ABSTRACT
Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the current global pandemic of the COVID-19, which has persisted partly through the emergence of new variants. A non-infectious, convenient, and reproducible in vitro system is needed to assess drug susceptibility of new variants of concern and potential drug resistance mutations. Method(s): The SARS-CoV-2 replicon protocol was adapted and optimized based on {Zhang 2021}. The replicon RNA was produced by in vitro transcription of full-length replicon DNA assembled by ligation of plasmid fragments encoding for the SARS-CoV-2 non-structural proteins (Nsps), nucleoprotein and gaussia luciferase reporter protein. Wild-type and mutant replicon RNAs were transfected into Huh7-1CN cells by electroporation and treated with remdesivir (RDV). To determine EC50 values, luciferase activity was determined at 48 hours post transfection. A recombinant SARS-CoV-2 virus rescue system {Xie 2020} was used to generate matching Nsp mutants for comparison with the replicon system. Result(s): The selected substitutions reflective of Omicron BA.5 sub-lineage BF.7 variant: the triple mutants (Nsp12 (P323L) +Nsp13 (R392C) + Nsp14 (I42V), and a single Nsp12 L247F mutant as well as several specific Nsp12 mutations identified by in vitro resistance selection with RDV or RDV parent nucleoside analog GS-441524 were cloned into the replicon and tested for susceptibility to RDV. RDV inhibited the SARS-CoV-2 wild-type replicon with a mean EC50 value of 14.7 +/- 3.5 nM (N=9). The Nsp12 P323L substitution, a common polymorphism in all major variants of concern including Omicron, was fully susceptible to RDV with a 0.6-fold change in EC50 from the wild-type. The Omicron BF.7 triple mutants and L247F were also fully susceptible to RDV with 0.5- and 0.4-fold changes, respectively. Nsp12 substitutions F480L, V557L, V792I, S759A+V792I, and C799F resulting from in vitro resistance selections with RDV showed minimal to moderate levels of reduced susceptibility to RDV (1.8 to 18.3-fold change) (Table 1). The RDV EC50 fold changes correlated between the noninfectious replicon and recombinant infection virus system (Table 1). Conclusion(s): The replicon system is a convenient and reproducible model to test the susceptibility of SARS-CoV-2 mutant variants to RDV and potentially other antivirals. The common Nsp12 polymorphisms in all variants including the highly transmissible Omicron variant were fully susceptible to RDV.