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\u27 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\u27 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\u27 termini resulted in efficient replication whereas substitution of the divergent (up to 58% difference in sequence) 5\u27 termini resulted in reduced but significant replication, generally in proportion to the extent of sequence divergence