NZ272307A

NZ272307A - Modified antiviral ribozyme and its use in preparing virus-resistant plants

Info

Publication number: NZ272307A
Application number: NZ272307A
Authority: NZ
Inventors: Peter Loss; Rudolf Schneider
Original assignee: Hoechst Schering Agrevo Gmbh
Priority date: 1994-06-10
Filing date: 1995-06-08
Publication date: 1996-03-26
Also published as: HUT72483A; ZA954772B; CA2151446A1; KR960001117A; IL114068A0; JPH0856678A; AU2059995A; DE4420298A1; AU695371B2; EP0691402A3; HU9501688D0; EP0691402A2

Description

<div class="application article clearfix" id="description"> a Patents Form 5 Oc.iw.,»): !P.||p}..^9r. j i N.Z. No. NEW ZEALAND Patents Act 1953 COMPLETE SPECIFICATION MODIFIED SATELLITE RNAs AND SATELLITE VIRUSES AS CARRIERS OF ADAPTED RIBOZYMES We, HOECHST-SCHERING AGREVO GMBH, a Joint Stock Company existing under the ia'vs of the Federal Republic of Germany, of D-13509 Berlin, Federal Republic of Germany, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement:- - 1 - (Followed by 1A) 27?rr7 Hoechst Schering AgrEvo GmbH ' -AOn Dl/M 819 "DgiAD/pp* Description Modified satellite RNAs and satellite viruses as carriers of adapted ribozymes 1. Introduction 5 The present invention relates to enzymic RNA molecules which are linked to satellite viruses or satellite RNA, tcuprocesses for their preparation, to the corresponding coding DNA fragments, and to cells transformed with this DNA. The invention also relates to the use of the novel 10 ribozymes as antiviral agents. The term "ribozymes" was coined to describe RNA sequences possessing enzymic activity which cam cleave RNA sequences intra- or intermolecularly (Symons, 1991; Edgington, 1992). The actual cleavage conBiBts of two 15 steps: the complementary sequence regions of the ribozyme first interact with the substrate RNA and the substrate sequence is then cleaved by the nucleolytic activity of a more or less conserved, so-called catalytic region. The interaction between ribozyme and substrate plays an 20 important role with regard to the efficacy of the ribozymes employed. This interaction is influenced by the intramolecular interactions of the ribozyme molecules and the substrate molecules among themselves and with each other. The compartmentation of eukaryotic cells also 25 plays an important role. The ribozyme and the substrate which is to be inhibited must be present in the same compartment for it to be possible for em interaction to take place at all. Owing to their properties, ribozymes present themselves 30 as a possible option for selectively destroying viral RNA in the interior of cells. Thus, the vast majority of the O - ■<?- Ci viruses which are pathogenic to plants use RNA as the information carrier. While this RNA is usually single-stranded, double-stranded RNA is also found. The viruses replicate in the host cell. Since viruses are not inde-5 pendent organisms, they make use of living cells and rely on this host cell for replicating their nucleic acid and synthesizing their protein coat. However, viral sequences or viruses also exist whose propagation is not only dependent on the host cell but on 10 the presence of cm additional virus as well. Thus, satellite RNAs are small RNA molecules which dc not encode their own proteins and which are not replicated by proteins which are endogenous to the host (Roossinck et al., 1992). Rather, their replication zo? lires the 15 presence of special, so-called helper viruse . which, on the one hand, make available the appropriate proteins for replication (RNA-dependent RNA polymerase; helicase) and, on the other hand, also encode the coat proteins which are required for the packaging and consequently the 20 systemic, and also extra-individual, dissemination of these molecules. While satellite viruses likewise do not encode the proteins which are required for replication, they do, in contrast to the satellite RNAs, encode a coat protein 25 (Kaper and Collmer, 1988). Satellite RNAs and satellite viruses were previously found in significant numbers in association with plant pathogens (Roosinck et al., 1992); however, since then, it has also been possible to detect, in addition to these, satellite viruses which are 30 pathogenic to humans and animals such as adeno-associated virus (AAV) (Bems, 1990), a DNA satellite virus, and hepatitis delta virus (HDV) (Taylor, 1990), an RNA satellite virus. 35 It is known that plant satellite RNAs not only require the presence of a so-called helper virus but are also - 3 - un able to modulate the pathogenicity of the helper virus to a greater or lesser extent (Kaper et al., 1988; Collmer and Howell, 1992). While this modulation can lead to complete attenuation of 5 the symptoms which would otherwise result from an infection with the virus on its own, it can also lead to a dramatic exacerbation of the symptoms. The nature and strength of the modulation is influenced by the combination of the three participating components - helper 10 virus, satellite RNA and host plant. For this reason, experiments have already been in progress for some time with the aim of achieving repression of viral symptoms by means of a supplementary infection with satellite RNAs of