Key result
Expression of viral antigenomic RNA under the control of an RNA Polymerase II promoter increased the rescue efficacy of Borna disease virus by nearly 20-fold and measles virus by approximately 100-fold compared to T7 or Pol I promoters.
Effect estimate: nearly 20-fold increase for BDV, ~100-fold increase for MV
Using an RNA Pol II promoter significantly enhances the reverse genetics rescue efficacy of negative-strand RNA viruses like BDV and MV from cDNA.
Pol II promoter may enhance reverse genetics rescue of negative-strand RNA viruses in vitro; leaves open broader applicability and in vivo validation.
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.
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Arnold et al. (2006) studied Viral rescue (Borna disease virus and measles virus). RNA Polymerase II promoter vs. T7 RNA polymerase or RNA Polymerase I promoters was evaluated on Rescue efficacy (number of BDV-positive cells or MV titers) (nearly 20-fold increase for BDV, ~100-fold increase for MV). Expression of viral antigenomic RNA under the control of an RNA Polymerase II promoter increased the rescue efficacy of Borna disease virus by nearly 20-fold and measles virus by approximately 100-fold compared to T7 or Pol I promoters.
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