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Effects of Modification of the Transcription Initiation Site Context on Citrus Tristeza Virus Subgenomic RNA Synthesis

María A. Ayllón,¹ Siddarame Gowda,¹ Tatineni Satyanarayana,¹ Alexander V. Karasev,^1, Scott Adkins,² Munir Mawassi,^1, José Guerri,³ Pedro Moreno,³ and William O. Dawson^1*

Department of Plant Pathology, University of Florida, Citrus Research and Education Center, Lake Alfred, Florida 33850,¹ U.S. Horticultural Research Laboratory, USDA Agricultural Research Service, Ft. Pierce, Florida 34945,² Instituto Valenciano de Investigaciones Agrarias, 46113 Moncada, Valencia, Spain³

Received 20 March 2003/ Accepted 11 June 2003

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 10 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 10 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 and 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.

University of Florida Agricultural Experiment Station Journal Series R-09453.

Present address: Department of Microbiology and Immunology, Thomas Jefferson University, Doylestown, PA 18901.

Present address: Department of Virology, Agricultural Research Organization, The Volcani Center, Bet-Dagan 50259, Israel.

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