Dual-Reporter SARS-CoV-2 Replicon for Screening Viral Polymerase Inhibitors

To design a safe cellular system for testing inhibitors targeting the RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2, a genetic construct was engineered containing viral cDNA with two blocks of reporter genes while the genes encoding structural S, E, and M proteins were absent. The first reporter block, consisting of Renilla luciferase and green fluorescent protein (Rluc-GFP), was located upstream of the SARS-CoV-2 5'-UTR. Meanwhile, the second block represented by firefly luciferase and red fluorescent protein (Fluc-RFP) was positioned downstream of the transcription regulatory sequence (TRS-N). While the first block of reporter genes can be transcribed by both viral RdRp and cellular polymerases, the second block can only be transcribed by the viral polymerase according to the Coronaviridae discontinuous transcription mechanism. This allowed us to accurately assess effectiveness of the viral RdRp inhibition. To facilitate the search for nucleoside RdRp inhibitors the cell line was obtained expressing herpes simplex virus thymidine kinase, which provides the first stage of nucleoside phosphorylation. When screening the ability of a number of compounds to inhibit catalytic activity of the SARS-CoV-2 RdRp, we discovered antiviral activity of 2'-amino-2'-deoxyadenosine and adenosine-N1-oxide, which exceeded activity of molnupiravir, a therapeutic agent used in the treatment of COVID-19.