequence identity, but the homology with other nepovirus satellite RNAs is more limited. With TBRV satellite RNA, for example, only short scattered stretches of homology occur in addition to the 5' end consensus sequence (Fuchs et al., 1989). Rubino et al. (1990) noticed a short region with about 50% conservation located between nucleotides 94 and 133 in the coding region of the CYMV satellite RNA, and between nucleotides 183 and 223 in RNA3 of GFLV. Comparison of the P3 amino acid sequences revealed 72% identity between GFLV and ArMV-L, but only

Grapevine fanleaf virus (GFLV) is a widely distributed nepovirus, causing serious damage in grapes. Its genome comprises two single-stranded polyadenylated RNAs which carry a genome-linked protein at their 5' ends (Pinck et al., 1988). RNA1 (7342 nucleotides) encodes polyprotein P1 (Mr 253K; Ritzenthaler et al., 1991) and RNA2 (3774 nucleotides) encodes polyprotein P2 (Mr 122K ; Serghini et al., 1990) (Fig. 1). An additional R N A of 1114 nucleotides with the same terminal structures as the genomic R N A [5' VPg and 3' poly(A)], named RNA3, is present in the F13 isolate of GFLV. This R N A is a satellite R N A because it requires the presence of a helper genome for its replication and encapsidation. RNA3 belongs to the class of large satellites (Fritsch & Mayo, 1989). It has coding capacity for a protein, P3, of M~ 37K and is translated in wheat germ extracts to produce a species of apparent Mr 39K (Pinck et al., 1988). Except for the consensus sequence of 10 nucleotides adjacent to the VPg described by Fuchs et al. (1989 and references therein), no significant sequence homology was found between the genomic RNAs and the satellite R N A of GFLV. This consensus sequence is also present at the 5' end of other large nepovirus satellite RNAs such t Present address: New York State Agricultural Experiment Station, Department of Plant Pathology, Cornell University, Geneva, New York 14456-0462, U.S.A. 0001-1073 © 1992 SGM

SI ~

RNA1 o-

(A)~ 7342nt

VPg~

RNA2

•

~q SlI 4 ~

(A). 3774 nt

VPg

RNA3 •

VPg

(A), 1114nt

Fig. 1. Localization of riboprobes on RNA1, RNA2 and RNA3 of GFLV. The sizes of the viral RNAs and riboprobes (double-headed arrows) are drawn to scale. Prl indicates the position of the primer on RNAI. The numbers on the double-headed arrows indicate the position in the sequence.

2518

F. Hans, M. Fuchs and L. Pinck

restricted homology with TBRV (residues 345 to 371). The high content of basic residues (K and R) in the N-terminal part of P3 may indicate that this domain is involved in interactions with nucleic acids (Fuchs et aL, 1989). In this paper, we describe the construction of a set of full-length cDNA clones of the GFLV satellite RNA. Transcripts of RNA3 were synthesized using an in vitro transcription system and Chenopodium quinoa protoplasts were used to test their biological activity. We report on the importance of the 5' and 3' non-viral nucleotides for the replication of the synthetic transcripts. The ability of several virus strains to act as helper for the replication of this satellite R N A was also investigated and the specificity of the helper virus-satellite R N A association is discussed.

