Inhibitory effects of bile acids and synthetic farnesoid X receptor agonists on rotavirus replication

Abstract

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.

Fig. 1.

Effects of various bile acids on the replication of SA11 and Wa rotaviruses in MA104 cells. (A and B) The inhibition of virus replication in MA104 cells treated with DMSO (Mock) or various bile acids was evaluated by the TCID₅₀ method. The final concentration of all compounds was 100 μM for results shown in panel A. Each bar represents the log₁₀ TCID₅₀/ml (mean ± standard error of the mean [SEM]) (*, P < 0.05 compared to mock treatment). (C and D) Expression of rotavirus VP6 protein in rotavirus-infected MA104 cells treated with DMSO or various bile acids was analyzed by Western blot analysis. β-Actin was loaded as an internal control. (E) Immunofluorescence of MA104 cells infected by SA11 rotavirus and treated with DMSO or various bile acids at 100 μM. Rotavirus VP6 protein was detected with a monoclonal antibody followed by a fluorescein isothiocyanate-labeled secondary antibody. Cells were fixed at 12 h after virus inoculation.

Fig. 2.

Effects of various FXR agonists on the replication of SA11 rotavirus in MA104 cells. (A) The inhibition of virus replication in MA104 cells treated with DMSO or various agonists was evaluated by the TCID₅₀ method. Each bar represents the log₁₀ TCID₅₀/ml (mean ± SEM) (*, P < 0.05). (B) Immunofluorescence of MA104 cells infected by SA11 rotaviruses. Panel a shows cells without virus infection, and panel b shows cells infected with SA11 rotavirus without treatment. Panels c and d show virus-infected cells treated with GW4064 (2 μM) and 6-ECDCA (100 μM), respectively. Cells were fixed at 12 h after virus inoculation. (C) Western blot analysis of SA11 virus-infected cell lysates treated with DMSO (0.1%) or various concentrations of GW4064 for 24 h. β-Actin was loaded as an internal control. (D) Chemical structures of FXR agonists CDCA, 6-ECDCA, GW4064, and fexaramine.

Fig. 3.

Effects of CDCA, GCDCA, and GW4064 on the expression of SHP mRNA in SA11-infected MA104, Huh-7, or Caco-2 cells treated for 2 h. The bar graphs show fold changes in SHP mRNA levels compared to levels seen with no treatment (mock) (means ± SEMs) (*, P < 0.05). (B) Western blot analysis of SHP and rotavirus VP6 expression. MA104 cells were transfected with different concentrations of SHP-expressing plasmid (pCI-SHP) (0, 0.5, or 3 μg) for 24 h prior to virus infection. Cell lysates were prepared 8 h after virus inoculation. β-Actin was loaded as an internal control.

Fig. 4.

(A) Effects of CDCA and GW4064 on triglyceride concentration in MA104 cells infected with rotavirus. The bar graph values shown are the means ± SEMs of triglyceride concentrations (*, P < 0.05 compared to mock treatment). (B) Nile red and NBD-cholesterol fluorescence of cells with or without virus infection and CDCA (100 μM) treatment. (C) Effects of CDCA and GW4064 on triglyceride concentration in Huh-7 cells (means ± SEMs) (*, P < 0.05 compared to mock treatment). (D) The fluorescence signals by NBD-cholesterol in rotavirus-infected MA104 cells at 0, 2, 4, 6, and 8 h postinfection were monitored by a fluorometer. Virus-infected cells were incubated in the presence of NBD-cholesterol (1 μg/ml) and trypsin with or without CDCA (100 μM), and the fluorescence signals were detected at the indicated times (*, P < 0.05 compared to no treatment).

Fig. 5.

(A) Fecal viral RNA equivalent to log₁₀ TCID₅₀/ml in mice orally inoculated with SA11 rotavirus of various doses. (B) Standard curve generated by plotting the C_T value versus log₁₀ titer of virus (TCID₅₀/ml). (C) Quantitation of virus shedding in feces in mice inoculated with 1 × 10^10.2 TCID₅₀ (in 150 μl) SA11 rotavirus and treated with PBS (mock) or CDCA. Animals received 150 mg/kg/day of CDCA in three divided doses by oral gavage. Each bar represents the log₁₀ TCID₅₀/ml (mean ± SEM) (*, P < 0.05 compared to mock treatment).