胆汁酸可以抑制肠道轮状病毒（Rotavirus)的复制

Inhibitory Effects of Bile Acids and Synthetic Farnesoid X Receptor Agonists on Rotavirus Replication

　

Yunjeong Kim，Kyeong-Ok Chang

 

轮状病毒（Rotaviruses ，A组轮状病毒）是全世界婴儿和儿童严重胃肠炎的最重要原因。目前，没有抗病毒药物，关于抗病毒药物开发的治疗靶标的信息仅限于轮状病毒感染。以前，研究表明脂质体内平衡在轮状病毒复制中很重要。由于法呢醇X受体（FXR）及其天然配体胆汁酸（如鹅去氧胆酸[CDCA]）在胆固醇和脂质稳态中起主要作用，我们检测了胆汁酸和合成FXR激动剂对轮状病毒复制与细胞脂质相关的作用水平。在轮状病毒感染的小鼠模型中，研究了口服CDCA对粪便轮状病毒脱落的影响。结果证明如下。首先，轮状病毒感染显着增加了甘油三酯的细胞内含量。其次，CDCA，脱氧胆酸（DCA）和其他合成的FXR激动剂，例如GW4064，以剂量依赖性方式显着减少细胞培养物中的轮状病毒复制。病毒复制的减少与FXR途径的激活和细胞甘油三酯含量的降低呈正相关（r 2 = 0.95）。第三，口服CDCA显着减少小鼠粪便病毒脱落（P <0.05）。我们得出结论，胆汁酸和FXR激动剂在抑制轮状病毒复制中起重要作用。提出抑制机制是轮状病毒感染引起的脂质合成的下调。

 

介绍

 

轮状病毒是无包膜的二十面体病毒，具有11段双链RNA基因组。轮状病毒颗粒含有六种结构蛋白，其包含核心（VP1至-3），内衣壳（VP6）和外衣壳（VP4和-7）。轮状病毒基于VP6分为7个形态学上无法区分但抗原性不同的血清群（A至G）（16）。 A组轮状病毒是全球婴儿和儿童严重胃肠炎的主要原因，与大约1.11亿次肠胃炎相关，需要2500万次就诊和200万次住院治疗，导致5岁以下儿童死亡超过500,000例（16）。即使有效的减毒活疫苗可用于人类轮状病毒感染（16），轮状病毒仍然是全世界婴儿和儿童胃肠炎的最重要原因。由于没有可用于轮状病毒感染的特异性抗病毒药物，轮状病毒介导的胃肠炎的治疗选择仅限于恢复和维持水合，直至感染消退（17）。因此，开发轮状病毒特异性药物对于降低疾病的严重性和轮状病毒相关住院治疗的持续时间是重要的。然而，关于轮状病毒感染的治疗靶标的信息是有限的。

 

以前，研究表明，通过抑制轮状病毒形态发生，脂筏和/或脂滴的破坏减少了感染性轮状病毒（7,10）。脂筏是富含鞘糖脂，胆固醇和蛋白质的专门膜结构域。脂滴是富含甘油三酯和胆固醇酯的主要脂质储存结构，并且在调节细胞脂质水平中起关键作用。病毒蛋白与这些亚细胞脂质体的相互作用对于某些病毒（包括人丙型肝炎病毒（30,32）和登革热病毒（38））中的感染性病毒颗粒形成也是重要的。这些发现表明，脂质体内平衡对某些病毒的复制很重要。

 