Methods Virus strains, multiplication and purification. GFLV and ArMV isolates were propagated on C. quinoa in a glasshouse and purified as described by Pinck el aL (1988) with the following modifications: the virus pellets from the polyethylene glycol (PEG) precipitation step and from the ultracentrifugation step were dissolved in 25 ra~-glycine buffer pH 9. The virus was further purified and the RNAs were extracted as described by Pinck et al. (1991). Bacterial strains. Escherichia coli strains JM109 and TG2 were transformed as described by Hanahan (1983) or by electroporation (Sambrook et al., 1989). All constructs were cloned in the phagemid BlueScribe M13+ (pBS+, Stratagene). Construction o f a full-length G F L V R N A 3 cDNA. Clone pA4 containing the cDNA of GFLV RNA3 inserted in a pUC9 vector was described by Fuchs et al. (1989). From this clone, the cDNA of RNA3 was excised by digestion with HindIII and EcoRI and cloned into the pBS + expression vector cut with the same enzymes. The resulting copy of RNA3 contained 33 non-viral nucleotides at the 5' end and 22 nonviral nucleotides at the 3' end arising from the cloning strategy. Positive-sense RNA synthesis was under control of the T3 promoter (Fig. 2). The transcription vector was named p45p4 (Fuchs, 1989). Site-directed mutagenesis (T) Elimination o f the 5" non-viral nucleotides. The non-viral nucleotides located between the T3 promoter and the first nucleotide of RNA3 were deleted by mutagenesis with a synthetic oligonucleotide complementary to nucleotides 1 to 20 of RNA3 and to the 12 3'terminal nucleotides of the T3 promoter region: 5' GTCCATAG A A A T T T T T C A T A C T T T A G T G A G G G 3'. The mutation procedure was carried out according to the manufacturer's instructions (Stratagene). Recombinant clones were recovered by hybridization on Colony/Plaque Screen membranes using 32p-labelled mutagenic oligonucleotide and further screened by restriction enzyme digestion. Selected clones were sequenced using the 18-mer 5' GATGGATCC A C G G T A A C G 3' complementary to nucleotides 26 to 43 of RNA3 as primer and the T7 modified R N A polymerase (Sequenase procedure). The resulting transcription vector was named p3S (Fig. 2). (ii) Elimination of the 3' non-viral nucleotides. The 22 non-viral nucleotides at the 3' end of plasmid p3S were substituted by a HindlII site so as to generate a cDNA of GFLV RNA3 with three extra nucleotides at the 3' end. The synthetic oligonucleotide used was 5" TATAGGGCGAATTCAAGC(T21) 3", complementary to the

T3

~

p45p4 ~

G

14 G

~

A --A K 3 C ~ ( ~ G ~ T C G A ( ] G G A ~ T T C / ~ : ; T

75 A

EcoRI

T_~3~ p3S

~:X~~

A__AtGca~'tcc.~Oc~tc~cc6c~aTFcAct 75 A

EcoR[

T3 ~ p3M

r x - ~ ~

a.,.~crr

75 AHindlIl

Fig. 2. Schematic representation of cDNA clones of GFLV RNA3 used for synthesis of in vitro transcripts (see Methods). The hatched box followed by one or two Gs corresponds to the T3 promoter. The noncoding regions of RNA3 cDNA are boxed and the P3 coding region is filled.