this nature which have been demonstrated to have symptom-15 attenuating activity (Tien et al., 1987; Montasser et al., 1991). Hitherto, satellite viruses having symptom-attenuating and symptom-exacerbating activity have been described in which the different effects on modulation of the symptoms 20 of the relevant helper virus have their roots solely in the exchange of a few nucleotides, and the mechanistics of the symptom modulation are still not precisely understood at the present time (Palukaitis, 1988; Kaper et al., 1990; Sleat and Palukaitis, 1990; Roossinck et 25 al., 1992). The different satellite RNAs of a helper virus differ from each other not only in respect to their sequence but also as regards their size, even if the variation in size only takes place within a narrow range. 2. Description of the invention 30 Surprisingly, it has been possible to demonstrate that both the problem of localization into the correct compartment and also that of controlling activity by inserting selected ribozyme sequences into a particular sequence environment can be solved. This sequence £' - 4 - environment is such that it naturally replicateB in the same compartment as the virus which is to be destroyed by it. The present invention relates to modified ribozymes which 5 contain (a) nucleolytically active RNA sequences which cleave viral genomic RNA, mRNA or hnRNA, and (b) satellite viruses or satellite RNA which are functionally linked to the nucleolytically active RNA 10 sequences. The present invention relates to a process for preparing modified ribozymes, which comprises (a) nucleolytically active RNA sequences which can cleave viral genomic RNA, mRNA or hnRNA being functionally 15 linked to (b) satellite viruses or satellite RNA. The invontion relates, in particular, to processes in which ribozyme sequences are integrated into satellite RNAs and also into the RNA of satellite viruses of plant-20 pathogenic, animal-pathogenic and human-pathogenic viruses. The previously known groups of ribozymes, such as, for example, the ribozymes which possess hammerhead or hairpin motifs, the ribozymes of the group-I intron type, 25 RNAseP-like sequences or hepatitis delta virus sequence motifB can be used as the starting point for preparing these modified ribozymes. The invention also relates to the DNA fragments which encode these modified ribozymes. 30 For eliminating RNA viruses, the ribozyme sequences are preferably directed against the genomic RNA of these viruses, particularly preferably against the RNA which is present in the host cell as & replication intermediate - 5 - 27 2 30 7 and has a sequence which is complementary to this genomic RNA. The novel ribozymes can be used as antiviral pharmaceuticals against plant, animal or human viruses. 5 The invention relates, in particular, to a process for treating plants, which comprises (a) plant cells being transformed with the DNA encoding the modified ribozymes of the invention, (b) the DNA being stably integrated into the genome of 10 the plants, and (c) plants being regenerated from these cells. The invention also relates to a process for treating plants, which comprises (a) plants being treated with the modified ribozymes. 15 In this context, the term treatment means that the modified ribozymes are not formed in the cell but are applied to the plants. However, the plants can also, as explained in 2.3, be both transgenic in relation to a ribozyme and also be treated with RNA sequences in 20 addition. The invention furthermore relates to the use of the novel ribozymes, or parts thereof, for treating plants against viruses. The crucial advantage as compared with using conventional 25 approaches in ribozyme technology is to be foufi.d in the following points: II 1. The ribozymes are present in the same compal the virus which is to be destroyed by them, s3 the satellite RNAs and satellite viruses are repli-30 cated by the replicases and the helicases of the corresponding helper viruses, with the replication of these virtues taking place in the cytoplasm of the host cell. The satellite RNA or the RNA of the satellite viruses is replicated for as long as the viral replicase is also present and the viral RNA is consequently replicated. The satellite RNA or the RNA of the satellite viruses is transferred together with the virus when a further host individual is freshly infected, and is then, as a result, also active in this individual. When the ribozymes are used together with satellite RNAs or satellite viruses, transgenic, prophylactic individual protection can be bypassed since it is still possible to introduce these molecules even after an infection. This can be effected in the form of in-vitro transcripts or in the form of "reassembled" virions. It is known that RNA viruses are subject to a relatively high rate of mutation. When the modified ribozymes are not used until an infection has taken place, the possibility presents itself, by means of a preceding sequence determination (using RT-PCR and then directly sequencing the PCR products), of adjusting the integrated ribozyme sequence to the current sequence of the viral RNAs. It is thus possible, by means of this approach, to