25-Hydroxyvitamin D Inhibits Hepatitis C Virus Production in Hepatocellular Carcinoma Cell Line by a Vitamin D Receptor-Independent Mechanism

Previously, we have reported that the active vitamin D metabolite, calcitriol and vitamin D(3) (cholecalciferol), both remarkably inhibit hepatitis C virus production. The mechanism by which vitamin D(3) exerts its effect is puzzling due to the low levels of calcitriol produced in vitamin D(3)-treated Huh7.5 cells. In this study, we aimed to explore the mechanism of vitamin D(3) anti-hepatitis C virus effect. We show that vitamin D(3) activity is not mediated by its metabolic conversion to calcitriol, but may be due to its primary metabolic product 25(OH)D(3). This is inferred from the findings that 25(OH)D(3) could inhibit hepatitis C virus production in our system, and that adequate concentrations needed to exert this effect are produced in Huh7.5 cells treated with vitamin D(3). Using the CRISPR-Cas9 editing technology to knockout the vitamin D receptor, we found that the antiviral activity of vitamin D(3) and 25(OH)D(3) was not impaired in the vitamin D receptor knockout cells. This result indicates that 25(OH)D(3) anti-hepatitis C virus effect is exerted by a vitamin D receptor-independent mode of action. The possibility that vitamin D(3) and 25(OH)D(3), being 3β-hydroxysteroids, affect hepatitis C virus production by direct inhibition of the Hedgehog pathway in a vitamin D receptor-independent manner was ruled out. Taken together, this study proposes a novel mode of action for the anti-hepatitis C virus activity of vitamin D(3) that is mediated by 25(OH)D(3) in a vitamin D receptor-independent mechanism.