poly(A) tail of the cDNA of RNA3 and to 17 nucleotides of pBS+ located after the cDNA of RNA3. The mutagenesis was performed using the procedure for efficient selection of mutated D N A developed by Kunkel (1985). Recombinant D N A molecules were screened by colony hybridization using 32p-labelled mutagenic oligonucleotide, by restriction analysis and by nucleotide sequence analysis as described above. This transcription vector was named p3M (Fig. 2). In vitro transcription. DNA was linearized by appropriate enzymes, phenol-chloroform-extracted and ethanol-precipitated. For uncapped transcripts, 101xg of linearized D N A was incubated in 100111 containing 40 mM-Tris-HC1 pH 7-5, 6 mM-MgCI, 2.2 mM-spermidine, 0.4 mM each of rATP, rGTP, rCTP and rUTP, 1-6 units/pl RNasin and 2 units/pl T3 RNA polymerase for 1 h at 37 °C. For synthesis of capped transcripts, GTP was decreased to 25 pM and 500 ~tM-cap analogue mTGpppG was added. After 30 rain at 37 °C, the GTP concentration was adjusted to 0-4 mM and the reaction continued for 1 h. The DNA was removed by treatment with RNase-free DNase I for 30 min at 37 °C. The transcripts were purified by phenol-chloroform and ether extractions followed by ethanol precipitation. The size and integrity of transcripts were checked by electrophoresis under denaturing conditions in a 1% formaldehyde-agarose gel. By convention, the transcripts derived from p45p4, p3S and p3M were named tr45p4, tr3S and tr3M, respectively. In vitro translation. The coding capacity of the transcripts was tested in a wheat germ system prepared as described by Godefroy-Colburn et al. (1985) and in the nuclease-treated rabbit reticulocyte lysate system (NT-RRLS) obtained from Promega, as described by Margis et al. (1991). The 3sS-labelled proteins synthesized were analysed on a 1 5 ~ SDS-polyacrylamide gel using the system of Laemmli (1970). C. quinoa protoplast preparation and inoculation. (i) Preparation. C. quinoa plantlets were conditioned in a growth chamber at 22 °C during a 16 h light period and at 18 °C during the night. The light was provided by a sodium lamp producing 8000 lux. Plants were regularly watered with a 1/1000 (v/v) solution of Lifan (Bayer). After 10 to 15 days, plants with four to six pairs of leaves were used for protoplast isolation. Leaves were rinsed briefly in a 7 0 ~ ethanol bath, sterilized in a 1% bleach batik' for 10 min and washed three times in water for 10 min per wash. They were cut into small squares and placed in 0.6 M-mannitol, 10 mM-CaClz, pH 5.6. The leaf fragments were digested overnight at 25 °C with 1.3 ~ cellulase, 0-3 % Macerozyme (R-10, Onozuka) and 0.1% BSA in culture medium containing 0.2mM-KH2PO4, 1mbI-KNO3, l mM-MgSO4, 10mMCaC12, 1 p.M-KI, 0.01 ~tM-CuSO4, pH5.6 (Rottier et al., 1979). Protoplasts were freed from tissue debris on a 100 ~tm filter, washed twice with 0.54 M-sucrose and three times with 0.6 M-mannitol, pH 5.6. Fluorescein diacetate (FDA) staining was used to determine the percentage of viable protoplasts (Widholm, 1972).

G F L V satellite R N A transcripts in protoplasts (ii) PEG inoculation. C. quinoaprotoplasts were inoculated according to the procedure of de Varennes et al. (1984). Protoplasts (1 x 106) concentrated in I00 Izl 0.6 M-mannitol pH 5-6 were mixed with 250 lal 30% PEG-6000, 3 mM-CaCI2, 0"4 M-mannitol, containing the RNA inoculum. The suspension was gently mixed for 15 s, diluted with 2-5 ml 0-6 M-mannitol, 10 mM-CaCI2, pH 5-6, kept on ice for 10 min and washed three times with 0-6 M-mannitol, 10 mM-CaCI2, pH 5-6. The protoplasts were adjusted to 3 x 105 per ml culture medium and were incubated in small Petri dishes with a bottom layer of 1% agarose (Seaplak LE, Seakem) containing culture medium at 22 °C under continuous diffuse lighting. In some experiments, 30 I~Ci[35S]methionine per 1 x l0 s protoplasts was added to the culture medium for labelling. (iii) Electroporation procedure for inoculation. Protoplasts were prepared as described above but the digestion time was reduced to 3.5 h, the temperature increased to 30 °C and the sucrose wash was omitted. The protoplasts were infected as described by Veidt et al. (1992). After the isolation step, protoplasts were kept overnight at 4 °C. Genomic viral helper RNA supplemented or not with the synthetic transcripts was added to 2 x l0 s protoplasts in 0.5 ml 0.6 M-mannitol, 0"1 mM-CaCIz,pH 5-6. The suspension was transferred to a cold 0-4 cm path-length cuvette and electroporated during a 12 ms high voltage pulse provided by discharge of a 125 IxF capacitor (Bio-Rad) set to 300 V. Protoplasts were incubated for 30 min on ice, washed once with 0.6 M-mannitol, 0"1 mM-CaCI2, pH 5-6, and cultivated as described for PEG inoculation, except that 0.3 mg/ml carbenicillin was added to the culture medium.

