A Toolkit for Rapid Modular Construction of Biological Circuits in Mammalian Cells

João Pedro Fonseca; Alain R. Bonny; G. Renuka Kumar; Andrew H. Ng; Jason Town; Qiu Chang Wu; Elham Aslankoohi; Susan Y. Chen; Patrick Harrigan; Lindsey C. Osimiri; Amy L. Kistler; Hana El-Samad

In the late 1990's a 6-plasmid BSL2 ribonucleoprotein (RNP) minigenome replicon system was developed for EBOV to accelerate research on the transcription and genome replication sub-lifecycle of the virus, and aid antiviral discovery efforts 25, 26 . This system requires expression of four viral proteins (NP, VP35, VP30, and Lpol), along with aT7 polymerase, and a T7-driven minigenome reporter construct 25 (Fig. 7a) . Transfection of these 6 plasmids into mammalian cells results in expression of NP, VP35, VP30, Lpol viral proteins, and the T7 polymerase. The viral proteins recognize and associate with the T7-transcribed minigenome reporter RNA template (vRNA (-)) then catalyze transcription (mRNA) and replication (aRNA (+)), resulting in measurable reporter gene expression (Fig. 7a) . While the 6-plasmid system has been a valuable tool for initial discoveries into EBOV replication and transcription, it is not readily amenable to systematic studies due to its transient nature, inefficiency (all 6 plasmids must be present in the same cell), and variability that limits scaling. More recently, a stable cell line version of this system has been developed via sequential integration of RNP viral proteins 27 .