The crucial role of bile acids in the entry of porcine enteric calicivirus

Abstract

Replication of porcine enteric calicivirus (PEC) in LLC-PK cells is dependent on the presence of bile acids in the medium. However, the mechanism of bile acid-dependent PEC replication is unknown. Understanding of bile acid-mediated PEC replication may provide insight into cultivating related human noroviruses, currently uncultivable, which are the major cause of viral gastroenteritis outbreaks in humans. Our results demonstrated that while uptake of PEC into the endosomes does not require bile acids, the presence of bile acids is critical for viral escape from the endosomes into cell cytoplasm to initiate viral replication. We also demonstrated that bile acid transporters including the sodium-taurocholate co-transporting polypeptide and the apical sodium-dependent bile acid transporter are important in exerting the effects of bile acids in PEC replication in cells. In summary, our results suggest that bile acids play a critical role in virus entry for successful replication.

Keywords: Bile acids; Bile transporters; Endosomal escape; Porcine enteric calicivirus; Virus entry.

Fig. 1

The effects of GCDCA on the one-step replication of PEC. (A) Confluent LLC-PK cells were infected with PEC at an MOI of 50 in the presence or absence of GCDCA (100 µM) at 37 °C for 1 h. Viral RNA was extracted from the cells at 5 min, 4 h, 8 h and 12 h PI for real-time qRT-PCR, and genome copy numbers were calculated by plotting Ct values against a standard curve generated using a series of dilutions of in-vitro transcribed PEC RNA genome. The graph shows PEC genome copy numbers in the samples collected at different time points. Numbers above the symbols indicate virus titers determined by the TCID₅₀ method (log₁₀ TCID₅₀/ml). (B) The TCID₅₀ values of the virus-infected cells incubated with or without GCDCA were determined at 16 h PI. An asterisk indicates a significant difference between the groups (p<0.05). (C) IFA in the cells infected with PEC with or without GCDCA (100 µM). Virus infected cells were fixed at 12 h PI and expression of viral proteins was determined by probing with antibodies specific to viral proteins 2AB, POL and VPg.

Fig. 2

Effects of bile acids on PEC replication with time-of-addition assay. Confluent LLC-PK cells were infected with PEC at an MOI of 50. GCDCA (100 µM) was added at different time points during virus replication. (A) A schematic drawing shows various time periods of GCDCA treatments. The bars indicate the time during which GCDCA was present in the media. (B) Virus replication was quantified by real time qRT-PCR at 6 h and 12 h PI. Asterisks indicate significant (p<0.05) difference in virus genome levels, compared to those of PEC infection without GCDCA.

Fig. 3

Transfection of PEC RNA into the permissible and non-permisible cells. (A) One-day old LLC-PK cells or MARC-145 cells were transfected with PEC RNA genome (0.5 µg/well) in the presence or absence of GCDCA (100 µM). At 16 h post-transfection, recovery of infectious virus was determined by the TCID₅₀ method. (B) One-day old LLC-PK cells were transfected with PEC RNA genome or medium (mock) and incubated without GCDCA. At 12 h post-transfection, the cells were fixed and probed with antibodies for viral proteins 2AB, POL and VPg in an IFA assay.

Fig. 4

Confocal laser scanning microscopic examination of PEC entry. Confluent LLC-PK cells grown on Lab-Tek II CC² chamber slides were infected either with mock (medium) or PEC (MOI 50) in the presence or absence of GCDCA (100 µM), fixed and stained at 1 h PI (A) or at 4 or 6 h PI (B) and observed under a confocal laser scanning microscope. (A) Top panels (a–d) show the mock-infected cells without GCDCA; middle panels (e to l) show the cells infected with PEC with or without GCDCA; bottom panels (m–p) show the cells infected with PEC in the presence of GCDCA and chloroquine. (B) Top panels (a–d) show the cells infected with PEC without GCDCA and observed at 4 h PI; bottom panels (e–h) show the cells infected with PEC without GCDCA and observed at 6 h PI. (C) Cells were infected with PEC (MOI 50) for 1 h, then treated with GCDCA for 5 min (a–d), 15 min (e–l), 30 min (i–l) or 60 min PI .(A–C) Cells were fixed and probed with rabbit polyclonal anti-Rab7 and swine polyclonal anti-PEC VLP primary antibodies and detected by PerCP-Cy5.5 labelled goat-anti-rabbit antibody (red) and FITC labelled goat-anti-swine antibody (green). Nuclei were stained with sytox orange (5 µM) (pseudo colored blue), and merged images for PEC and Rab7 were prepared. In the merged images, colocalization of PEC (green) and Rab7 (red) appears in white by using ImageJ.

Fig. 4

Confocal laser scanning microscopic examination of PEC entry. Confluent LLC-PK cells grown on Lab-Tek II CC² chamber slides were infected either with mock (medium) or PEC (MOI 50) in the presence or absence of GCDCA (100 µM), fixed and stained at 1 h PI (A) or at 4 or 6 h PI (B) and observed under a confocal laser scanning microscope. (A) Top panels (a–d) show the mock-infected cells without GCDCA; middle panels (e to l) show the cells infected with PEC with or without GCDCA; bottom panels (m–p) show the cells infected with PEC in the presence of GCDCA and chloroquine. (B) Top panels (a–d) show the cells infected with PEC without GCDCA and observed at 4 h PI; bottom panels (e–h) show the cells infected with PEC without GCDCA and observed at 6 h PI. (C) Cells were infected with PEC (MOI 50) for 1 h, then treated with GCDCA for 5 min (a–d), 15 min (e–l), 30 min (i–l) or 60 min PI .(A–C) Cells were fixed and probed with rabbit polyclonal anti-Rab7 and swine polyclonal anti-PEC VLP primary antibodies and detected by PerCP-Cy5.5 labelled goat-anti-rabbit antibody (red) and FITC labelled goat-anti-swine antibody (green). Nuclei were stained with sytox orange (5 µM) (pseudo colored blue), and merged images for PEC and Rab7 were prepared. In the merged images, colocalization of PEC (green) and Rab7 (red) appears in white by using ImageJ.

Fig. 5

Role of bile acid transporters NTCP, ASBT and receptors FXR and TGR5 in PEC replication. (A) One-day old LLC-PK cells were transfected with siRNA of NTCP, ASBT, FXR, or irrelevant control. After incubation for 48 h following transfection, cells were infected with PEC at an MOI of 50, and incubated in the presence of GCDCA (100 µM) for 1 h. Virus infected cells were then further incubated following washing with PBS, and total RNA was collected at 12 PI to assess viral replication and gene knockdown by qRT-PCR. Asterisks indicate a significant (p<0.05) difference in the PEC genome levels, compared to mock (PEC). (B) The effects of an agonist of FXR (GW4064) or TGR5 (oleanolic acid) in PEC replication. Individual or combinations of agonists was added at various concentrations, and viral replication was monitored by real time qRT-PCR at 12 h PI.

Fig. 6

A proposed model for bile acid-mediated PEC replication in LLC-PK cells. In this model, PEC enters the cells via endocytic pathway [through unidentified receptor(s)] and reaches the late endosomes. In the presence of bile acids and bile acid transporters, PEC escapes from the late endosomes into the cytoplasm to initiate virus replication. In the absence of bile acids or bile acid transporters, PEC remains in the late endosomes/lysosomes and is destined to be degraded.