Detection of viral RNAs in total RNA from infected protoplasts. Antisense RNA probes (riboprobes) specific for the GFLV genome were used to detect the presence of GFLV RNA synthesized in protoplasts. The PstI-EcoRI fragment of eDNA clone pA87 of RNA 1 (nucleotides 2176 to 3100), the EcoRI-HindIII fragment of eDNA clone pG38 of RNA2 (nucleotides 147 to 1020) and the SphI-SalI fragment of eDNA clone pA5 of RNA3 (nucleotides 129 to 753) were subcloned into compatible sites of pBS +. The resulting plasmid, pSI, pSII and pSIII, respectively, were used for the synthesis of the corresponding SI, SII and SIII riboprobes, pSI was linearized by HindIII digestion and transcribed by T3 RNA polymerase, pSII and pSIII were linearized by EcoRI treatment and transcribed by T7 RNA polymerase. Transcription was performed on 1 ~tg of template DNA as described above except that rUTP was replaced by 60~tCi [~-32p]rUTP. Protoplasts were harvested at various times post-inoculation, pelleted and disrupted in 2001al 50 mM-Tris-HCl pH 7-5, 100 mMNaCI, 1 mM-EDTA, 1~ SDS. Total nucleic acid was twice extracted with phenol-chloroform (1:1, v/v) and ethanol-precipitated in the presence of 0.3 M-sodium acetate pH 5.2. To recover RNA free of DNA, the pellet was twice washed with 3 M-sodium acetate pH 5-2. Total protoplast RNAs were separated by electrophoresis on a 1% formaldehyde-agarose gel and blotted for 3 h with 10 mM-NaOH and 1 mM-EDTA to a Hybond N ÷ membrane. Hybond N ÷ membrane was prehybridized for 1 to 4 h in 50% formamide, 0-5% non-fat milk, 1-5 x SSPE, 1% SDS, 200 ~tg/ml yeast RNA and 500 ~tg/ml sonicated salmon sperm DNA. After addition of the labelled probes, hybridization was continued overnight at 50 °C. Membranes were washed twice for 15 min in 2 x SSPE, 0.1% SDS and for 15min in 0.5 x SSPE, 0.1% SDS at 50°C. lmmunoprecipitation. To detect the coat protein of GFLV, healthy and infected C. quinoa protoplasts were cultured in the presence of 30~tCi [3sS]methionine per 1 x l0 s protoplasts and harvested at various times post-inoculation. The coat protein was detected by immunoprecipitation as described by Demangeat et al. (1990) in aliquots containing 1 x 10s protoplasts.

2519

Results and Discussion Transcription o f full-length G F L V R N A 3 c D N A T h e t r a n s c r i p t tr45p4 p r o d u c e d b y in vitro run-off t r a n s c r i p t i o n o f EcoRI-linearized p 4 5 p 4 using R N A p o l y m e r a s e T3 c o n t a i n e d 33 a n d 22 n o n - v i r a l n u c l e o t i d e s at the 5' a n d 3' ends, r e s p e c t i v e l y (Fig. 2). T o d e t e r m i n e the influence o f n o n - v i r a l n u c l e o t i d e s on i n f e c t i v i t y o f tr45p4, the n o n - v i r a l n u c l e o t i d e s at the 5' e n d were first e l i m i n a t e d b y s i t e - d i r e c t e d m u t a g e n e s i s to give clone p3S. T h e t r a n s c r i p t tr3S o b t a i n e d f r o m this clone c o n t a i n e d only one n o n - v i r a l G n u c l e o t i d e at the 5' e n d (nucleotide + 1) a n d 22 n o n - v i r a l n u c l e o t i d e s at the 3' end. T h e p l a s m i d p 3 M was c o n s t r u c t e d b y s u b s t i t u t i o n o f a HindIII site for t h e 22 n o n - v i r a l n u c l e o t i d e s at the 3' e n d o f clone p3S. T h e t r a n s c r i p t t r 3 M o b t a i n e d f r o m HindIIIl i n e a r i z e d p 3 M differed f r o m n a t u r a l R N A 3 only in the a b s e n c e o f VPg, a n d the p r e s e n c e o f one n o n - v i r a l G at the 5' e n d a n d t h r e e n o n - v i r a l residues at t h e 3' e n d o f the p o l y ( A ) tail. T h e yield o f t r a n s c r i p t tr45p4 was fivefold t h a t o f tr3S a n d t r 3 M p r e s u m a b l y b e c a u s e the T3 p r o m o t e r in p 4 5 p 4 c o n t a i n e d t w o G s at its 5" t e r m i n u s , w h e r e a s tr3S a n d t r 3 M c o n t a i n e d only one G (Fig. 2). It is k n o w n t h a t the efficiency o f in vitro t r a n s c r i p t i o n w i t h such a system is affected by the c o n t e x t n e a r t h e t r a n s c r i p t i o n i n i t i a t i o n site ( E g g e n et al., 1989). I n all cases, the yield o f c a p p e d t r a n s c r i p t s was t w o f o l d lower t h a n the yield o f u n c a p p e d t r a n s c r i p t s ( d a t a n o t shown).

