Bile acids and ceramide overcome the entry restriction for GII.3 human norovirus replication in human intestinal enteroids

Abstract

Human noroviruses (HuNoVs) cause sporadic and epidemic outbreaks of gastroenteritis in all age groups worldwide. We previously reported that stem cell-derived human intestinal enteroid (HIE) cultures support replication of multiple HuNoV strains and that some strains (e.g., GII.3) replicate only in the presence of bile. Heat- and trypsin-treatment of bile did not reduce GII.3 replication, indicating a nonproteinaceous component in bile functions as an active factor. Here we show that bile acids (BAs) are critical for GII.3 replication and replication correlates with BA hydrophobicity. Using the highly effective BA, glycochenodeoxycholic acid (GCDCA), we show BAs act during the early stage of infection, BA-dependent replication in HIEs is not mediated by detergent effects or classic farnesoid X receptor or Takeda G protein-coupled receptor 5 signaling but involves another G protein-coupled receptor, sphingosine-1-phosphate receptor 2, and BA treatment of HIEs increases particle uptake. We also demonstrate that GCDCA induces multiple cellular responses that promote GII.3 replication in HIEs, including enhancement of 1) endosomal uptake, 2) endosomal acidification and subsequent activity of endosomal/lysosomal enzyme acid sphingomyelinase (ASM), and 3) ceramide levels on the apical membrane. Inhibitors of endosomal acidification or ASM reduce GII.3 infection and exogenous addition of ceramide alone permits infection. Furthermore, inhibition of lysosomal exocytosis of ASM, which is required for ceramide production at the apical surface, decreases GII.3 infection. Together, our results support a model where GII.3 exploits rapid BA-mediated cellular endolysosomal dynamic changes and cellular ceramide to enter and replicate in jejunal HIEs.

Genetic Manipulation of Human Intestinal Enteroids Demonstrates the Necessity of a Functional Fucosyltransferase 2 Gene for Secretor-Dependent Human Norovirus Infection

“…To assess whether these isogenic cell lines support HuNoV replication, we evaluated virus binding and subsequent replication of GII.4, GII.3, GII.17, and GI.1 HuNoV strains that we previously demonstrated replicate in HIEs (2). Since all of these viruses but GII.4 require bile for replication, all infections were performed in the presence of glycochenodeoxycholic acid (GCDCA), a bile acid that supports replication (21).…”

“…Similarly, HIEs derived from nonsecretor individuals are not susceptible to GII.4 HuNoV infection (2). HIEs and gastrointestinal epithelial cells of secretor-positive individuals also bind norovirus virus-like particles (VLPs) (17,21). However, while HIEs from secretors are permissive to HuNoV replication, FUT2 expression in conventional cancer-derived cell lines is not sufficient to make cells susceptible to HuNoV replication, suggesting that HBGAs function primarily as an initial attachment factor (18,19).…”

Vero Cells as a Mammalian Cell Substrate for Human Norovirus

“…Cell culture media supplemented with bile acids have been shown to aid porcine enteric calicivirus [88][89][90], murine NoV [91], and HuNoV [38,92,93]. Bile acids are thought to assist the virus to escape from endosomes into the cytosol [90,93,94]. Also, bile acids have been shown to bind to murine NoV [91] and HuNoVs in a genotype-or strain-specific manner [95].…”

Grand Challenge in Human/Animal Virology: Unseen, Smallest Replicative Entities Shape the Whole Globe

“…On one hand, more general subjects covering various pathogenic viruses and related research areas have been targeted as well. Of socially important pathogenic viruses, some are solely tropic for humans, HIV-1 as an example (Hatziioannou et al, 2006(Hatziioannou et al, , 2009Kamada et al, 2006;Nomaguchi et al, 2008Nomaguchi et al, , 2013b, and some like human norovirus are known not to replicate in cultured cells (Duizer et al, 2004;Herbst-Kralovetz et al, 2013;Ettayebi et al, 2016;Murakami et al, 2020). Indeed, these viral properties hamper and limit our experimental strategies, and are representing major study projects to overcome in the virology.…”

“…Alternative practical experimental systems, irrespective of being commonly useful and applicable to numbers of viruses or specifically to certain viruses, to circumvent the observed difficulties are definitely required. Expert researchers on the viruses have been making every effort to achieve the primary purposes (Kamada et al, 2006;Nomaguchi et al, 2011Nomaguchi et al, , 2013bSoll et al, 2013;Hatziioannou et al, 2014;Ettayebi et al, 2016;Doi et al, 2018;Schmidt et al, 2019;Murakami et al, 2020). The trend described above is understandable and wellrecognized by published articles and RTs in the human and animal virus field in the Virology section.…”