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.

中文翻译：

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胆汁酸和神经酰胺克服了 GII.3 人诺如病毒在人肠内复制的进入限制。

人类诺如病毒 (HuNoVs) 在全世界所有年龄组中引起胃肠炎的散发性和流行性暴发。我们之前曾报道，干细胞衍生的人肠小肠 (HIE) 培养物支持多种 HuNoV 菌株的复制，并且某些菌株（例如 GII.3）仅在胆汁存在的情况下进行复制。胆汁的加热和胰蛋白酶处理不会减少 GII.3 的复制，表明胆汁中的非蛋白质成分是一种活性因子。在这里，我们表明胆汁酸 (BA) 对 GII.3 复制至关重要，并且复制与 BA 疏水性相关。使用高效的 BA，糖鹅脱氧胆酸 (GCDCA)，我们显示 BAs 在感染的早期阶段起作用，HIE 中的 BA 依赖性复制不是由洗涤剂效应或经典的法尼醇 X 受体或 Takeda G 蛋白偶联受体 5 信号介导，而是涉及另一种 G 蛋白偶联受体、1-磷酸鞘氨醇受体 2，并且 BA 处理 HIE 会增加颗粒吸收。我们还证明 GCDCA 诱导促进 HIE 中 GII.3 复制的多种细胞反应，包括增强 1) 内体摄取，2) 内体酸化和随后的内体/溶酶体酶酸性鞘磷脂酶 (ASM) 活性，以及​​ 3) 神经酰胺水平顶膜。内体酸化抑制剂或 ASM 可减少 GII.3 感染，而仅外源性添加神经酰胺可导致感染。此外，抑制 ASM 的溶酶体胞吐作用，这是在顶端表面产生神经酰胺所必需的，减少 GII.3 感染。总之，我们的结果支持一个模型，其中 GII.3 利用快速 BA 介导的细胞内溶酶体动态变化和细胞神经酰胺进入并在空肠 HIE 中复制。