Resistance to viruses and viroids in transgenic plants and animals expressing dsRNA-binding protein

Abstract

The invention provides a method for imparting resistance in animals to viruses, and in plants to viruses and viroids, that express double-stranded RNA-like structures (dsRNAs). This method enables the binding of pathogenic dsRNAs during the infection process by expression of dsRNA-binding protein in transgenic animal and plant hosts, thus interrupting the infection cycle and inhibiting disease. The presence of a dsRNA-binding protein in a transgenic host renders the transgenic host resistant to the phenotypic symptoms of viral infection and/or decreased pathogen replication. Accordingly, the present invention provides a genetically engineered animal and plant, stably transformed to express a dsRNA-binding protein, such that the transgenic host displays resistance to virus and/or viroid challenge.

1 . A method of providing resistance in a host from an infecting virus or viroid having a double-stranded RNA-like (dsRNA) structure, said method comprising the step of transforming said host with a recombinant DNA molecule comprising a gene encoding a dsRNA-binding protein, such that dsRNA binding protein is expressed in said host at a level sufficient to render said host resistant to infection by any virus or viroid having a dsRNA-like structure.  
     
     
         2 . The method of  claim 1  wherein said virus is a plant virus or a mammalian virus.  
     
     
         3 . The method of  claim 2  wherein said plant virus is selected from the group consisting of the phytoreovirus group, the tymovirus group, luteovirus group, tombusvirus group, Southern bean mosaic virus group, tobacco necrosis virus group, maize chlorotic dwarf virus group, closterovirus group, carlavirus group, potyvirus group, potexvirus group, tobamovirus group, nepovirus group, pea enation mosaic virus group, comovirus group, tobravirus group, cucumovirus group, bromovirus group, ilarvirus group, alfalfa mosaic virus group, and hordeivirus group.  
     
     
         4 . The method of  claim 2  wherein said mammalian virus is selected from a group consisting of the picornavirus group, togavirus group, bunyavirus group, hantaan virus group, arenavirus group, rhabdovirus group, orthomyxovirus group, paramyxovirus group, coronavirus group, hepatitis delta group and retrovirus group.  
     
     
         5 . The method of  claim 4  wherein said mammalian virus is a human immuno-deficiency virus.  
     
     
         6 . The method of  claim 1  wherein said viroid is selected from the group consisting of the potato spindle tuber viroid, the coconut cadang-cadang viroid group, avocado sunblotch viroid group, and hop latent viroid group.  
     
     
         7 . The method of  claim 1  wherein said dsRNA-binding protein is selected from the group consisting of the human and mouse interferon-induced, dsRNA-dependent protein kinases; the vaccinia virus E3L protein;  Escherichia coli  RNase III protein; the human TAR/RRE binding protein; the Drosophila staufen gene product; the  Saccharomyces pombe  Pac1 protein; Xenopus RNA-binding protein A; porcine rotavirus group C P8 protein; the reovirus σ3 protein; and the human son-a protein  
     
     
         8 . The method of  claim 1  wherein said step of transforming said host is carried out using a means selected from the group comprising a prokaryotic transformation system, liposomes, electroporation, diffusion, particle bombardment, gene gun, microinjection, calcium phosphate coprecipitation, and a viral vector  
     
     
         9 . The method of  claim 1  wherein said host is an animal or a plant.  
     
     
         10 . The method of  claim 9  wherein said animal is a human.  
     
     
         11 . The method of  claim 9  wherein said plant is a dicot or a monocot.  
     
     
         12 . The method of  claim 11  wherein said step of transforming said plant is carried out using a prokaryotic transfection system.  
     
     
         13 . The method of  claim 9  wherein said step of transforming said animal is carried out using a viral vector.  
     
     
         14 . The method of  claim 13  wherein said viral vector is a retrovirus.  
     
     
         15 . The method of  claim 1  wherein said dsRNA-binding protein is expressed constitutively and/or inducibly.  
     
     
         16 . The method of  claim 1  wherein said expression of dsRNA-binding protein is induced by said viral or viroid infection of said host.  
     
     
         17 . The method of  claim 1  wherein said dsRNA binding protein comprises a dsRNA-binding domain having a consensus sequence of: 
 R/K-E-F-X-X-G/A-X-G-R/K-S/T K/R-K/R-E/D-A-K-N/Q-A-A-A-K L/V-A-L/V-D/E.  
 
     
     
         18 . The method of  claim 1  wherein said gene encoding a dsRNA-binding protein is modified to comprise an expression enhancing modification selected from the group consisting of: 
 alteration of a translational initiation site located within the gene or in an inappropriate reading frame to a non-initiator codon;  
 insertion of a TAA termination at the end of said dsRNA-binding protein structural gene; and  
 utilization of plant preferred codons.  
 