I n vitro translation o f R N A 3 transcripts T h e m e s s e n g e r p r o p e r t i e s o f tr45p4, tr3S a n d t r 3 M were a n a l y s e d in w h e a t g e r m e x t r a c t s a n d in N T - R R L S . T h e p r o t e i n s t r a n s l a t e d f r o m all t r a n s c r i p t s c o m i g r a t e d w i t h the P3 p r o t e i n s y n t h e t i s e d f r o m the n a t u r a l R N A 3 ( d a t a n o t shown) ( P i n c k et al., 1988). T h i s i n d i c a t e d t h a t the full-length clones o f R N A 3 were n o t a l t e r e d d u r i n g the cloning a n d m u t a g e n e s i s steps.

Replication o f G F L V R N A in C. q u i n o a protoplasts A yield o f 2 × 106 to 4 x 106 C. quinoa p r o t o p l a s t s p e r g o f leaves was r e g u l a r l y o b t a i n e d . F D A s t a i n i n g i n d i c a t e d t h a t 98 to 100% o f the p r o t o p l a s t s were v i a b l e after t h e isolation step a n d 50 to 6 0 % a f t e r P E G t r e a t m e n t . E i g h t y a n d 6 0 % o f the p r o t o p l a s t s s u r v i v e d a f t e r 48 h a n d 72 h o f culture, respectively. S i m i l a r p r o t o p l a s t v i a b i l i t y w a s o b t a i n e d by de V a r e n n e s et al. (1984). T h e c a p a c i t y o f freshly p r e p a r e d C. quinoa p r o t o p l a s t s to r e p l i c a t e G F L V R N A was tested a f t e r i n f e c t i o n b y t h e P E G i n o c u l a t i o n m e t h o d . P r o t o p l a s t s (1 × 10 6) were

F. Hans, M. Fuehs and L. Pinck

2520

1

2

3

4

5

6

7

8

9

RNAI - RNA2

RNA3 - Fig. 3. Northern blot analysis of the time course of GFLV RNA synthesis in protoplasts inoculated with GFLV RNA. Protoplasts (1 × 106)were inoculatedwith 10 ~tgRNA by the PEG method. RNAs were extracted from I x l0 s protoplasts at 0 (lane 3), 12 (lane 4), 24 (lane 5), 48 0ane 6), 72 (lane 7) and 96 (lane 8) h post-inoculation.Total protoplast RNAs were separated by electrophoresis on a 1X formaldehyde-agarosegel, blotted on a Hybond N ÷ membrane and hybridizedwith a mixture of the three 32p-labelledriboprobes specific for each GFLV RNA. Lanes 1 and 9, GFLV RNA as control; lane 2, RNAs from healthy protoplasts.