counteract the genetic drift of the viral RNA. In addition to employing only one updated ribozyme sequence, it is also possible to employ several different sequences in one organism. The use of ribozyme-containing satellite RNAs or satellite viruses is not limited to the destruction of corresponding helper viruses. Instead, a / 7 - transgenic organism which contains the nucleic acid for synthesizing the necessary proteins (of the helper virus) as a chromosomal constituent can be infected, in the manner described above (point 4, 5 sentence 2), with the modified satellite RNA or the modified RNA of a satellite virus. This then leads to the cleavage of specific, endogenously encoded RNA sequences in the cytoplasm of the host cells. In this way, it is possible to influence develop-10 mentally specific processes, such as, for example, the maturation processes in fruit-forming plants of agricultural importance, starting at any time which is considered optimal. This application renders it possible to bypass the compartmentation problem of 15 the two interacting partners (the ribozyme-encoding RNA and the substrate RNA which is to be cleaved). The only thing which is essential in this case is that the viral genome sequences which are integrated in the host genome and which encode the virus 20 proteins are not complete, that is that the synthesized viral RNA lacks the sequences which are necessary for its own replication and for packaging. The plants can be trans formed using the known methods as described, for example, in Maniatis et al. (Molecular 25 Cloning: A Laboratory Manual, 1982, New York) . These methods include transformation of plant cells with T-DNA using Agrobacterium technology (EP 116 718), direct protoplast transformation (EP 164 575), electroporation or ballistic methods. 30 The novel DNA sequences can be altered and modified by insertions, deletions and substitutions if this does not alter any sequences which are relevant to the ribozyme itself. Suitable expression vectors are described in the literature. The vectors should expediently contain a 35 selection marker gene. Neither the choice of the transformation method nor the choice of the vectors has any crucial influence on the invention. /> // - 8 - It is possible to insert foreign RNA into the non-coding RNA regions both in the case of the satellite RNAs and in the case of the satellite viruses. In the case of the satellite RNAs, these insertions can be distributed over 5 the RNA, and the respective insertion must be tested, in each individual case, for any possible negative interactions with the replication and the stability of this RNA. In the case of the satellite viruses, the non-coding region of the RNA can be used as the site for cloning in 10 any possible foreign nucleic acid sequences. In the known plant satellite viruses, the coding sequences for the capsid protein are located at the 5' end of the RNA, so that additional RNA should preferably be cloned into the region which is situated further 15 towards the 3' end. The novel ribozymes may advantageously be employed for the following purposes: the cleavage of elementary viral RNA sequences with the aim of completely inhibiting viral replication and the cleavage of endogenously encoded 20 host-specific RNA transcripts. 2.1 Use of satellite RNAs for inhibiting the growth of viruses The novel ribozymes are preferably prepared using the plant satellite RNAs listed in Table 1, with the 25 ribozyme-encoding sequences being linked to the DNA sequences encoding the satellite RNAs. Table 1 gives the helper viruses (virus family in square brackets) which have been demonstrated to have satellite RNAs, and also the sizes (number of nucleotides) of the 30 corresponding satellite RNAs. ar Iw1. Table 1: Plant satellite RNA Helper virus Size of the satellite RNA (nt) Cucumber mosaic virus (CMV) [Cucumoviruses] Peanut stunt virus (PSV) [Cucumoviruses] 5 Tobacco ringspot virus (TobRV) [Nepoviruses] Strain S Strain F Tomato blackring virus (TBRV) [Nepoviruses] Chicory yellow mottle virus (CYMV) [Nepo-10 viruses] Arabis mosaic virus (ArMV) [Nepoviruses] Strawberry latent ringspot virus (SLRV) [Nepoviruses] Myrobalan latent virus (MLRV) [Nepoviruses] 15 Grapevine Bulgarian latent virus (GBLV) [Nepoviruses] Grapevine £anlea£ virus (GFLF) [Nepoviruses] Pea enantion virus (PEV) Velvet tobacco mottle virus (VTMoV) [Sobemo-20 viruses] Solanum nodiflorum mottle virus (SNMV) [Sobemo-viruses] Lucerne transient streak virus (LTSV) [Sobemo-viruses] 25 Subterranean clover mottle virus (SCMoV) [Sobemoviruses] Turnip crinkle virus (TCV) Tomato bushy stunt virus (TBSV) [Tomboviruses] Artichoke mottled crinkle virus (AMCV) [Tombo-30 viruses] Carnation Italian ringspot virus (CIRV) [Tomboviruses] Cymbidium ringspot virus (CyRSV) [Tomboviruses] 35 Petunia asteroid mosaic virus (PAMV) [Tomboviruses] Pelargonium leaf curl virus (PLCV) [Tomboviruses] Groundnut rosette virus (GRV) 40 Strain MG Strain NG Bett necrotic yellow vein virus (BNYW) 333-390 369-393 359 -340, -750 1375 1145 457 1104 300 -1200 -1400 - 1500 1114 -900 365, 366 377 324 332, 388 194, 230, 355 -700 -700 -700 621 -700 -700 -900 -900 10 Helper virus Size o£ the