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Journal of Virology, February 2003, p. 2116-2123, Vol. 77, No. 3
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.3.2116-2123.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Replication of Carnation Italian Ringspot Virus Defective Interfering RNA in Saccharomyces cerevisiae

Vitantonio Pantaleo, Luisa Rubino, and Marcello Russo*

Dipartimento di Protezione delle Piante e Microbiologia Applicata, Università degli Studi and Istituto di Virologia Vegetale del CNR, Bari, Italy

Received 3 September 2002/ Accepted 29 October 2002

Two plasmids from which the sequences coding for the 36- and 95-kDa proteins of Carnation Italian ringspot virus (CIRV) could be transcribed in vivo in the yeast Saccharomyces cerevisiae under the control of the ADH1 promoter and terminator were constructed. The two proteins, which constitute the viral replicase, were correctly translated and integrated into membranes of the yeast cells. An additional plasmid was introduced in yeasts expressing the CIRV replicase, from which a defective interfering (DI) RNA (DI-7 RNA) could be transcribed under the control of the GAL1 promoter and terminated by the Tobacco ringspot virus satellite ribozyme, which cleaved 19 nucleotides downstream of the 3' end of DI RNA. The DI-7 RNA transcripts were amplified by the viral replicase as demonstrated by the restoration of the authentic 3' end, the requirement of a specific cis-acting signal at this terminus, the preferential accumulation of molecules with the authentic 5' terminus (AGAAA), the synthesis of head-to-tail dimers, the presence of negative strands, and the incorporation of 5-bromo-UTP. Additionally, transformation with a dimeric construct of DI-7 RNA led to the synthesis of monomers, mimicking the activity of the viral replicase in plant cells.

* Corresponding author. Mailing address: Dipartimento di Protezione delle Piante, Via Amendola 165/A, 70126 Bari, Italy. Phone: 39 080 5442934. Fax: 39 080 5442911. E-mail: csvvmr01{at}area.ba.cnr.it.

Journal of Virology, February 2003, p. 2116-2123, Vol. 77, No. 3
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.3.2116-2123.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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