Submitted to: Journal of Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/11/2003
Publication Date: 9/1/2003
Citation: Ayllon, Maria A., Gowda, Siddarame, Saryanarayana, Tatineni, Karasev, Alexander, Adkins, S., Mawassi, Munir, Guerri, Jose, Moreno, Pedro, Dawson, William. 2003. Effects of Modification of the Transcription Initiation site context on Citrus Tristeza Virus Subgenomic RNA Synthesis. Journal of Virology. 77:9232-9243. Interpretive Summary: The objective of this research was to further elucidate the mechanism by which citrus tristeza virus (CTV) regulates its replication (reproduction). Data collected permit a comparison to established methods of viral replication and suggest that CTV uses a different mechanism. This report continues a collaborative citrus virology research effort between ARS and the University of Florida. It also provides basic virology information to university, state and Federal research and regulatory scientists studying CTV, an important pathogen of citrus worldwide.
Technical Abstract: Citrus tristeza virus (CTV), a member of the Closteroviridae, has a positive-sense RNA genome of about 20-kb organized into 12 open reading frames (ORFs). The last ten ORFs are expressed through 3´-coterminal subgenomic RNAs (sgRNAs) regulated in both amounts and timing. Additionally, relatively large amounts of complementary sgRNAs are produced. We have been unable to determine whether these sgRNAs are produced by internal promotion from the full-length template minus strand or by transcription from the minus-stranded sgRNAs. Understanding the regulation of ten sgRNAs is a conceptual challenge. In analyzing commonalities of a replicase complex in producing so many sgRNAs, we examined initiating nucleotides of the sgRNAs. We mapped the 5´ termini of intermediate (CP, p13) and low (p18) produced sgRNAs that like the two highly abundant sgRNAs (p20 and p23) previously mapped all initiate with an adenylate. We then examined modifications of the initiation site, which has been shown to be useful in defining mechanisms of sgRNA synthesis. Surprisingly, mutation of the initiating nucleotide of the CTV sgRNAs did not prevent sgRNA accumulation. Based on our results, the CTV replication complex appears to initiate sgRNA synthesis with purines, preferably with adenylates, and is able to initiate synthesis using a nucleotide a few positions 5¿ or 3¿ of the native initiation nucleotide. Furthermore, the context of the initiation site appears to be a regulatory mechanism for levels of sgRNA production. These data do not support either of the established mechanisms for synthesis of sgRNAs, suggesting that CTV sgRNA production utilizes a different mechanism.