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Journal of Virology, November 2006, p. 10900-10906, Vol. 80, No. 21
0022-538X/06/$08.00+0     doi:10.1128/JVI.00385-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Construction of a Severe Acute Respiratory Syndrome Coronavirus Infectious cDNA Clone and a Replicon To Study Coronavirus RNA Synthesis{triangledown}

Fernando Almazán, Marta L. DeDiego, Carmen Galán, David Escors, Enrique Álvarez, Javier Ortego, Isabel Sola, Sonia Zuñiga, Sara Alonso, Jose L. Moreno, Aitor Nogales, Carmen Capiscol, and Luis Enjuanes*

Centro Nacional de Biotecnología, CSIC, Department of Molecular and Cell Biology, Darwin 3, Campus Universidad Autónoma, Cantoblanco, Madrid, Spain

Received 23 February 2006/ Accepted 15 August 2006

The engineering of a full-length infectious cDNA clone and a functional replicon of the severe acute respiratory syndrome coronavirus (SARS-CoV) Urbani strain as bacterial artificial chromosomes (BACs) is described in this study. In this system, the viral RNA was expressed in the cell nucleus under the control of the cytomegalovirus promoter and further amplified in the cytoplasm by the viral replicase. Both the infectious clone and the replicon were fully stable in Escherichia coli. Using the SARS-CoV replicon, we have shown that the recently described RNA-processing enzymes exoribonuclease, endoribonuclease, and 2'-O-ribose methyltransferase were essential for efficient coronavirus RNA synthesis. The SARS reverse genetic system developed as a BAC constitutes a useful tool for the study of fundamental viral processes and also for developing genetically defined vaccines.

* Corresponding author. Mailing address: Department of Molecular and Cell Biology, Centro Nacional de Biotecnología, CSIC, Darwin 3, Campus Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain. Phone: 34 91 585 4555. Fax: 34 91 585 4915. E-mail: L.Enjuanes{at}cnb.uam.es.

{triangledown} Published ahead of print on 23 August 2006.

Journal of Virology, November 2006, p. 10900-10906, Vol. 80, No. 21
0022-538X/06/$08.00+0     doi:10.1128/JVI.00385-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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