Bovine coronavirus enters HRT-18 cells via membrane fusion and clathrin-mediated endocytosis in a low pH-, dynamin-, cholesterol-, microtubule-, Rab7-, and Rab11-dependent manner.

Bovine coronavirus (BCoV) infection poses a significant threat to the global cattle industry due to its dual tropism for the respiratory and intestinal systems, causing substantial economic losses. Elucidating the molecular mechanisms of viral entry is critical for developing targeted interventions against BCoV. This study systematically investigates the entry mechanisms of BCoV in HRT-18 cells through different methods. Our data reveal that BCoV entry into HRT-18 cells is dependent on membrane fusion and clathrin-mediated endocytosis (CME). This process is dependent on dynamin, cholesterol, microtubules, cathepsins, and low pH. In contrast, caveolin-mediated endocytosis, micropinocytosis, and TMPRSS2 do not contribute to BCoV entry. Furthermore, we identified Rab7 and Rab11 as key regulators of BCoV endocytosis. Silencing Rab7 and Rab11 significantly inhibited BCoV entry, while silencing Rab5 had no discernible effect. Confocal microscopy confirmed the co-localization of BCoV particles with Rab7 and Rab11, further supporting their role in the viral entry process. Collectively, our findings provide the first evidence that BCoV enters HRT-18 cells via membrane fusion and CME in a low pH-, dynamin-, cholesterol-, microtubule-, cathepsin-, Rab7-, and Rab11-dependent manner. These findings advance our understanding of BCoV pathogenesis and may facilitate the development of novel antiviral strategies against this pathogen.IMPORTANCEEmerging and re-emerging coronaviruses are causing severe epidemics in both humans and animals worldwide. Bovine coronavirus (BCoV) is a major pathogen causing severe diarrhea and respiratory disease in cattle, leading to substantial economic losses in the livestock industry. However, the molecular mechanism of BCoV entry into cells remains poorly understood. Here, we reveal that BCoV enters HRT-18 cells via membrane fusion and clathrin-mediated endocytosis, and acidic environment, dynamin, cholesterol, microtubules, cathepsins, Rab7, and Rab11 are also required. This study represents the first report on the mechanism of BCoV cell entry, which advances the understanding of BCoV infection pathogenesis and provides potential targets for the development of novel antiviral drugs.