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 le ast nine 3' coterminal subgenomic RNAs (sgRNAs) in infected tissue, Th e organization and expression of the 19,296-nucleotide (nt) CTV genome resembles that of coronaviruses, with polyprotein processing, transla tional frameshifting, and multiple sgRNA formation, but phylogenetical ly the CTV polymerase, like polymerases of other closteroviruses, belo ngs to the Sindbis virus-like lineage of RNA virus polymerases. Both p ositive-strand RNA virus supergroups, coronaviruses and Sindbis-like v iruses, utilize different mechanisms of transcription. To address the mechanism of CTV transcription, 5' termini for the two most abundant s gRNAs, 1.5 and 0.9 kb, respectively, were mapped by runoff reverse tra nscription. The two sgRNAs were demonstrated to have 48- and 38-nt 5' untranslated regions (5'-UTRs), respectively. The 5'-UTR for the 1.5-k b RNA was cloned, sequenced, and demonstrated to be colinear with the 48-nt genomic sequence upstream of the initiator codon of the respecti ve open reading frame 10, i.e., to be of continuous template origin. T he data obtained suggest that the sgRNA transcription of CTV is dissim ilar from the coronavirus transcription and consistent with the transc riptional mechanism of other Sindbis-like viruses. Thus, the Sindbis v irus-like mechanism of transcription of the positive-strand RNA genome s might be successfully utilized by the closterovirus genome of up to 19.3 kb with multiple sgRNAs.