SARS-CoV-2 ORF8 Exploits Host miRNA Networks to Rewire Post-Transcriptional Regulation

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) encodes several accessory proteins that modulate host cellular pathways and contribute to immune evasion and disease severity. Among them, ORF8 has emerged as a key immunomodulatory factor; however, its impact on host post-transcriptional regulatory networks remains poorly defined. In this study, we investigated how ORF8 expression reshapes the microRNA (miRNA) landscape of lung epithelial cells and how these changes translate into functional proteomic alterations. Using A549 cells transduced with ORF8 and control cells, we performed small RNA sequencing to identify differentially expressed miRNAs, followed by integrative analysis with quantitative proteomic data. ORF8 expression induces extensive remodeling of the miRNA profile, with coordinated upregulation and downregulation of miRNAs involved in inflammatory signaling, interferon regulation, apoptosis, autophagy and epithelial homeostasis. Integration of miRNA and proteomic datasets revealed inverse regulatory relationships linking ORF8-associated miRNAs to proteins involved in immune defense, stress responses and epithelial integrity. Selected miRNA alterations were validated by RT-qPCR, and the junctional protein plakoglobin (JUP) was experimentally confirmed as a representative downregulated target. Collectively, our findings identify ORF8 as a factor associated with coordinated miRNA-mediated post-transcriptional reprogramming in lung epithelial cells. This network-based regulatory framework provides new insight into how SARS-CoV-2 accessory proteins may influence host responses and highlights miRNA networks as potential contributors to viral pathogenesis and host immune dysregulation.