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

To further characterize the ZEBOV-4cis stable RNP cell line system, we also tested their susceptibility to previously identified EBOV small molecule inhibitors: Gedunin, an inhibitor of heat shock protein 90, and 6-Azauridine, a nucleoside analog 31, 32 . ZEBOV-4cis stable cells were transfected with the ZEBOV nLuc minigenome reporter and treated with DMSO (1%, control), Gedunin (5 µM), or 6-Azauridine (5 µM) for 2 days. Luciferase assays revealed that both compounds inhibited minigenome activity by >50%, with minimal impact on cell viability (Fig. 7g ). These data indicate that the ZEBOV-4cis stable cell lines generated here are similarly susceptible to known inhibitors identified in transient RNP minigenome systems or recombinant virus systems 31, 32 . Therefore, our approach implemented through the MTK introduces a robust workflow for generating BSL2 systems for filoviruses and other viral agents as they emerge, providing a critical opportunity to implement rapid chemical compound susceptibility screens using these reagents.