Abstract
The roles of host cell miRNAs have not been well studied in the context of BCoV replication and immune regulation. The main aim of this study was to identify miRNA candidates that regulate essential host genes involved in BCoV replication, tissue tropism, and immune regulation. To achieve these goals, we used two isolates of BCoV (enteric and respiratory) to infect bovine endothelial cells (BECs) and Madine Darby Bovine Kidney (MDBK) cells. This is in addition to the ex vivo model using peripheral bovine blood mononuclear cells (PBMCs). We determined the miRNA expression profiles of these cells after BCoV infection. The expression of miR-16a is differentially altered during BCoV infection. Our data show that miRNA-16a is a significantly downregulated miRNA in both in vitro and ex vivo models. We confirmed the miRNA-16a expression profile by qRT‒PCR. Overexpression of pre-miRNA-16a in the BEC and MDBK cell lines markedly inhibited BCoV infection, as determined by the viral genome copy numbers measured by qRT‒PCR, viral protein expression (S and N) measured by Western blot, and virus infectivity using a plaque assay. Our bioinformatic prediction showed that Furin is a potential target of miRNA-16a. We compared the Furin protein expression level in pre-miRNA-16a-transfected/BCoV-infected cells to that in pre-miRNA-scrambled-transfected cells. Our qRT‒PCR and Western blot data revealed marked inhibition of Furin expression at the mRNA level and at the protein level, respectively. BCoV-S protein expression was markedly inhibited at both the mRNA and protein levels. To further confirm the impact of the downregulation of the Furin enzyme on the replication of BCoV, we transfected cells with specific Furin-siRNAs parallel to the scrambled siRNA. Marked inhibition of BCoV replication was observed in the Furin-siRNA-treated group. To further validate Furin as a novel target for miRNA-16a, we cloned the 3'UTR of bovine Furin carrying the seed region of miRNA-16a in a dual luciferase vector. Our data showed that luciferase activity in pre-miRNA-16a-transfected cells decreased by more than 50% compared to that in cells transfected with the construct carrying the mutated Furin seed region. Our data confirmed that miRNA-16a inhibits BCoV replication by targeting the host cell line Furin and the BCoV-S glycoprotein. It also enhances the host immune response, which contributes to the inhibition of viral replication. To our knowledge, this is the first study to confirm that Furin is a valid target of miRNA-16a. Our findings highlight the clinical applications of host miRNA-16a as a potential miRNA-based vaccine/antiviral therapy.