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Journal of Virology, June 2006, p. 5708-5715, Vol. 80, No. 12
0022-538X/06/$08.00+0     doi:10.1128/JVI.02389-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

RNA Polymerase II-Controlled Expression of Antigenomic RNA Enhances the Rescue Efficacies of Two Different Members of the Mononegavirales Independently of the Site of Viral Genome Replication

Arnold Martin, Peter Staeheli, and Urs Schneider*

Department of Virology, University of Freiburg, D-79104 Freiburg, Germany

Received 14 November 2005/ Accepted 14 March 2006

De novo generation of negative-strand RNA viruses depends on the efficient expression of antigenomic RNA (cRNA) from cDNA. To improve the rescue system of Borna disease virus (BDV), a member of the Mononegavirales with a nuclear replication phase, we evaluated different RNA polymerase (Pol) promoters for viral cRNA expression. Human and mouse Pol I promoters did not increase the recovery rate of infectious BDV from cDNA compared to the originally employed T7 RNA polymerase system. In contrast, expression of viral cRNA under the control of an RNA Pol II promoter increased the rescue efficacy by nearly 20-fold. Similarly, rescue of measles virus (MV), a member of the Mononegavirales with a cytoplasmic replication phase, was strongly improved by Pol II-controlled expression of viral cRNA. Analysis of transcription levels derived from different promoters suggested that the rescue-enhancing function of the Pol II promoter was due mainly to enhanced cRNA synthesis from the plasmid. Remarkably, correct 5'-terminal processing of Pol II-transcribed cRNA by a hammerhead ribozyme was not necessary for efficient rescue of BDV or MV. The correct 5' termini were reconstituted during replication of the artificially prolonged cRNA, indicating that the BDV and MV replicase complexes are able to recognize internal viral replication signals.

* Corresponding author. Mailing address: Department of Virology, University of Freiburg, Hermann Herder Strasse 11, D-79104 Freiburg, Germany. Phone: 49-761-203-6622. Fax: 49-761-203-5350. E-mail: urs.schneider{at}uniklinik-freiburg.de.

Journal of Virology, June 2006, p. 5708-5715, Vol. 80, No. 12
0022-538X/06/$08.00+0     doi:10.1128/JVI.02389-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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