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bioRxiv · 10.1101/2024.05.29.596392

The dual actions of the host miRNA-16a in restricting Bovine coronavirus (BCoV) replication through targeting host cell Furin and in enhancing the host immune response.

Shah, A. U.·Hemida, M. G.
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Abstract

1.The roles of host cell miRNAs have not been studied well in the context of BCoV replication and immune regulation. The main aim of this study was to identify some 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 the bovine endothelial cells (BEC) and Madine Darby Bovine Kidney (MDBK) cells. This is in addition to the ex vivo model using the peripheral bovine blood mononuclear cells (PBMC). We determined the miRNA expression profiles in these cells after BCoV infection. miRA-16a is one of the differentially altered during BCoV infection. Our data shows that miRNA-16a is a significantly downregulated miRNA in both in vitro and ex vivo models. We confirmed the miRNA-16a expression profile by the qRT-PCR. Overexpression of the pre-miRNA-16a in BEC and MDBK cell lines resulted in marked inhibition of BCoV infection based on the viral genome copy numbers measured by qRT-PCR, the viral protein expression (S and N) measured by Western blot, and the virus infectivity using plaque assay. Our bioinformatic prediction showed that Furin is a potential target for the miRNA-16a. We checked the Furin protein expression level in the pre-miRNA-16a transfected/BCoV infected cells compared to the pre-miRNA scrambled to validate that. Our data shows marked inhibition of the Furin expression levels on the mRNA levels by qRT-PCR and the protein level by Western blot. The BCoV-S protein expression was markedly inhibited on both the mRNA and protein levels. To further confirm the impacts of the downregulation of the Furin enzyme on the replication of BCoV, we used transfected cells with specific Furin-siRNA parallel to the scrambled siRNA. A 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 3UTR of the bovine Furin carrying the seed region of the miRNA-16a in the dual luciferase vector. Our data shows luciferase activity in the pre-miRNA-16a transfected cells decreased by more than 50% compared to the cells transfected with the construct carrying the mutated Furin seed region. Our data confirms miRNA-16a inhibits BCoV replication by targeting the host cell Furin and the BCoV-S glycoprotein. It will also enhance 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 for the miRNA-16a. Our findings highlight the clinical applications of the host miRNA-16a as a potential miRNA-based vaccine/antiviral therapy.

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Shah, A. U., Hemida, M. G.. 2024-05-29. The dual actions of the host miRNA-16a in restricting Bovine coronavirus (BCoV) replication through targeting host cell Furin and in enhancing the host immune response.. https://doi.org/10.1101/2024.05.29.596392

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