satellite RNA (nt) Strain 3 (Designation of the satellite RNA) 1774 Strain 4 (Designation of the satellite RNA) 1467 Strain 5 (Designation of the satellite RNA) -1400 Strain 6 (Designation of the satellite RNA) -1000 Sizes of from 333 to 390 nucleotides have been reported for the satellite RNA of cucumber mosaic virus (CMV), which is designated CARNA 5 in the literature (Roossinck et al., 1992) . However, it is clear, from a comparison of all previously described CARNA 5 satellite RNAs, that it is possible, in all the subspecies, to differentiate between highly conserved, partially variable and highly variable sequence regions. In Fig. 1, different satellite RNA subspecies are compared, with the symptom-determining regions being specially emphasized (Fig. 1A) . At the same time, the highly variable sequence region of the satellite RNAs is shown as a broken line. The cloning of the ribozyme sequences into such satellite RNAs is depicted diagram-matically in Fig. IB using CARNA 5 as an example. Any arbitrary sequence which is able to cleave a sequence region of the helper virus can be inserted into the cDNA of the satellite RNA/WL1 at a single cleavage site (BsmI) which is not present on the chosen vector. It is clear that, while the resulting RNA of the CARNA 5/WL1/RIB is somewhat longer than the RNA of the CARNA 5/Y RNA, it iB still horter them the longest CARNA 5 RNAs, which have been reported to have 390 nucleotides. Prior to the ribozyme cloning, the CARNA 5 cDNA was inserted into a transcription vector which does not possess any BsmI cleavage site (pBLUESCRIPT SK). - 11 - The cDNA is cloned into the transcription vector in a directed manner using the Xhol/Smal cleavage sites, it being possible for in-vitro transcription to take place following linearization with Smal. Such in-vitro tran-5 scripts are infectious and can be introduced into the host plant using standard methods which have been described. In this connection, it is important that the 3' end of the resulting transcripts should not contain any additional nucleotides whereas additional nucleotides 10 can be present at the 5' end of the resulting transcripts (Kurath and Palukaitis, 1987). The cloning of a ribozyme, taken as an example, against RNA2 of the CMV Fny strain into the cDNA of CARNA 5/WL1 is depicted in Table 1 in terms of the sequences 15 involved. Table 1 firstly shows the sequence of the CARNA 5/WL1 wild type, in which the cloning site for inserting any ribozyme sequences is underlined. Table 1: Scheme for cloning any ribozyme sequences into the RNA of CARNA 5/WL1 using a ribozyme directed against 20 CMV/Fny RNA2 as an example. GUUOTJGOUUG ABCGAGAAUU GCGUAGAGGC CUUAUAUCUA CCUGACGAUC COUCACUCGG 60 COGUGUOGGU UACCUCCCUG CUACSGCGGG CUGAGUUGAC GCACCUCGGA CUCGGGGACC 120 CCUUGGUUUO CGAGOAPCOU CCCCAPUCPU AGCACUACGC CCCAAUUUOA CCCCCCCCCU 280 AGUUUCCTAG CAGCACACGC UCAUGCUUUO CCGUUACCAU QGAAUUUCGA AAGAAACACU 240 CUCUUAG6UQ CUAUCOUOGA UCACGCACCC ACCCAGAGGC UUAOACUUAO GUUAUGCUOA 300 UCOCCGUSAA UGUCUACACA 0UCCUCUACA GGACCC 3' 336 f> T <••••. . c / .' - 12 - Table 2: Cloned ribozyme sequence, taken as an example catroucuuuG ADGCAGAAUU GCGUAGAGGC CUUAUAUCUA COUOASGAUC CGUCACOCCC 60 CSGUCnCGCD CACCOCCCUO CUACGGCGGG UUGAGUUGAC GCACCUCGGA CUCGGGGACC 120 ■vTauGGOTTOO CGAGUAUCGU CCCNfiNNNNN HHHMWHNWMW MWMOmWMN mWWNHWHHN 180 JfJt .^SKVINNN CAUUCUUAGC ACUACCCCCC AAOUUCAGCC CCCCCCUACC UUGCUAGCAO 240 s 1 I CCCUUUSCCG UUACCAUGCA AUUUCGAAAG AAACACUCUS UUAGGUGGUA 300 UCGUGGAUPA C6CAC0CACC GAGAGGCtJUA GACUUAGCUU AUGCUOAOCU CCGUGAAUCU 360 CUACACAUUC CUCOACAGC& CCC 383 (N represents the ::ibozyme sequence cloned into the CARNA 5/WL1 sequence) Table 3: Example of a ribozyme sequence 5 CUCOACGACO CTJSAUOAQU CCCUQAOOAC OAAACCAUTJU UAOCQ 45 The nucleotides which are added at the 5' end during the in-vitro transcription are not shown. The particular advantage of introducing ribozyme sequences into virus-infected plants in this manner is that it is possible, by 10 means of this post-infestation superinfection, to react in a flexible manner to the particular sequence of the viral RNA which is to be destroyed. This is particularly important since it is especially RNA viruses which are subject to a particularly high rate of mutation (Drake, 15 1993). Once a virus infection has been ascertained, so-called RT PCR can be used to determine the dominant sequence of a particular region of the viral RNA. Following on from this, it is possible to generate the ribozyme sequence, introduce the latter into the basic 20 WLl vector and protect the plants which are to be protected from further spread of the virus infection by simply applying the adapted in-vitro transcripts. Since the satellite RNAs can be disseminated as virions which are packaged in helper virus proteins, these modified, 25 ribozyme-encoding satellite RNAs will be immediately available in newly infected plants for interacting with the RNA of the helper virus. - 13 - In addition to applying the modified satellite RNA in the form of an in-vitro transcript in this manner, there is the possibility of transgenically integrating the modified satellite RNA into the host plant organism which 5 is to be protected. Stable integration of this nature, and the consequences of the existence of satellite RNA in a host plant which is not infected with virus, have been amply investigated in Baulcombe's research group (Baulcombe et al., 1986). 