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

Autor: Korolev SP; Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119992, Russia. spkorolev@mail.ru.; Faculty of Chemistry, Lomonosov Moscow State University, Moscow, 119992, Russia.; Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Moscow, 119992, Russia., Shulepova AA; Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119992, Russia.; Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Moscow, 119992, Russia., Anisenko AN; Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119992, Russia.; Faculty of Chemistry, Lomonosov Moscow State University, Moscow, 119992, Russia.; Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Moscow, 119992, Russia., Galkin SO; Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Moscow, 119992, Russia., Alexandrova LA; Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991, Russia., Jasko MV; Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991, Russia., Matyugina ES; Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991, Russia., Novikov MS; Department of Pharmaceutical & Toxicological Chemistry, Volgograd State Medical University, Volgograd, 400131, Russia., Khandazhinskaya AL; Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991, Russia., Kochetkov SN; Faculty of Chemistry, Lomonosov Moscow State University, Moscow, 119992, Russia.; Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991, Russia., Gottikh MB; Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119992, Russia.; Faculty of Chemistry, Lomonosov Moscow State University, Moscow, 119992, Russia.
Jazyk: angličtina
Zdroj: Biochemistry. Biokhimiia [Biochemistry (Mosc)] 2024 Nov; Vol. 89 (11), pp. 2037-2050.
DOI: 10.1134/S0006297924110166
Abstrakt: 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.
Databáze: MEDLINE
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