RNA Folding Energy Regulates Discontinuous Transcription in Coronaviruses

Coronaviruses use discontinuous transcription to generate subgenomic RNAs (sgRNAs) that encode structural and accessory proteins. However, the factors regulating sgRNA abundance remain unclear. Here, we combined strand-specific RNA sequencing, RNA:RNA interaction mapping, thermodynamic modeling, and targeted mutagenesis to define the regulation of (-) sgRNA synthesis in SARS-CoV-2 infection. We demonstrate that the relative (-) sgRNA abundance across viral genes is stable throughout infection and strongly correlates with corresponding (+) sgmRNA levels. Through meta-analysis of published SPLASH data, we found that the frequency of long-range interactions between the 5′ genomic transcription regulatory sequence (TRS)-Leader and downstream TRS-Body sequences correlates with sgRNA abundance. Notably, the thermodynamic stability (ΔG) of these duplexes quantitatively predicts (-) sgRNA transcript levels. Mutations in non-coding regulatory regions that altered the ΔG resulted in corresponding changes in (-) sgRNA expression, establishing a causal role for TRS duplex stability in transcriptional regulation. Analysis of naturally occurring mutations near regulatory sites further suggests that modulation of duplex stability may also serve as an evolutionary mechanism to fine-tune viral gene expression. Together, our findings identify TRS-Leader:TRS-Body thermodynamics as a key determinant of discontinuous transcription and reveal how RNA structure encodes gene expression levels in the coronavirus genome.