在发生轮状病毒复制的小肠中，脂质的从头合成和膳食脂质的吸收影响上皮细胞中的细胞脂质含量（19）。在肠腔中，胆汁酸乳化脂肪以形成胶束以帮助其吸收。胆汁酸由肝脏中的胆固醇合成，储存在胆囊中，并释放到十二指肠中。初级胆汁酸，胆酸（CA）和鹅去氧胆酸（CDCA）在肝脏中由胆固醇通过酶（包括胆固醇7α-羟化酶）合成，随后与牛磺酸或甘氨酸缀合以增强对酸和碱的亲和力。原发性胆汁酸被肠道细菌转化为二级胆汁酸，脱氧胆酸（DCA），石胆酸（LCA）和熊去氧胆酸（UDCA）（11）。当分泌的胆汁酸通过肠道时，它们在回肠中被重新吸收并通过门静脉返回肝脏（25）。这种肠肝循环对维持胆汁酸和胆固醇体内平衡的有效浓度至关重要。胆汁酸受体之一是法尼醇X受体（FXR）（12,27,34）。胆汁酸对FXR的激活诱导各种蛋白质的表达，包括小异二聚体配偶体（SHP），其抑制胆固醇7α-羟化酶（胆汁酸合成中的限速酶）的表达（27,34）。 FXR / SHP途径在肝细胞，肠细胞和肾细胞中发育良好，并且还参与脂肪酸（包括胆固醇）代谢和葡萄糖稳态的调节（40,41,44）。此前，我们小组报道胆汁酸在丙型肝炎病毒（HCV)和猪肠道杯状病毒的复制中起重要作用（5,6）。然而，胆汁酸和其他FXR激动剂在轮状病毒复制中的作用尚不清楚。

 

在这里，我们报告轮状病毒感染显着增加甘油三酯的细胞内含量。此外，我们证明生理浓度的CDCA和DCA以及低微摩尔浓度的合成FXR激动剂GW4064以剂量依赖性方式显着降低细胞中的轮状病毒复制。病毒复制的减少与FXR / SHP途径的激活和细胞脂质含量（甘油三酯）的减少正相关。与未治疗组相比，口服CDCA显着降低轮状病毒感染小鼠模型中病毒脱落的峰值量（P <0.05）。我们得出结论，胆汁酸和FXR激动剂在抑制轮状病毒复制中起重要作用，并且提出抑制机制是轮状病毒感染诱导的脂质合成的下调。

Pathogenesis and Immunity
Inhibitory Effects of Bile Acids and Synthetic Farnesoid X Receptor Agonists on Rotavirus Replication
Yunjeong Kim, Kyeong-Ok Chang

Rotaviruses (group A rotaviruses) are the most important cause of severe gastroenteritis in infants and children worldwide. Currently, an antiviral drug is not available and information on therapeutic targets for antiviral development is limited for rotavirus infection. Previously, it was shown that lipid homeostasis is important in rotavirus replication. Since farnesoid X receptor (FXR) and its natural ligands bile acids (such as chenodeoxycholic acid [CDCA]) play major roles in cholesterol and lipid homeostasis, we examined the effects of bile acids and synthetic FXR agonists on rotavirus replication in association with cellular lipid levels. In a mouse model of rotavirus infection, effects of oral administration of CDCA on fecal rotavirus shedding were investigated. The results demonstrate the following. First, the intracellular contents of triglycerides were significantly increased by rotavirus infection. Second, CDCA, deoxycholic acid (DCA), and other synthetic FXR agonists, such as GW4064, significantly reduced rotavirus replication in cell culture in a dose-dependent manner. The reduction of virus replication correlated positively with activation of the FXR pathway and reduction of cellular triglyceride contents (r 2 = 0.95). Third, oral administration of CDCA significantly reduced fecal virus shedding in mice (P < 0.05). We conclude that bile acids and FXR agonists play important roles in the suppression of rotavirus replication. The inhibition mechanism is proposed to be the downregulation of lipid synthesis induced by rotavirus infection.


