Optimizing SARS-CoV-2 Mini-Genome Replication and Modeling Discontinuous Transcription

Abstract

SARS-CoV-2 is a constantly evolving positive-sense virus that replicates and transcribes its genome through an RNA-dependent RNA polymerase (RdRp) composed of nonstructural proteins (nsp) nsp7, nsp8, and nsp12. This RdRp synthesizes both full-length genomic RNA during viral replication and subgenomic mRNAs during transcription. Subgenomic RNAs are produced via a template switching mechanism regulated by transcription regulation sequences (TRS). However, other viral nsps contribute to SARS-CoV-2 RNA synthesis, and their exact impact on efficient SARS-CoV-2 RNA replication remains unclear. Additionally, there is currently no high-throughput assay to detect template switching events and address the relationship between TRS sequence and TRS structure and template switching. To address this, we sought to optimize a negative-sense SARS-CoV-2 mini-genome assay to determine the minimal components for efficient RNA replication and transcription and to develop a reporter assay to investigate the role of TRS homology in template switching. Using a nanoluciferase assay, I found that the pPOLI-driven mini-genome system produces higher replication efficiency than the T7-driven system. Using this optimal system, I report that nsp7 and nsp12 form a minimal replicase unit capable of supporting subgenomic RNA replication and that replication efficiency is influenced by the stoichiometry of nsp7, nsp8, and nsp12. However, the template-switching assay did not produce sufficient activity above background, and RT-PCR analysis confirmed a lack of detectable mini-genome transcription. Overall, these findings establish an optimized system for more sensitive and efficient research into SARS-CoV-2 replication and transcription and contribute to a deeper understanding of the functional activity of the SARS-CoV-2 RdRp.