10 In this case, so-called head-to-tail multimers are cloned downstream of a strong plant promoter (for example the 35S promoter). These multimeric transcripts mimic the replication intermediates which are produced in accordance with the current model and, in the presence of the 15 helper viruses, are correctly processed to form monomers. In non-infected host plants, the multimeric transcripts which are produced do not give rise to any abnormalities as regards growth, flowering and seed production (Baulcombe et al., 1986). 20 The ribozyme sequences which are integrated into the satellite molecules are then able to interact with the corresponding sequence of the helper virus. The satellite RNA which is transcribed in transgenic plants was also demonstrated to be packaged into virions (Baulcombe et 25 al., 1986). The test for the presence of inhibition of CMV replication was carried out by infecting tobacco plants with a CMV isolate, as a rule at eight weeks after sowing, when from four to six leaves had formed. The lowest leaf 30 was rubbed with a brush which had been dipped in carborundum. 5 fig or 25 fig of virus from a purified virus suspension was then pipetted onto this site. In the control plants, the treatment was carried out using water. The plants were stored in climatic chambers. The 35 temperature was set to a day cycle of 22°C and a night cycle of 18°C. The plants received light for 18 hours and */ - 14 - then stood in the dark for 8 hours. Assessment of the plants indicated that the virus-infected plants into which modified satellite RNAs were introduced transgeni-cally, or to which these RNAs were subsequently applied, 5 exhibited a significantly greater resistance than did the untreated, virus-infected control plants. Isolated plant protoplasts (200,000/sample) were transformed (coelectroporated) with satellite RNA and viral genomic RNAs using the methods described below. 10 Protoplast isolation The protoplasts were isolated from tobacco SRI plants or samsun plants. The plant container, which contained tobacco plants which had been grown under sterile conditions, was stored overnight in the dark at 27°C. It was 15 then incubated for 6-8 hours in a refrigerator. The necessary 25 ml of enzyme solution, comprising 150 mg of cellulysin (1560 /x) and 25 mg of maceras© (80 fi) , were prepared in parallel. After the ingredients had dissolved completely, the solution had to be sterilized by filtra-20 tion. 4 g of leaf material were cut into small pieces under sterile conditions and incubated in a petri dish together with 20 ml of pre-plasmolysis solution (see below) at 20°C for 20-30 minutes in the absence of light. The pre-plasmolysis solution was then carefully drawn 25 off. The enzyme solution (see above) was added and the leaf pieces were then incubated in the absence of light at 20°C for 12 hourB. After adding 20 ml of rinsing solution (see below), the remaining solution was filtered. The filtrate was divided between 4 centrifuge 30 tubes. Centrifugation then took place at 600 rpm for 4 minutes and at a temperature of 20°C. The resulting supernatant was discarded and the pellet was resuspended in the rinsing solution. The total solution from all 4 tubes was transferred into 1 new centrifuge tube and the 35 volume was made up to 10 ml. Centrifugation then took place at 600 rpm for 4 minutes and at a temperature of 10 - 15 - 20°C. The resulting pellet was dissolved in 10 ml of sucrose solution and carefully overlaid with 1 ml of rinsing solution. Centrifugation then took place at 400 rpm for 15 minutes and at a temperature of 20°C. The protoplast band was carefully drawn off and the harvested protoplasts were transferred into a new tube; rinsing solution was added to make the volume up to 10 ml and the tube was then centrifuged at 600 rpm for 4 minutes and at a temperature of 20°C. The pellet was taken up in 3 ml of VKM*~. The protoplasts which were obtained were quantified using a Thoma chamber. Electroporation The volume containing 200,000 protoplasts w&d stored on ice in a mannitol/MES buffer with the final volume being 15 160 i*l. The nucleic acid for the transformation was stored on ice in & final 160 pi volume of KCl/mannitol. The cuvettes for the electroporation were washed with 70% ethanol and dried. The protoplasts and the nucleic acids were added together directly prior to the electro-20 poration. The standard electroporation conditions were 200 V and 25 pa (apparatus manufacturer: Dialog; apparatus type: Electroporator II) . The samples were subsequently incubated for 10 minutes on ice and then for 5 minutes at 20°C. The contents of the cuvette were 25 transferred to Eppendorf tubes and the cuvettes were rinsed with 200 fil of mannitol/MES. Centrifugation then took place at 500 rpm for 4 minutes. The supernatant was removed and the protoplasts were taken up once again in 200 fil of mannitol/MES. The centrifugation was repeated 30 once. The protoplasts were then incubated at 20°C