Transcriptional strategy of closteroviruses: mapping the 5' termini of the citrus tristeza virus subgenomic RNAs.

Citrus tristeza virus (CTV) induces formation of a nested set of at least nine 3' coterminal subgenomic RNAs (sgRNAs) in infected tissue. The organization and expression of the 19,296-nucleotide (nt) CTV genome resembles that of coronaviruses, with polyprotein processing, translational frameshifting, and multiple sgRNA formation, but phylogenetically the CTV polymerase, like polymerases of other closteroviruses, belongs to the Sindbis virus-like lineage of RNA virus polymerases. Both positive-strand RNA virus supergroups, coronaviruses and Sindbis-like viruses, utilize different mechanisms of transcription. To address the mechanism of CTV transcription, 5' termini for the two most abundant sgRNAs, 1.5 and 0.9 kb, respectively, were mapped by runoff reverse transcription. The two sgRNAs were demonstrated to have 48- and 38-nt 5' untranslated regions (5'-UTRs), respectively. The 5'-UTR for the 1.5-kb RNA was cloned, sequenced, and demonstrated to be colinear with the 48-nt genomic sequence upstream of the initiator codon of the respective open reading frame 10, i.e., to be of continuous template origin. The data obtained suggest that the sgRNA transcription of CTV is dissimilar from the coronavirus transcription and consistent with the transcriptional mechanism of other Sindbis-like viruses. Thus, the Sindbis virus-like mechanism of transcription of the positive-strand RNA genomes might be successfully utilized by the closterovirus genome of up to 19.3 kb with multiple sgRNAs.