Assays

(A) Bicistronic SARS-CoV-2 Reporter System for RdRp Activity Monitoring.
This schematic illustrates the p(+)RLuc–(−)UTR–FLuc reporter construct used to assess SARS-CoV-2 RdRp activity in cells. The construct includes two luciferase genes: Renilla luciferase (RLuc), driven by a CMV promoter in the positive-sense orientation, serves as a constitutive internal control. In contrast, the Firefly luciferase (FLuc) gene is placed under the regulation of the SARS-CoV-2 5′ and 3′ untranslated regions (UTRs) and flanked by hepatitis delta virus (HDV) ribozyme sequences to generate precise RNA ends. FLuc expression depends on replication by the viral replicase, composed of co-expressed NSP5 (3CLpro), NSP7, NSP8, and NSP12 (RdRp), allowing quantification of polymerase activity by luminescence. Variations in FLuc levels reflect changes in RdRp function due to compounds or genetic perturbations.

(B) Alphavirus-Based Reporter for RdRp Functional Screening.
This panel depicts an analogous alphavirus-based system for monitoring polymerase activity. The alphavirus replicon encodes FLuc and Gaussia luciferase (GLuc) under the control of a subgenomic promoter (P_SG), with N* indicating a truncated nucleocapsid. The template vector is transcribed by the cellular RNA polymerase II (HSPolII), while a separate replicase vector expresses the alphavirus nonstructural proteins (nsP1–nsP4) necessary for RNA replication. Ribozymes ensure precise transcript ends. Luciferase signal reflects RdRp-driven expression of the subgenomic RNA, offering a complementary platform for screening viral polymerase inhibitors.