1

2

3

4

5

6

7

Fig. 4. Detection of the 56K coat protein in 1 x 106 protoplasts infected with 10 ~tgGFLV RNA using the PEG method. Immunoprecipitation was carried out on total proteins of I x 105 protoplastswith anti-viral serum diluted 1/1000,at 0 (lane 2), 12 (lane 3), 24 0ane 4), 48 (lane 5), 72 (lane 6) and 96 (lane 7) h post-inoculation. The immunoprecipitateswere analysedon a 10H SDS-polyacrylamidegel. Lane 1, uninfected protoplasts as control.

inoculated with 10 ~tg G F L V R N A and 1 x 105 protoplasts were collected at various times post-inoculation. Total protoplast RNAs were extracted and analysed by Northern blotting for the presence of viral R N A using the SI, SII and SIII riboprobes. Newly synthesized G F L V R N A s were detected 48 h post-inoculation (Fig. 3, lane 6) and the intensity of the signal increased with time to reach a maximum 96 h post-inoculation (Fig. 3, lane 8). In a separate experiment, the coat protein could be detected by immunoprecipitation 24 h post-inoculation (Fig. 4). These results demonstrate that the C. quinoa protoplasts permit the replication of G F L V RNA.

In order to determine the minimum level of R N A inoculum necessary for infection, 1 x 106 protoplasts were inoculated with increasing amounts of G F L V R N A by using the P E G method. Total R N A s from 1 × 105 protoplasts were extracted 72 h post-inoculation and the presence of viral R N A was detected by primer extension (Dore et al., 1989) using the Prl primer complementary to nucleotides 77 to 94 of RNA1 (Fig. 1). Protoplasts inoculated with 1 ~tg of G F L V R N A were not infected, whereas protoplasts inoculated with 5 or 10 ~tg of viral R N A were infected (data not shown). In all further infection experiments, the ratio of 10 ~tg G F L V R N A per 1 x 10 6 protoplasts was used for infection assays.

Replication of the RNA3 transcripts in C. quinoa protoplasts Experiments to demonstrate the biological activity of the RNA3 transcripts in vitro were performed in C. quinoa protoplasts inoculated by the P E G method with ArMVS, an isolate free of satellite R N A and originating from grapevine (Fuchs et al., 1991), as helper because no satellite-free G F L V was available as shown later. Protoplasts (5 x 105) were inoculated with 5 ~tg ArMV-S, supplemented with uncapped and capped tr45p4, tr3S and tr3M obtained from 20 ~tg of the corresponding linearized DNA. Total R N A s from 5 x 105 protoplasts were extracted 72 h post-inoculation and tested for the presence of satellite R N A using the SIII riboprobe. The signal corresponding to RNA3 was very faint after a 12 day autoradiographic exposure of the membrane, indicating only very low replication of the uncapped and capped tr45p4 transcripts (data not shown). Similarly, the uncapped and capped tr3S and tr3M transcripts were poorly replicated. These results suggested that the inoculation method used was responsible for the low efficiency obtained. To improve the inoculation efficiency the electroporation method was used: 2 x 105 protoplasts were electroporated with 2 txg ArMV-S R N A as a helper, supplemented with uncapped and capped tr45p4, tr3S and tr3M. Total R N A s were extracted from 1 x 105 protoplasts 72 h post-inoculation and analysed with the three riboprobes previously used for G F L V R N A detection. Under these conditions the detection signal corresponding to ArMV-S R N A 2 was higher than for RNA1 (Fig. 5, lanes 2 to 7); this is related to the large amount of R N A 2 in ArMV-S and to the similarity of the SII probe with the sequence of ArMV-S R N A 2 (unpublished results). In contrast with the previous results, unambiguous signals corresponding to R N A 3 were visible after an overnight exposure. The reduced loss of protoplasts after electroporation (approx. 10~o) and the rapidity of the infection step resulting in limited degradation of the inoculated R N A may explain the

G F L V satellite R N A transcripts in protoplasts

tr45p4 1

2

3

tr3S 4

5

tr3M 6

7

8

RNA1 --

RNA2 --

RNA3-Fig. 5. Northern blot showingthe replicationof the differentRNA3 in vitro transcripts in protoplasts 72 h post-inoculation. Protoplasts (2 × 105)were electroporatedwith 2 ktg ArMV-S RNA as helper plus different transcripts: uncapped (lane 2) and capped (lane 3) tr45p4; uncapped (lane 4) and capped (lane 5) tr3S; uncapped (lane 6) and capped (lane 7) tr3M. Lane 8, 2 × 105 protoplasts electroporatedwith 2 ~tg GFLV RNA without added transcript; lane 1, GFLV RNA as control. Northern blot analysis was performed as described in the legend to Fig. 3.

