Establishment of a stable SARS-CoV-2 replicon system for application in high-throughput screening

Abstract

Experiments with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are limited by the need for biosafety level 3 (BSL3) conditions. A SARS-CoV-2 replicon system rather than an in vitro infection system is suitable for antiviral screening since it can be handled under BSL2 conditions and does not produce infectious particles. However, the reported replicon systems are cumbersome because of the need for transient transfection in each assay. In this study, we constructed a bacterial artificial chromosome vector (the replicon-BAC vector) including the SARS-CoV-2 replicon and a fusion gene encoding Renilla luciferase and neomycin phosphotransferase II, examined the antiviral effects of several known compounds, and then established a cell line stably harboring the replicon-BAC vector. Several cell lines transiently transfected with the replicon-BAC vector produced subgenomic replicon RNAs (sgRNAs) and viral proteins, and exhibited luciferase activity. In the transient replicon system, treatment with remdesivir or interferon-β but not with camostat or favipiravir suppressed the production of viral agents and luciferase, indicating that luciferase activity corresponds to viral replication. VeroE6/Rep3, a stable replicon cell line based on VeroE6 cells, was successfully established and continuously produced viral proteins, sgRNAs and luciferase, and their production was suppressed by treatment with remdesivir or interferon-β. Molnupiravir, a novel coronavirus RdRp inhibitor, inhibited viral replication more potently in VeroE6/Rep3 cells than in VeroE6-based transient replicon cells. In summary, our stable replicon system will be a powerful tool for the identification of SARS-CoV-2 antivirals through high-throughput screening.

Development of SARS-CoV-2 replicons for the ancestral virus and variant of concern Delta for antiviral screening

“…Spontaneous mutations in nsp4 and nsp10 were identified in almost all replicon RNA populations isolated for cells stabling maintaining the replicons, which could be suggestive of adaptive mutations that allowed for non-cytopathic replication in BHK-21 cells (37). Furthermore, a study reporting the isolation of Vero E6 populations stably maintaining a minimal replicon did not include the nsp1 mutations in the replicon genome, although the sequences of the stably maintained replicons were not determined (59).…”

Reverse genetic systems of SARS-CoV-2 for antiviral research

“…Interestingly, Ricardo-Lax et al failed to select a stable cell line containing the YAC-derived SARS-CoV-2 replicon with the Nsp1 K164A/H165A mutations in both Huh-7.5 and BHK-21 cells. In contrast, Tanaka et al successfully obtained a stable VeroE6 cell line that harbors the BAC plasmid-derived SARS-CoV-2 replicon without introducing any mutations into the SARS-CoV-2 genome ( Tanaka et al, 2022 ), arguing different cell backgrounds and multiple adaptive mutations may be necessary. These two stable SARS-CoV-2 replicons have been proven effective in screening inhibitors against SARS-CoV-2 replication.…”

Reverse genetics systems for SARS‐CoV‐2

“…The first reverse genetics system for coronavirus was successfully constructed with a targeted RNA recombination strategy. 21 , 22 , 23 Homologous RNA recombination is common in regions with high sequence similarity during viral RNA replication. 24 Lai et al first reported that RNA recombination mediates the fusion of RNA of different mouse hepatitis virus (MHV) strains during genome replication.…”

“…The first reverse genetics system for coronavirus was successfully constructed with a targeted RNA recombination strategy 21–23 . Homologous RNA recombination is common in regions with high sequence similarity during viral RNA replication 24 .…”