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

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Abstract

To prepare a safe cellular system for testing inhibitors targeting the RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2, a genetic construct containing viral cDNA with two blocks of reporter genes while lacking genes encoding the structural S, E and M proteins was engineered. The first reporter block, consisting of renilla luciferase and green fluorescent protein (Rluc-GFP), 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). Although 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, aligning with the Coronaviridae discontinuous transcription mechanism. This allowed us to accurately assess the effectiveness of 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 the catalytic activity of SARS-CoV-2 RdRp, we first discovered the antiviral activity of 2′-amino2′-deoxyadenosine and adenosine-N1-oxide, exceeding the activity of molnupiravir, a therapeutic agent used in the treatment of COVID-19.

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About the authors

S. P. Korolev

Lomonosov Moscow State University

Author for correspondence.
Email: spkorolev@mail.ru
Russian Federation, 119991, Moscow

A. A. Shulepova

Lomonosov Moscow State University

Email: spkorolev@mail.ru
Russian Federation, 119991, Moscow

A. N. Anisenko

Lomonosov Moscow State University

Email: spkorolev@mail.ru
Russian Federation, 119991, Moscow

S. O. Galkin

Lomonosov Moscow State University

Email: spkorolev@mail.ru
Russian Federation, 119991, Moscow

L. A. Alexandrova

Engelhardt Institute of Molecular Biology of the Russian Academy of Sciences

Email: spkorolev@mail.ru
Russian Federation, 119991, Moscow

M. V. Yasko

Engelhardt Institute of Molecular Biology of the Russian Academy of Sciences

Email: spkorolev@mail.ru
Russian Federation, 119991, Moscow

E. S. Matyugina

Engelhardt Institute of Molecular Biology of the Russian Academy of Sciences

Email: spkorolev@mail.ru
Russian Federation, 119991, Moscow

M. S. Novikov

Volgograd State Medical University

Email: spkorolev@mail.ru
Russian Federation, 400131, Volgograd

A. L. Khandazhinskaya

Engelhardt Institute of Molecular Biology of the Russian Academy of Sciences

Email: spkorolev@mail.ru
Russian Federation, 119991, Moscow

S. N. Kochetkov

Lomonosov Moscow State University; Engelhardt Institute of Molecular Biology of the Russian Academy of Sciences

Email: spkorolev@mail.ru
Russian Federation, 119991, Moscow; 119991, Moscow

M. B. Gottikh

Lomonosov Moscow State University

Email: spkorolev@mail.ru
Russian Federation, 119991, Moscow

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2. Fig. 1. Model of a full-length SARS-CoV-2 replicon with two reporter gene blocks. The first Rluc-GFP reporter gene block is located within ORF1a under the control of the CMV promoter, while the second Fluc-RFP block is located between the protein N gene and the 3′-UTR and can only be transcribed by viral RdRp via a discontinuous transcription mechanism. Below is a schematic of the 6 virus cDNA fragments from which the full-length replicon was assembled and cloned into the pBAC_lacZ plasmid using the Golden Gate approach

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3. Fig. 2. Schematic of the functioning of the replicon. Expression of the first reporter (Rluc) can occur either through transcription from vector DNA (BAC) by cellular RNA polymerase II (RNAPII) or through replication of viral RNA under the action of RdRp. The second reporter (Fluc) can be transcribed only by viral RdRp through the mechanism of interrupted transcription

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4. Fig. 3. Comparison of expression levels of renilla (Rluc) and firefly (Fluc) luciferases in Vero E6 cells transfected with REP and REP_mut replicons. Mean values from at least 3 experiments ± standard deviation are presented

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5. Fig. 4. Validation of the inhibitory activity of zidovudine (a) and acyclovir (b) in Vero E6 (solid line) and Vero E6_TK (dashed line) cell lines transduced with a replication-incompetent HIV-1-based vector encoding a firefly luciferase reporter protein. Mean values from at least 3 experiments ± standard deviation are presented

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6. Fig. 5. Structures of the studied compounds

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7. Fig. 6. Results of the ability of compounds, molnupiravir (a), acyclovir (b), 2′-adenosine-N1-oxide (c), amino-2′-deoxyadenosine (d), and 3′-O-methyladenosine (e), stavudine (f), LN140 (g), and Z876 (h), inhibit RdRp activity of SARS-CoV-2 using the REP replicon in Vero E6 (solid line) and Vero E6_TK (dashed line) cell lines. Mean values from at least 4 experiments ± standard deviation are presented

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