Satyanarayana, T., Department of Plant Pathology, University of Florida, Citrus Research and Education Center, Lake Alfred, FL 33850, United States Gowda, S., Department of Plant Pathology, University of Florida, Citrus Research and Education Center, Lake Alfred, FL 33850, United States Boyko, V.P., Department of Plant Pathology, University of Florida, Citrus Research and Education Center, Lake Alfred, FL 33850, United States Albiach-Marti, M.R., Department of Plant Pathology, University of Florida, Citrus Research and Education Center, Lake Alfred, FL 33850, United States Mawassi, M., Department of Plant Pathology, University of Florida, Citrus Research and Education Center, Lake Alfred, FL 33850, United States Navas-Castillo, J., Department of Plant Pathology, University of Florida, Citrus Research and Education Center, Lake Alfred, FL 33850, United States Karasev, A.V., Department of Plant Pathology, University of Florida, Citrus Research and Education Center, Lake Alfred, FL 33850, United States Dolja, V., Dept. of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331, United States Hilf, M.E., U.S. Hort. Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Orlando, FL 32803, United States Lewandowski, D.J., Department of Plant Pathology, University of Florida, Citrus Research and Education Center, Lake Alfred, FL 33850, United States Moreno, P., Inst. Valenciano de Invest. Agrarias, 46113 Moncada, Valencia, Spain Bar-Joseph, M., Volcani Institute, Bet-Dagan 5020, Israel Garnsey, S.M., U.S. Hort. Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Orlando, FL 32803, United States Dawson, W.O., Department of Plant Pathology, University of Florida, Citrus Research and Education Center, Lake Alfred, FL 33850, United States, CREC, 700 Experiment Station Road, Lake Alfred, FL 33850, United States
Citrus tristeza virus (CTV) populations in citrus trees are unusually complex mixtures of viral genotypes and defective RNAs developed during the long-term vegetative propagation of the virus and by additional mixing by aphid transmission. The viral replication process allows the maintenance of minor amounts of disparate genotypes and defective RNAs in these populations. CTV is a member of the Closteroviridae possessing a positive-stranded RNA genome of ≃20 kilobases that expresses the replicase-associated genes as an ≃400-kDa polyprotein and the remaining 10 3' genes through subgenomic mRNAs. A full-length cDNA clone of CTV was generated from which RNA transcripts capable of replication in protoplasts were derived. The large size of cDNA hampered its use as a genetic system. Deletion of 10 3' genes resulted in an efficient RNA replicon that was easy to manipulate. To investigate the origin and maintenance of the genotypes in CTV populations, we tested the CTV replicase for its acceptance of divergent sequences by creating chimeric replicons with heterologous termini and examining their ability to replicate. Exchange of the similar 3' termini resulted in efficient replication whereas substitution of the divergent (up to 58% difference in sequence) 5' termini resulted in reduced but significant replication, generally in proportion to the extent of sequence divergence.
An engineered closterovirus RNA replicon and analysis of heterologous terminal sequences for replication
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Satyanarayana, T., Department of Plant Pathology, University of Florida, Citrus Research and Education Center, Lake Alfred, FL 33850, United States Gowda, S., Department of Plant Pathology, University of Florida, Citrus Research and Education Center, Lake Alfred, FL 33850, United States Boyko, V.P., Department of Plant Pathology, University of Florida, Citrus Research and Education Center, Lake Alfred, FL 33850, United States Albiach-Marti, M.R., Department of Plant Pathology, University of Florida, Citrus Research and Education Center, Lake Alfred, FL 33850, United States Mawassi, M., Department of Plant Pathology, University of Florida, Citrus Research and Education Center, Lake Alfred, FL 33850, United States Navas-Castillo, J., Department of Plant Pathology, University of Florida, Citrus Research and Education Center, Lake Alfred, FL 33850, United States Karasev, A.V., Department of Plant Pathology, University of Florida, Citrus Research and Education Center, Lake Alfred, FL 33850, United States Dolja, V., Dept. of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331, United States Hilf, M.E., U.S. Hort. Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Orlando, FL 32803, United States Lewandowski, D.J., Department of Plant Pathology, University of Florida, Citrus Research and Education Center, Lake Alfred, FL 33850, United States Moreno, P., Inst. Valenciano de Invest. Agrarias, 46113 Moncada, Valencia, Spain Bar-Joseph, M., Volcani Institute, Bet-Dagan 5020, Israel Garnsey, S.M., U.S. Hort. Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Orlando, FL 32803, United States Dawson, W.O., Department of Plant Pathology, University of Florida, Citrus Research and Education Center, Lake Alfred, FL 33850, United States, CREC, 700 Experiment Station Road, Lake Alfred, FL 33850, United States
An engineered closterovirus RNA replicon and analysis of heterologous terminal sequences for replication
Citrus tristeza virus (CTV) populations in citrus trees are unusually complex mixtures of viral genotypes and defective RNAs developed during the long-term vegetative propagation of the virus and by additional mixing by aphid transmission. The viral replication process allows the maintenance of minor amounts of disparate genotypes and defective RNAs in these populations. CTV is a member of the Closteroviridae possessing a positive-stranded RNA genome of ≃20 kilobases that expresses the replicase-associated genes as an ≃400-kDa polyprotein and the remaining 10 3' genes through subgenomic mRNAs. A full-length cDNA clone of CTV was generated from which RNA transcripts capable of replication in protoplasts were derived. The large size of cDNA hampered its use as a genetic system. Deletion of 10 3' genes resulted in an efficient RNA replicon that was easy to manipulate. To investigate the origin and maintenance of the genotypes in CTV populations, we tested the CTV replicase for its acceptance of divergent sequences by creating chimeric replicons with heterologous termini and examining their ability to replicate. Exchange of the similar 3' termini resulted in efficient replication whereas substitution of the divergent (up to 58% difference in sequence) 5' termini resulted in reduced but significant replication, generally in proportion to the extent of sequence divergence.