An Engineered Closterovirus RNA Replicon and Analysis of Heterologous Terminal Sequences for Replication

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 mino...

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Published in:Proceedings of the National Academy of Sciences - PNAS Vol. 96; no. 13; pp. 7433 - 7438
Main Authors: Satyanarayana, T., Gowda, S., Boyko, V. P., Albiach-Marti, M. R., Mawassi, M., Navas-Castillo, J., Karasev, A. V., Doija, V., Hile, M. E., Lewandowski, D. J., Moreno, P., Bar-Joseph, M., Garnsey, S. M., Dawson, W. O.
Format: Journal Article
Language:English
Published: United States National Academy of Sciences of the United States of America 22-06-1999
National Acad Sciences
The National Academy of Sciences
Subjects:
Biological Sciences
Citrus tristeza virus
Closterovirus
Closterovirus - physiology
Complementary DNA
Genetic Engineering
Genomes
Genotypes
Hybridity
Nucleotides
Open reading frames
Protoplasts
Replicon - genetics
RNA
RNA, Viral - genetics
Sequence Analysis
Virions
Virus Replication - genetics
Viruses
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Summary: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.
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To whom reprint requests should be addressed at: CREC, 700 Experiment Station Road, Lake Alfred, FL 33850. e-mail: wodtmv@icon.lal.ufl.edu.
Communicated by George Bruening, University of California, Davis, CA
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.96.13.7433