better performance of electroporation compared with the PEG inoculation method. As noticed previously after PEG-mediated infection, capped and uncapped tr45p4 were not infectious in electroporated protoplasts (Fig. 5, lanes 2 and 3). This is in contrast to the results of Liu et aL (1991) who demonstrated that, in C. quinoa plants, replication of the synthetic transcripts of ArMV-L satellite R N A was not inhibited despite the presence of six or 29 non-viral nucleotides at the 5" end. Similady, Fuchs (1989) found that the uncapped tr45p4 transcript was able to replicate in C. quinoa plants. The failure of tr45p4 to replicate in C. quinoa protoplasts may be due to the limited number of sites present in protoplasts available for successful initiation of infection. The transcript tr3S with one non-viral G at the 5' end and 22 non-viral nucleotides at the 3' end was infectious (Fig. 5, lanes 4 and 5). The transcript tr3M with three non-viral nucleotides at the 3' end was markedly more infectious than tr3S (Fig. 5, lanes 6 and 7) when the RNA3 signal is compared to the amount of R N A 2 detected in each case. Thus, the presence of extra nucleotides at the 5' end has a dramatic effect on transcript infectivity, but their presence at the 3' end is less inhibitory. An inhibitory effect of 5' non-viral extensions has also been demonstrated for synthetic satellite R N A transcripts of TBRV (Greif et al., 1990) and cucumber mosaic virus (CMV; Masuta et al., 1988) and for synthetic transcripts of genomic R N A from

2521

several animal and plant viruses (van der Werf et al., 1986; Dawson et al., 1986; Janda et al., 1987; ZieglerGraft et al., 1988; Heaton et al., 1989). As was the case for the in vitro transcripts of ArMV-L and TBRV satellite RNA, uncapped tr3S and tr3M were infectious. In contrast, in vitro transcripts of tobacco vein mottling virus (Domier et al., 1989) and plum pox virus (Riechmann et al., 1990) c D N A clones were infectious only if they were capped. In our case, although not strictly necessary, capping improved the infectivity of the transcripts considerably. The signal observed on the Northern blot for the capped transcripts was always significantly higher than for uncapped trancripts (Fig. 5, lanes 4 and 5). Taking into account that the amount of capped transcripts used to infect protoplasts was two times less than that of uncapped transcripts, since the capping procedure reduced the yield of transcripts, it can be concluded that transcripts were notably more infectious when capped. Similar results were also obtained for cowpea mosaic virus (Vos et al., 1988), barley yellow dwarf virus (Young et al., 1991) and beet western yellows virus (Veidt et al., 1992). Although the role of the cap is not precisely known, it probably protects the natural or synthetic RNAs from degradation by exonucleases and increases their biological activity (Shimotohno et al., 1977). It is also possible that capped transcripts are more efficiently translated immediately after inoculation if the P3 protein somehow plays a role in the replication of the satellite RNA. Ability o f different nepovirus strains to act as helper

Two G F L V isolates were expected to be potential helpers, the GFLV-Tu strain shown to be free of satellite R N A when multiplied on C. quinoa (Fuchs et al., 1991) and the GFLV-24 strain originating from Malta, kindly provided by Professor G. P. Martelli (Bari, Italy) which was also free of satellite. Both isolates are serologically related to GFLV-F13. Each was tested for its ability to replicate tr3M, ArMV-S being used as control. In these experiments, the same batch of protoplasts, the same preparation of capped tr3M and the same ratio of tr3M helper R N A were used for the infection. A control experiment revealed that when GFLV-Tu R N A which contained no RNA3 detectable with the SIII riboprobe was used alone to inoculate C. quinoa protoplasts, an R N A nearly comigrating with the natural GFLV-F13 RNA3 (Fig. 6, lane 8) was detected which would interfere with detection of the R N A derived from the inoculated transcript (Fig. 6, lane 9), rendering this strain unusable as a helper. The presence of this presumed satellite R N A was unexpected since it was not detected with c D N A probes in GFLV-Tu-infected plants (Fuchs et al., 1991 ). Valverde et al. (1991) reported

