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

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Sergey P. Korolev^1,2,3,a*, Aleksandra A. Shulepova^1,3, Andrey N. Anisenko^1,2,3, Simon O. Galkin³, Liudmila A. Alexandrova⁴, Maxim V. Jasko⁴, Elena S. Matyugina⁴, Mikhail S. Novikov⁵, Anastasiya L. Khandazhinskaya⁴, Sergey N. Kochetkov^2,4, Marina B. Gottikh^1,2

¹Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119992 Moscow, Russia

²Faculty of Chemistry, Lomonosov Moscow State University, 119992 Moscow, Russia

³Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, 119992 Moscow, Russia

⁴Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia

⁵Department of Pharmaceutical & Toxicological Chemistry, Volgograd State Medical University, 400131 Volgograd, Russia

^* To whom correspondence should be addressed.

Received: September 10, 2024; Revised: October 14, 2024; Accepted: October 15, 2024

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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.

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KEY WORDS: dual-reporter replicon, inhibitors, RNA-dependent RNA polymerase, SARS-CoV-2, viruses

DOI: 10.1134/S0006297924110166

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