title: 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.
abstract: 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.
authors.affiliation: Department of Virology, University of Freiburg, Hermann Herder Strasse 11, D-79104 Freiburg, Germany.
authors.forename: Arnold
authors.initials: A
authors.lastname: Martin
authors.forename: Peter
authors.initials: P
authors.lastname: Staeheli
authors.forename: Urs
authors.initials: U
authors.lastname: Schneider
mesh_terms: D001891:Borna disease virus / Q000235:genetics; D016679:Genome, Viral; D018096:Mononegavirales / Q000235:genetics*; D018701:Mononegavirales Infections; D011401:Promoter Regions, Genetic; D012319:RNA Polymerase II / Q000502:physiology*; D016372:RNA, Antisense / Q000235:genetics*; D012367:RNA, Viral / Q000096:biosynthesis / Q000235:genetics*; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism; D014158:Transcription, Genetic; D014779:Virus Replication
chemical_list: D016372:RNA, Antisense; D012367:RNA, Viral; D012319:RNA Polymerase II; D012324:RNA-Dependent RNA Polymerase