Investigating the Antiviral Properties of Berbamine Against Murine β-coronavirus

Abstract

COVID‑19 remains a major cause of global morbidity and mortality despite widespread deployment of vaccines and direct‑acting antivirals, whose impact is limited by viral evolution, resistance, narrow treatment windows, and inequitable access. A host‑directed, broad‑spectrum antiviral could complement existing tools by targeting conserved host pathways and providing affordable, orally deliverable options for current and future β‑coronavirus threats. Berbamine, a plant‑derived bisbenzylisoquinoline alkaloid previously identified as a potent SARS‑CoV‑2 entry inhibitor with host‑modulatory activity, has not been fully characterized in β‑coronavirus systems or in relation to peroxisome‑linked antiviral immunity.This study investigates the antiviral properties of Berbamine against murine hepatitis virus strain A59 (MHV‑A59), a biosafety level‑2 model of β‑coronavirus infection that recapitulates key features of severe disease, including pneumonia and hepatitis, in mice. It also leverages prior evidence that MHV‑based platforms can predict natural‑product antivirals later shown to be active against human coronaviruses. The study further delineates peroxisome‑associated host pathways modulated by Berbamine to inform its translational development as a host‑directed antiviral. In vitro assays using murine cell lines assessed Berbamine cytotoxicity, prophylactic and therapeutic antiviral activity across a range of concentrations, and effects on viral replication and cell viability. Global transcriptomic profiling by RNA sequencing of infected cells with and without Berbamine treatment identified differentially expressed genes and enriched pathways. Candidate peroxisome‑related and innate immune genes, as well as organelle‑level phenotypes, were validated using immunofluorescence microscopy, RT‑qPCR, Western blotting, and reactive oxygen species assays. Berbamine reduced MHV replication in a dose‑dependent manner at non‑toxic concentrations and retained antiviral efficacy when administered before or shortly after infection, indicating pre‑exposure and early‑treatment antiviral potential. Transcriptomic and validation data demonstrate that Berbamine reprograms infection‑induced gene expression, modulates peroxisome‑associated and interferon‑stimulated gene networks, partially preserves peroxisome abundance and integrity in infected cells, and enhances expression of peroxisome‑linked antiviral genes. These findings support a dual mechanism of action that combines direct antiviral effects with host‑directed modulation of peroxisome‑dependent antiviral immunity and redox homeostasis. Collectively, this study establishes an MHV‑based experimental platform that integrates virology, transcriptomics, and organelle‑level readouts for preclinical evaluation of host‑directed antivirals. It positions Berbamine as a promising, orally deliverable host‑directed candidate and identifies peroxisome‑related pathways and genes as putative pharmacodynamic biomarkers to guide future in vivo studies and early‑phase clinical trials. These results have implications for pandemic preparedness, particularly for advancing equitable antiviral strategies that can be rapidly adapted to emerging and re‑emerging β‑coronaviruses and Disease X scenarios.