2522

F. Hans, M. Fuchs and L. Pinck

1

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GFLV-24

ArMV-S

4

6

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GFLV-Tu 8

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RNA1

RNA2

RNA3

Fig. 6. Northern blot showing the replication of tr3M using different genomic viral RNAs as helper. Protoplasts (2 x l0 s) were electroporated with 2 lag GFLV-:24 R N A (lane 4), 2 lag GFLV-24 RNA plus 4 lag tr3M (lane 5), 2 lag ArMV-S R N A (lane 6), 2 lag ArMV-S RNA plus 4 lag Tr3M (lane 7), 2 lag GFLV-Tu RNA (lane 8), 2 lag GFLV-Tu RNA plus 4 lag tr3M (lane 9). Lane 1, GFLV RNA as control; lane 2, healthy protoplasts; lane 3, 2 x 105 protoplasts inoculated with 10 lag GFLV RNA. Northern blot analysis was performed as described in the legend to Fig. 3.

capacity to replicate its satellite depends only on the origin of RNA1 (Murant & Mayo, 1982). Using pseudorecombinants constructed from the Fny-CMV and Sny-CMV strains, Roossink & Palukaitis (1991) mapped the ability to support the replication of the WL 1CMV satellite R N A in zucchini plants to RNA1. In our case, it would be interesting to construct chimeric infectious synthetic RNA1 transcripts containing sequences of GFLV-F13, GFLV-24 and ArMV-S RNA1. Their capacity to replicate tr3M in C. quinoa protoplasts should permit mapping of the sequences involved in the interactions between RNA1 and the satellite of GFLVF13. The development of a C. quinoa protoplast system and the obtaining of infectious transcripts will greatly help investigations on the mechanisms of satellite R N A replication, the helper virus-satellite association and the role of the P3 protein. The authors are grateful to Dr S. Bouzoubaa for his help concerning preparation of protoplasts and to Dr K. Richards for improving the manuscript. F. Hans was supported by a grant from CNRS and R6gion Alsace.

References the reappearance of a satellite of the tobacco mosaic virus U5 strain (TMV-U5) after several passages through Nicotiana tabacum for some subcultures of TMV-U5 which were initially free of detectable satellite but which had been derived from isolates containing satellite RNA. A similar situation may occur with GFLV-Tu. It is also possible that satellite R N A multiplication in protoplasts is more efficient than in whole plant cells. When GFLV-24 genomic RNAs were used as helper, no replication of the transcripts was detected 72 h postinoculation (Fig. 6, lane 5). Thus, although GFLV-24 is serologically related to GFLV, it cannot replicate the RNA3 transcripts of GFLV-F13. Liu et al. (1991) demonstrated that GFLV was not a helper for ArMV-S satellite RNA; they also showed that the ivy and ash isolates of ArMV were not helpers for ArMV-L satellite R N A in contrast to hop and sugar-beet isolates of the same virus. Similarly, TBRV satellite R N A from serotypes S or G multiplied only with TBRV of the same serotype (Murant & Mayo, 1982). These results illustrate the close relationship between a satellite R N A and its helper virus. When protoplasts were electroporated with ArMV-S R N A and tr3M, F13 satellite R N A was detected 72 h post-inoculation. The ArMV-S helper RNAs were able to replicate tr3M in spite of the low homology of the R N A sequence between ArMV-S and GFLV-F13, indicated by the low rate of hybridization of the ArMV genomic R N A with the SI and SII riboprobes (Fig. 6, lane 7). It has been demonstrated for TBRV that the

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(Received 28 April 1992; Accepted 7 July 1992)