25-Hydroxycholesterol restricts human norovirus replication in human intestinal enteroids

Norovirus is the leading cause of acute gastroenteritis worldwide. Since the development of human intestinal enteroid (HIE) cell culture systems, significant advancements have been made in exploring and evaluating therapeutic strategies to combat human norovirus infections. Here, we investigate whether 25-hydroxycholesterol (25-HC), a key regulator of cholesterol homeostasis and bile acid production, can restrict human norovirus replication in jejunal HIE monolayers. Jejunal HIE monolayers derived from two donors (J2 and J3 cell lines) were treated with increasing concentrations of 25-HC (ranging from 0.0001 µM to 5 µM) for 24 hours. Monolayers were then infected with norovirus GII.1[P33] or GII.4 Sydney[P31] and incubated for 24 hours at 37°C and 5% CO₂. Viral RNA was quantified by reverse transcription-quantitative PCR at 1 hour post-infection (hpi) and 24 hpi. Regardless of HIE cell line or norovirus genotype, a dose-dependent restriction of human norovirus replication was observed after 25-HC treatment (0.07-1.23 log decrease). The top upregulated pathways associated with the 25-HC treatment included innate immunity and pro-inflammatory pathways, while the top downregulated pathways included cholesterol biosynthesis pathways. The 25-HC resulted in upregulation of genes associated with innate immune response, including C-C motif chemokine 2 (CCL2), CCL20, intercellular adhesion molecule 1 (ICAM1), and interleukin 23A (IL23A), and downregulation of genes related to cholesterol biosynthesis, including proprotein convertase subtilisin/kexin type 9 and acetyl-CoA acetyltransferase (ACAT2). Our data suggest that modulating the downstream signaling pathway of 25-HC has therapeutic potential as a strategy to control human norovirus infections and enhance antiviral immune responses. IMPORTANCE Human noroviruses are a leading cause of both epidemic and endemic acute gastroenteritis worldwide. Currently, there are no licensed vaccines or antiviral treatments available. The immune response to human norovirus infection remains incompletely understood. The development of the human intestinal enteroid (HIE) system has revolutionized norovirus research by enabling detailed investigations into viral replication, evaluation of potential control strategies, and analysis of host cellular responses involving both innate and adaptive immunity. In recent years, 25-hydroxycholesterol (25-HC) has emerged as a key regulator of cellular metabolism and antiviral defense. In this study, we investigated the antiviral effects of 25-HC on human norovirus using the HIE culture system. Our findings highlight the therapeutic potential of 25-HC in controlling human norovirus infections and enhancing antiviral immune responses, thereby contributing to a deeper understanding of host restriction mechanisms in viral infections.