for 24 hours. VKM nutrient stock solution Macronutrients: KNO, 35 MgS04*7H20 CaCl2*2H20 73 mM 20 xoM 25 mM Micronutrients: H3BO3 240 juM MnS04*H20 300 fM ZnS04*7H20 35 fM kh2po4 FeS04*7H20 Na2EDTA*2H20 2.5 mM 0.5 mM 0.5 mM 16 - Na2Mo04*2H20 CuS04*5H20 C0C12*6H20 / ^ 5 /xM 0.5 /xM 23 ftM Vitamins: Pantothenic acid Choline chloride Ascorbic acid 20 fM 36 /xM 57 /xM p-Ainiuobenzoic acid Nicotinic acid 10 Vitamin B6-HC1 Thiamine HC1 Folic acid Biotin Vitamin A acetate 15 Vitamin B, Vitamin B 12 0.7 /xM 40 fiM 34 fi M :<50 fiK 5 ,mM 0.2 /xM 0.5 fiM 0.1 /xM 73 nM VKM stock solution: D (-) -mannitol 7 mM D(-)-sorbitol 7 mM 20 Sucrose 4 mM D(-)-fructose 7 mM D(-)-ribose 8 mM D(-)-xylose 8 mM Mannose 7 mM 25 L (-f)-rhamnose znonohydrate 7 mM Cellobiose 4 mM Casein hydrolysate 1.25 g/1 Myoinositol 3 mM Pyruvic aeid 1 mM 30 Fumaric acid 2 zaM Citric acid 1 mM Malic acid 1.5 mM 35 VKM* culture medium: Nutrient stock solution Glucose 100 ml/1 27 mM D(-) -maniiitol Sucrose MES 230 mM 30 mM 3 mM BAP 2.2 mM 5 Osmolarity 460 mOsmol/kg pH 5.8 The culture medium was sterilized by filtering through a 0.2 im. filter. VKM rinsing medium; 10 Nutrient stock solution 100 ml/1 KC1 0.2 M MES 3 mM Osmolarity 460 mOsmol/kg pH 5.8 15 Pre-plasmolysis VKM medium: Nutrient stock solution 100 ml/1 Mannitol 0.4 M MES 3 mM Osmolarity 490 mOsmol/kg 20 pH 5.8 The culture medium was sterilized by filtering through a 0.2 fm. filter. VKM sucrose solution: Nutrient stock solution 100 ml/1 25 Sucrose 0.3 M Osolarity 460 mOsmol/kg pH 5.8 The culture medium was sterilized by filtering through a 0.2 fun. filter. 30 VKM enzyme solution: Cellulysin 6 g dissolved in 1 1 of VKM culture medium - 18 - Macerase 1 g dissolved in 1 1 of VKM culture medium The osmolarity and the pH were tested after the substances had dissolved. A subsequent incubation was carried out for a period of 5 8-48 hours at a temperature of 25°C. The effects of the ribozyme-bearing satellite RNA on replication of the CMV within the protoplasts were quantified firstly using antibodies which were directed against the viral coat protein and then by means of RNA isolations and subse-10 quent Northern blot analysis. Antibody test A kit from Agdia Inc. (30380 County Road 6, Elkhart, IN 46514) was used for the antibody detection. The antibodies contained in this kit are directed against the 15 CMV coat protein. The total protoplast population used in each case per electroporation was employed for each respective antibody test. Northern blots The whole protoplast population employed for the electro-20 poration was employed for the RNA extraction. The protoplasts were mixed with an equal volume of a disruption buffer (200 mM NaCl, 100 mM Tris-HCl, pH 7.5, 1 mM EDTA) , and this mixture was shaken briefly, after the aqueous phase had been adjusted to a final SDS concentration of 25 1%, with a phenol/chloroform solution and then centri-fuged in an Eppendorf centrifuge for 5 minutes. An equal volume of chloroform was added to the aqueous phase and this mixture was shaken and centrifuged once again for 5 minutes. A 1/10 volume of a 3 M solution of sodium 30 acetate, pH 4.8, and then 3 volumes of 96% analytical grade ethanol were added to the aqueous phase. After a precipitation period of 1 hour at -80°C, the mixture was centrifuged for 15 minutes. The pellet was then washed in f) - 19 - a 70% solution of ethanol, dried and then dissolved in a 100 pel volume of TE, pH 7.5. I» case this solution still contained too much DNA, it was treated with DNase (RNase-free DNase). Following an extraction with phenol/ 5 chloroform (see above), the nucleic acid was electro-phoresed on an agarose/formaldehyde gel. It was then transferred to a nylon membrane and hybridized with radioactively labeled transcripts having a sequence complementary to the viral genomic RNA. 10 By removing protoplasts at different time points, it was possible, as well as quantifying the RNA, also to monitor the chronological course of the inhibition. In addition to this inhibition of viral replication demonstrated here using the example of CARNA 5 satellite 15 RNA and the corresponding CMV helper virus, it is also possible, naturally, to conceive of other helper virus/satellite RNA combinations which likewise provide protection against a systemic infection and thus protection against further dissemination of a particular virus. 20 2.2 Application of the technology using satellite viruses In contrast to the satellite RNAs, which do not encode any gene products of their own, the satellite viruses have an open reading frame which is located at the 5' end of the genomic RNA. This open reading frame encodes a 25 coat protein of these satellite viruses. Thus, while these satellite viruses rely on the presence of so-called helper viruses, they are not packaged into the capsid of the latter viruses. The 3' end of the genomic RNA does not encode any open reading frame and is therefore 30 available for the insertion of additional RNA sequences. Due to the fact that they can encode a protein, satellite viruses are also somewhat larger than satellite RNAs. It was demonstrated in the case of tobacco necrosis virus satellites, STNV, that relatively small