An engineered closterovirus RNA replicon and analysis of heterologous terminal sequences for replication

Citrus tristeza virus (CTV) populations in citrus trees are unusually compl ex 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 r amounts of disparate genotypes and defective RNAs in these populations. C TV is a member of the Closteroviridae possessing a positive-stranded RNA ge nome of approximate to 20 kilobases that expresses the replicase-associated genes as an approximate to 400-kDa polyprotein and the remaining 10 3' gen es through subgenomic mRNAs. A full-length cDNA clone of CTV was generated from which RNA transcripts capable of replication in protoplasts were deriv ed. The large size of cDNA hampered its use as a genetic system. Deletion o f 10 3' genes resulted in an efficient RNA replicon that was easy to manipu late. To investigate the origin and maintenance of the genotypes in CTV pop ulations, we tested the CTV replicase for its acceptance of divergent seque nces 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% dif ference in sequence) 5' termini resulted in reduced but significant replica tion, generally in proportion to the extent of sequence divergence.