A cell-penetrating NS5B-specific nanobody inhibits bovine viral diarrhea virus replication

Bovine viral diarrhea virus (BVDV) causes one of the significant devastating diseases for the cattle industry worldwide. The virus can cross the placenta and result in the persistent infection of the fetus, which has hampered the efficacy and the development of vaccines. Hence, efficient antiviral strategies are urgently needed. In our previous work, a specific nanobody Nb1 against nonstructural protein 5 (NS5B) was successfully isolated, and the replication of BVDV was significantly reduced in the MDBK cell line stably expressing Nb1. Nevertheless, the Nb1 protein itself cannot enter the cells autonomously, which has severely hampered the application of the Nb1. In this work, Nb1fuses with a trans-activating transduction (TAT) peptide to form the TAT-Nb1. The TAT-Nb1 was expressed in Escherichia coli, and then purified by Ni-nitrilotriacetic acid (Ni-NTA) resin. We showed that TAT successfully delivered Nb1 into the MDBK cells, and the TAT-Nb1 efficiently inhibited the replication of BVDV in a dose-and time-dependent manner. Furthermore, the recognition site of Nb1 to NS5B was identified as NS5B_aa186-487 by the yeast two-hybrid assay. In summary, this study indicates that the TAT-Nb1 can potentially to be an antiviral drug against BVDV infection, and this research may accelerate the process of Nb1 for clinical use.