insertions into the non-coding 3' region of the RNA of the satellite virus did not, in most cases, have any effect on the RNA production of the satellite virus as far as replication 5 was concerned. Such integrated sequences are then replicated and stably inherited, as a permanent constituent, in the progeny. In contrast to the possible insertion of short sequences into the non-coding region, an insertion into the coding 10 5' region should be avoided since this destroys the open reading frame (Van Emmelo et al., 1987). While this change does not have any effect on the appearance of the replication intermediates, it does affect dissemination of the satellite viruses since the coat protein which is 15 necessary for packaging them cannot be formed in a proper manner. Figure 2 shows the construction of a satellite virus in diagrammatic outline, using the example of STNV. The thickened, bar-shaped region corresponds to the coat 20 protein ORF. The neutral insertion sites which have been identified are indicated by the positions of arrows (van Emmelo et al., 1987). Thus, it should be possible to insert a ribozyme at these positions, with this ribozyme then also 25 being stably inherited in the progeny and being systemically disseminated by being packaged in satellite virus virions. Either plants which are infected with the helper virus can be inoculated directly with the satellite virus cDNA 30 containing a ribozyme sequence, or else in-vitro transcripts are prepared from the cloned cDNA and these transcripts can then be employed for an inoculation. In addition to post-infestation superinfection, it is also possible, in the case where a ribozyme sequence is - 21 - cloned into the cDNA of a satellite virus, to obtain genomic integration of such a modified cDNA, with consequent permanent transcription of the modified satellite virus RNA resulting in increased resistance to the helper 5 virus. The cloning is carried out using a binary plant vector which has a strong promoter, for example the CaMV 35S promoter, and the corresponding termination sequences. In addition to the STNV satellite virus of tobacco 10 necrosis virus (TNV) described here, the following viruses can also, with preference, be used for preparing the novel ribozymes: the satellite virus of maize white line mosaic virus (MWLMV), SV-MWLMV (Zhang et al., 1991) or the satellite virus of panicum mosaic virus (PMV), 15 SPMV (Masuta et al., 1987). These satellite viruses are also suitable for having ribozyme sequences incorporated into non-coding regions of their RNA even if they also all differ somewhat from each other as regards their genome structure and their 20 size. 2.3 Inactivation of endogenously encoded gene products In contrast to the use of satellite RNAs and satellite viruses as coinfaction of a helper virus, it is also possible to conceive of employing modified, ribozyme-25 containing satellite RNAs and satellite viruses as a specific means of inactivating endogenously encoded RNAs. For this purpose, the viral RNAs which are necessary for replication and packaging are stably integrated, as cDNA, into the genome of the host organism, with the upstream 30 presence of a strong promoter ensuring a high level of transcription of the RNA encoding the essential viral proteins. However, these DNA sequences lack the 5'-terminal and 3'-terminal sequence regions since, if 0 1 - 22 - ' they did not, these viral RNAs could also be replicated and packaged in virions thereby leading to systemic infection of the host organism and to transfer to other organisms. However, the satellite RNAs and also the 5 satellite viruses can be systemically disseminated and thus lead to the inactivation of specific gene products in the whole of the organism concerned. The advantage as compared with simple transgenic organisms which encode a ribozyme is that the single basic provision with the 10 necessary genes of the particular helper virus is always identical and this construction therefore only needs to be carried out once. Modified satellite RNAs or RNA molecules of satellite viruses which are adapted to the particular purposes or requirements are subsequently 15 introduced into the organism and then strongly amplified, at the site of action, by the functional molecules of the helper virus. All satellite viruses are suitable for use as cloning vehicles for applying this invention in the plant, animal 20 and human spheres, it being possible also to use modified satellite RNAs for this purpose for applications in the plant sphere. 