     
     
         19 . A transgenic plant resistant from an infecting virus or a viroid having a dsRNA-like structure, said transgenic plant comprising a recombinant DNA molecule comprising a gene encoding a dsRNA-binding protein and expressing a dsRNA-binding protein at a level sufficient to render said transgenic plant resistant to infection from any virus or viroid having a dsRNA-like structure.  
     
     
         20 . The transgenic plant of  claim 19  wherein said dsRNA-binding protein is expressed constitutively.  
     
     
         21 . The transgenic plant of  claim 19  wherein said gene is derived from a mammalian gene, a plant gene, a microorganismic gene or is a synthetic gene.  
     
     
         22 . The transgenic plant of  claim 19  wherein said dsRNA-binding protein comprises a dsRNA-binding protein domain having a consensus sequence of: 
 R/K-E-F-X-X-G/A-X-G-R/K-S/T K/R-K/R-E/D-A-K-N/Q-A-A-A-K L/V-A-L/V-D/E.  
 
     
     
         23 . The transgenic plant of  claim 19  wherein said transgenic plant is selected from the group consisting of tobacco, potato, tomato, rice, wheat, barley, maize, cucumber, soya, sweet potato, pepper, grapes, rapeseed, sugar beet, cotton, tea, sunflower, strawberry, rose and chrysanthemum.  
     
     
         24 . A plant or progeny thereof which is resistant to any virus or viroid having a dsRNA-like structure and which is characterized as having a genome comprising a DNA copy of a gene encoding a dsRNA-binding protein.  
     
     
         25 . A transgenic animal resistant to infection from any virus having a dsRNA-like structure, said transgenic animal comprising a recombinant DNA molecule comprising a gene encoding a dsRNA-binding protein and expressing a dsRNA-binding protein at a level sufficient to render said transgenic animal resistant to infection from any virus having a dsRNA-like structure.  
     
     
         26 . The transgenic animal of  claim 25  wherein said dsRNA-binding protein is expressed constitutively.  
     
     
         27 . The transgenic animal of  claim 25  wherein said gene is derived from a mammalian gene, a plant gene, a microorganismic gene or is a synthetic gene.  
     
     
         28 . The transgenic animal of  claim 25  wherein said dsRNA-binding protein comprises a dsRNA-binding protein domain having a consensus sequence of: 
 R/K-E-F-X-X-G/A-X-G-R/K-S/T K/R-K/R-E/D-A-K-N/Q-A-A-A-K L/V-A-L/V-D/E.  
 
     
     
         29 . The transgenic animal of  claim 25  wherein said dsRNA-binding protein is selected from the group consisting of the human and mouse interferon-induced, dsRNA-dependent protein kinases; the vaccinia virus E3L protein; Escherichia coli RNase III protein; the human TAR/RRE binding protein; the Drosophila staufen gene product; the  Saccharomyces pombe  Pac1 protein; Xenopus RNA-binding protein A; porcine rotavirus group C P8 protein; the reovirus σ3 protein; and the human son-a protein.  
     
     
         30 . An animal or progeny thereof which is resistant to any virus having a dsRNA-like structure and which is characterized as having a genome comprising a DNA copy of a gene encoding a dsRNA-binding protein.  
     
     
         31 . A method for providing to a human infected with a virus having a dsRNA-like structure with a dsRNA-binding protein, comprising the step of introducing into said human a human-compatible vector comprising a recombinant DNA molecule comprising a gene encoding a dsRNA-binding protein, such that a therapeutically effective amount of dsRNA-binding protein is expressed in said human.  
     
     
         32 . The method according to  claim 31  wherein said virus is a retrovirus.  
     
     
         33 . The method according to  claim 32  wherein said retrovirus is a human immuno-deficiency virus.  
     
     
         34 . The method according to  claim 31  wherein said dsRNA-binding protein is a vaccinia virus E3L protein or a rotavirus group C P8 protein.  
     
     
         35 . The method according to  claim 31  wherein said recombinant DNA molecule is introduced into said human using a retroviral vector system.  
     
     
         36 . A method of detecting in a host cell an infection from a virus or viroid having a dsRNA-like structure comprising the steps of (a) transforming said host cell with a recombinant nucleic acid molecule comprising a gene encoding a dsRNA-binding protein and (b) measuring the level of dsRNA in said host cell complexed to said dsRNA-binding protein expressed in said host cell, said measure of the level of dsRNA in said host cell being indicative of the level of infection from said virus or viroid.  
     
     
         37 . The method of  claim 36  wherein said virus is a retrovirus.  
     
     
         38 . The method of  claim 37  wherein said retrovirus is a human immunodeficiency virus.  
     
     
         39 . The method of  claim 36  wherein said host cell is plant or animal.  
     
     
         40 . The method of  claim 36  wherein said host cell is a human cell.  
     
     
         41 . A recombinant virus comprising a deletion of a segment of the virus genome and an insertion of a nucleic acid segment comprising a sequence encoding a dsRNA-binding protein.