Cell entry of Bovine herpesvirus-1 through clathrin- and caveolin-mediated endocytosis requires activation of PI3K-Akt-NF-κB and Ras-p38 MAPK pathways as well as the interaction of BoHV-1 gD with cellular receptor nectin-1

Bovine herpesvirus-1 (BoHV-1) can infect all breeds of cattle and cause severe respiratory organs and genital tract diseases. However, the mechanism of BoHV-1 entering the cells remains unclear. In this study, we explored the mechanism of BoHV-1 entering MDBK cells. We found that the entry of BoHV-1 was blocked by NH4Cl and bafilomycin A1, indicating that BoHV-1 entry is dependent on the acidic environment of endosome. Specific inhibitor dynasore and small interfering RNA (siRNA) knockdown of dynamin-2 inhibited BoHV-1 entry, showing that dynamin is required in BoHV-1 entry. The results of specific inhibitor, siRNA knockdown and co-localization indicating clathrin- and caveolin- mediated endocytosis play a role in BoHV-1 entry. BoHV-1 infection was not affected by EIPA which is a specific inhibitor of macropinocytosis. In addition, we found that BoHV-1 triggered PI3K-Akt-NF-κB and Ras-p38 MAPK signaling pathways to induce clathrin-mediated and caveolin-mediated endocytosis at the early stage of BoHV-1 infection. BoHV-1 binding was sufficient to activate the endocytic signaling pathways and promote viral entry. These two signaling pathways were activated by transfection of viral gD protein, and were inhibited by deletion of viral gD protein and the siRNA knockdown of cellular receptor nectin-1. The results of co-localization indicating the entered BoHV-1 is traced to late endosomes via early endosomes. Our results suggested the interaction of viral gD protein and cellular receptor nectin-1 triggered the PI3K-Akt-NF-κB and Ras-p38 MAPK signaling pathways and induced clathrin-mediated and caveolin-mediated endocytosis to promote BoHV-1 entry into MDBK cells at the early stage of BoHV-1 infection.