3. Legends to the figures Figure 1: Comparison of different satellite RNA 25 subspecies 1A: Y-satRNA (large, necrotic, chlorotic) B5-satRNA (small, chlorotic) D/WLl/II7N-satRNAs (small, necrotic) IB: Example of CARNA 5 30 Figure 2: Construction of a satellite virus, shown in diagrammatic outline using the example of STNV. The thickened bar-shaped region corresponds to the coat protein ORF. 23 SEQUENCE LISTING 27 2 3 (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: Hoechst Schering AgrEvo GmbH (B) STREET: - (C) CITY: Frankfurt (D) STATE: - (E) COUNTRY: Germany (F) POSTAL CODE: 65926 (G) TELEPHONE: 069-305-5596 (H) TELEFAX: 069-357175 (I) TELEX: 4 1234 700 ho d (ii) TITLE OF APPLICATION: Modified satellite RNAs and satellite viruses as carriers of adapted ribozymes (iii) NUMBER OF SEQUENCES: 2 (iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version #1.25 (EPA) (2) INFORMATION FOR SEQ ID No: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 336 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single 27230 (ix) FEATURES: (A) NAME/KEY: exon (B) LOCATION: 1..336 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: GUUUUCUUUG AUGGAGAAUU CCGUAGAGGG GUUAUAUC'JA CGUGAGGAUC CCUCACJCGG 60 CGCUGUCCGU UACCUCCCUC CUACGCCGGG UUCACUUCAC CCACCUCGCA CUCGGGGACC 120 GCUUGGUUUG CGAGUAUCGU CCGCAUUCUU ACCACUACGC GCCAAUUUGA CCCCCCCCCU 180 AGUUUGCUAG CAGCACACGC UCAUGCUUUG CCCUUACCAU GGAAUUUCGA AAGAAACACU 240 CUGUUAGGUG GUAUCGUCGA USACGCACCC AGGGAGAGGC UUAGACUUAG CUUAUGCUCA 300 UCUCCGUCAA UGUCUACACA UUCCUCUACA GGACCC 3' 336 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 383 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: RNA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM: virus (ix) FEATURES: (A) NAME/KEY: exon (B) LOCATION: 1..383 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: GUUUUCUUUC AUGGAGAAUU GCGUAGAGGC CUUAUAUCUA CGUGAGGAUC CCUCACUCGC 60 CCGUGUCGGU UACCUCCCUC CUACGCCGGG UUCACUUCAC CCACCUCGCA CUCGGGGACC 120 GCUUGGUUUG CGAGUAUCCU CCCNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 180 NNNNNNNNNN CAUUCUUAGC ACUACGCGCC AAUUUGACCC CCCCCCUAGU UUGCUAGCAG 240 CACACCCUCA UGGUUUCCCC UUACCAUGGA AUUUCGAAAG AAACACUCUC UUAGGUGGUA 300 UCGUGGAUGA CGCACCCAGG CAOAGGCUUA GACUUAGCUU AUCCUGAUCU CCGUGAAUGU 360 CUACACAUUC CUCUACACGA CCC . 25. 27 2 30 4. References Baulcombe, D.C., Saunders, 6.R., Bevan, M.W., Mayo, M.A., Harrison, B.D. (1986), Nature, 321, 446-449 Bems, K.I.,(1990), Microbiological Reviews, 54, 316-329 5 Collmer, C.W., Howell, S.H. (1992), Ann. Rev. Phytopathol. 30, 419-442 Drake, J.W. (1993), Proc. Natl. Acad. Sci. USA, 90, 4171-4178 Edgington, S.M. (1992), Biotechnology, 10, 256-262 10 Kaper, J.M., Collmer, C.W. (1988), In: RNA Genetics, Vol. Ill, 317-343 Eds: Domingo, J., Holland, J., Ahlquist, P., CRC Press, Boca Raton Kaper, J.M., Tousignant, M.U., Steen, M.T. (1988), Virology, 163, 284-292 15 Kaper, J.M., Tousignant, M.E., Geletka, L.M. (1990), Res. Virol., 14, 487-503 Montasser, M.S., Tousignant, M.E., Kaper, J.M. (1991), Plant Dis. 75, 86-92 Palukaitis, P. (1988), Mol. Plant-Microbe Interact. 1, 20 175-181 Roossinck, M.J., Sleat, D., Palukaitis, P. (1992), Microbiological Reviews, 56, 256-279 Sleat. D.E., Paulkaitis, P. (1990), Proc. Natl. Acad. Sci. USA, 87, 2946-2950 25 Symons, R.H. (1991), Critical Reviews in Plant 10, 189-234 • - 26 - Taylor, J. .1990), Sem. Virol., 1, 135-141 27 2 30 Tien. P., Zhang, X., Oui, B., Qin, B., Wu, G. (1987), Ann, Appl. Biol,, Ill, 143-152 Van Esanelo, J., Ameloot, P., Fiers, W. (1987), Virology, 5 157, 480-487 Zhang, L., Zitter, T.A., Palukaitis, P. (1991), Virology, 180, 467-473 </div>

Claims

<div class="application article clearfix printTableText" id="claims"> - 27 - §723 ,agn oi/ii oi3—. WHAT WE CLAIM '•IS: 1.
A modified ribozyme which contains (a) a nucleolytically active RNA sequence which cleaves viral genomic RNA, snRNA or hnRNA, and 5 (b) a satellite virus or a satellite RNA which is functionally linked to the nucleolytically active RNA sequence. 2.
A process for preparing modified ribozymes, which comprises 10 (a) nucleolytically active RNA sequences which can cleave viral genomic RNA, mRNA or hnRNA being functionally linked to (b) satellite viruses or satellite RNA. 3.
The process as claimed in claim 2, wherein the 15 nucleolytically active RNA sequences are integrated into satellite RNAs and also into the RNA of satellite viruses of plant-pathogenic, animal-pathogenic and human-pathogenic viruses. 4.
The process as claimed in claim 2, wherein nucleoly-20 tically active RNA sequences are used which possess hammerhead or hairpin motifs, possess RNAseP-like sequences or hepatitis delta virus sequence motifs, or can be assigned to the ribozymes of the group-I intron type. 25 5.
A process for preparing virus-resistant plants, which comprises (a) plant cells being transformed with the DNA encoding the modified ribozyme of claim 1, (b) the DNA being stably integrated into the genome 30 of the plants, and (c) plants being regenerated from these cellte. A process for treating plants, which compr , - 28 plants being treated externally with the modified ribozymes according to claim 1.
7. DMA fragments which encode the modified ribozymes as claimed in claim 1. 5
8. Use of the modified ribozymes according to claim 1 as antiviral pharmaceuticals.
9. Use of the modified ribozymes according to claim 1 as antiviral agents against plant-pathogenic viruses.
10. A modified ribozyme according to claim 1 substantially as herein described or exemplified.
11. A process according to anyone of claims 2, 5 or 6 substantially as herein described or exemplified.
12. DNA fragments according to claim 7 substantially as herein described or exemplified.
13. Use according to claim 8 or claim 9 substantially as herein described or exemplified. HOECHST-SCHEERING AGREVO GMBH By Their At; torneys </div>