INTRODUCTION

Rotaviruses are nonenveloped, icosahedral viruses with an 11-segment double-stranded-RNA genome. Rotavirus particles contain six structural proteins, which comprise a core (VP1 to -3), an inner capsid (VP6), and an outer capsid (VP4 and -7). Rotaviruses are divided into 7 morphologically indistinguishable but antigenically distinct serogroups (A to G) based on VP6 (16). Group A rotaviruses are the leading cause of severe gastroenteritis in infants and children worldwide, associated with approximately 111 million episodes of gastroenteritis that have required 25 million clinic visits and 2 million hospitalizations and have resulted in over 500,000 deaths in children younger than 5 years of age (16). Even though effective live-attenuated vaccines are available for human rotavirus infection (16), rotavirus still remains the most important cause of gastroenteritis in infants and children worldwide. Since there are no specific antiviral drugs available for rotavirus infection, treatment options for rotavirus-mediated gastroenteritis are limited to restoration and maintenance of hydration until the infection resolves (17). Therefore, development of a rotavirus-specific drug is important to reduce severity of disease and duration of rotavirus-related hospitalization. However, information on the therapeutic targets for rotavirus infection is limited.

Previously, it was shown that disruption of lipid rafts and/or lipid droplets decreased infectious rotaviruses by inhibition of rotavirus morphogenesis (7, 10). Lipid rafts are specialized membrane domains enriched in glycosphingolipids, cholesterol, and protein. Lipid droplets are the major lipid storage structure enriched in triglycerides and cholesterol ester and play a crucial role in regulating cellular lipid levels. The interaction of virus proteins with these subcellular lipid bodies is also important for infectious virus particle formation in some viruses, including human hepatitis C virus (30, 32) and dengue virus (38). These findings suggest that lipid homeostasis is important for the replication of certain viruses.

In the small intestines, where rotavirus replication occurs, de novo synthesis of lipids and absorption of dietary lipids affect cellular lipid contents in the epithelial cells (19). In the intestinal lumen, bile acids emulsify fats to form micelles to aid their absorption. Bile acids are synthesized from cholesterol in the liver, stored at the gallbladder, and released into the duodenum. The primary bile acids, cholic acid (CA) and chenodeoxycholic acid (CDCA), are synthesized in the liver from cholesterol by enzymes, including cholesterol 7α-hydroxylase, and subsequently conjugated with taurine or glycine to enhance affinity to both acids and bases. Primary bile acids are transformed by intestinal bacteria into secondary bile acids, deoxycholic acid (DCA), lithocholic acid (LCA), and ursodeoxycholic acid (UDCA) (11). While the secreted bile acids travel through the intestinal tracts, they are reabsorbed in the ileum and returned to the liver via the portal vein (25). This enterohepatic circulation is essential in maintaining an effective concentration of bile acids and cholesterol homeostasis. One of the bile acid receptors is farnesoid X receptor (FXR) (12, 27, 34). The activation of FXR by bile acids induces the expression of various proteins, including small heterodimer partner (SHP), which represses the expression of cholesterol 7α-hydroxylase, the rate-limiting enzyme in bile acid synthesis (27, 34). The FXR/SHP pathway is well developed in hepatic, intestinal, and renal cells and also participates in the regulation of fatty acid (including cholesterol) metabolism and glucose homeostasis (40, 41, 44). Previously, our group reported that bile acids play an important role in the replication of hepatitis C viruses and porcine enteric caliciviruses (5, 6). However, the role of bile acids and other FXR agonists in rotavirus replication has been unknown.

Here, we report that the intracellular contents of triglycerides increased significantly by rotavirus infection. Furthermore, we demonstrate that CDCA and DCA at physiologic concentrations and a synthetic FXR agonist, GW4064, at a low micromolar concentration significantly reduced rotavirus replication in cells in a dose-dependent manner. The reduction of virus replication correlated positively with activation of the FXR/SHP pathway and reduction of cellular lipid contents (triglycerides). Oral administration of CDCA significantly reduced the peak quantities of virus shedding in the mouse model of rotavirus infection compared to those for the no-treatment group (P < 0.05). We conclude that bile acids and FXR agonists play important roles in the suppression of rotavirus replication, and the inhibition mechanism is proposed to be the downregulation of lipid synthesis induced by rotavirus infection.

https://jvi.asm.org/content/85/23/12570.full