Ahmed Eisa Elhag, Institute of Biochemistry and Biophysics, Polish Academy of Science

Ahmed Eisa Elhag-1, Katarzyna Grelewska-Nowotko-1, Vadim Shchepachev-2, David Tollervey-2, Tomasz W. Turowski-1. 1-Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland. 2-Wellcome Centre for Cell Biology, The University of Edinburgh, Edinburgh, Scotland

Expressing & Multiplying

Coronaviruses (CoVs) are positive-sense single-stranded RNA (ssRNA⁺) viruses with ~30 kb genomes encoding an RNA-dependent RNA polymerase (RdRP). Viral subgenomic RNAs are generated through discontinuous transcription, complicating strand-specific RNA quantification. Although RdRP is essential for replication, the early regulation of transcription and the initiation of negative-sense RNA (ssRNA⁻) synthesis remain poorly understood. While later replication occurs within double-membrane vesicles (DMVs), it is unclear whether DMV formation is required for early ssRNA⁻ synthesis. This study aimed to determine the timing of ssRNA⁻ and ssRNA⁺ initiation, examine their temporal relationship with replication complex formation, and identify host factors involved in early ssRNA⁻ synthesis. We analyzed time-resolved RNA-seq data from SARS-CoV-2–infected Vero cells and developed a strand-specific qPCR and digital PCR (ddPCR) workflow for Murine Hepatitis Virus (MHV) infection in LR-7 cells, enabling absolute RNA quantification from 2 hours post-infection (hpi). Replication complex formation was assessed by confocal immunofluorescence microscopy targeting NSP3, NSP4, and NSP6. The host factor SND1 was investigated using siRNA-mediated knockdown. RNA-seq revealed strong accumulation of positive-sense genomic RNA between 8–17 hpi, while negative-sense RNA remained comparatively low. In the MHV model, strand-specific assays detected both ssRNA⁻ and ssRNA⁺ as early as 2 hpi. NSP3, NSP4, and NSP6 co-localized at 2 hpi, indicating rapid establishment of replication compartments. SND1 depletion significantly reduced early ssRNA⁻ and subsequent ssRNA⁺ synthesis. These findings demonstrate that ssRNA⁻ synthesis is initiated early during CoV infection and identify SND1 as a critical host factor supporting early viral transcription, highlighting early replication events as potential antiviral targets.