Development and optimization of a novel T7 polymerase-independent Marburg virus minigenome system

Marburg virus (MARV) is the only known pathogenic filovirus not belonging to the genus Ebolavirus. Minigenomes have proven a useful tool to study MARV, but all existing MARV minigenomes are dependent on the addition of an exogenous T7 RNA polymerase to drive minigenome expression. However, exogenous expression of a T7 polymerase is not always feasible and acts as a confounding factor in compound screening assays. We have developed an alternative minigenome that is controlled by the natively expressed RNA polymerase II. We demonstrate here the characteristics of this new system and its applicability in a wide range of cell types. Our system shows a clear concentration-dependent activity and outperforms the existing T7 polymerase-based system at higher concentrations, especially in difficult-to-transfect cell lines. In addition, we show that our system can be used for high-throughput compound screening in a 96-well format, thereby providing an attractive alternative to previously developed MARV minigenomes.