Spatio-temporal proteomic profiling of SARS-CoV-2 replication organelles

Abstract Coronaviruses establish replication organelles (ROs) to synthesize their RNA while evading host defenses. To systematically profile the host factors and the dynamic interplay between viral and host factors during viral replication, we developed an antibody-based proximity labeling strategy tailored to profile the proteome at the coronavirus RO. Applying this to SARS-CoV-2 and HCoV-OC43, we revealed both conserved requirements and unique adaptations in each virus. Temporal profiling further uncovered dynamic recruitment patterns: an early enrichment of ER proteins, mitochondrial proteins and RNA processing factors, and a progressive exclusion of stress granule components. Functional analyses of RNA-binding proteins revealed regulatory host factors, including the double-stranded RNA-binding protein STAU1, which plays a pro-viral role on SARS-CoV-2 at the early infection stage. Collectively, these findings establish a robust proteomic framework for investigating coronavirus replication organelle biology and uncover virus-specific strategies to manipulate host cellular machinery.