25-Hydroxycholesterol Mediates Cholesterol Metabolism to Restrict Porcine Deltacoronavirus Infection via Suppression of Transforming Growth Factor beta 1

Porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus in pigs, is one of the major pathogens for lethal watery diarrhea in piglets and poses a threat to public health because of its potential for interspecies transmission to humans. 25-Hydroxycholesterol (25HC), a derivative of cholesterol, exhibits multiple potential modulating host responses to pathogens, including viruses and bacteria, as well as pathogeninduced inflammation, while its antiviral effect on PDCoV and how it mediates the biological process of host cells to counter against infections remain poorly understood. Here, we thoroughly explored the antiviral effect of 25HC on PDCoV infection and tried to elucidate the underlying mechanisms. 25HC showed no toxic effect in LLC-PK1 cells and exerted antiviral ability against PDCoV infection in vitro. The viral cycle and time-of-addition analyses showed that 25HC mainly restricted the early and middle periods of the PDCoV postentry stage to inhibit infection. 25HC regulated disordered cholesterol metabolism induced by PDCoV infection and stimulated interferon-related lipid droplet accumulation. Transforming growth factor beta 1 (TGF-beta 1), screened by bioinformatic analyses, seemed to play an important role in PDCoV infection and was downregulated by 25HC One interesting finding is that inhibition of TGF-beta 1 with the inhibitor asiaticoside exhibited a similar antiviral capacity to 25HC and demonstrated regulation of cholesterol metabolism. Taking all of the findings together, we verified the antiviral effect of 25HC on PDCoV through interference with cholesterol metabolism, which may be related to its suppression of TGF beta 1. IMPORTANCE As an emerging enteropathogenic coronavirus in pigs, porcine deltacoronavirus (PDCoV) causes giant economic loss in the pig industry because of lethal diarrhea and possesses the potential for transmission from animals to humans. Several pieces of evidence have suggested the antiviral potential of cholesterol-25-hydroxylase and importance of cholesterol in viral infection. This study reports that 25-hydroxycholesterol (25HC) significantly restricted PDCoV infection through modulation of cholesterol metabolism, and we identified that lipid droplets play important roles in interferon response against virus infection. Moreover, this study identified the importance of TGF-beta 1 in CoV infection by bioinformatic analysis and verified that the inhibition of TGF-beta 1 showed anti-PDCoV capacity. Moreover, we uncovered the relationship between TGF-beta and cholesterol metabolism initially. Given that the importance of cholesterol in viral infection, 25HC has a great potential to treat PDCoV infection and TGF-beta 1 can be a crucial antiviral target.