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Bile acids promote the caveolae-associated entry of swine acute diarrhea syndrome coronavirus in porcine intestinal enteroids [1]

['Qi-Yue Yang', 'Key Laboratory Of Animal Virology Of Ministry Of Agriculture', 'Center For Veterinary Sciences', 'Zhejiang University', 'Hangzhou', 'People S Republic Of China', 'Yong-Le Yang', 'Department Of Veterinary Medicine', 'Yi-Xin Tang', 'Pan Qin']

Date: 2022-08 

Intestinal microbial metabolites have been increasingly recognized as important regulators of enteric viral infection. However, very little information is available about which specific microbiota-derived metabolites are crucial for swine enteric coronavirus (SECoV) infection in vivo. Using swine acute diarrhea syndrome (SADS)-CoV as a model, we were able to identify a greatly altered bile acid (BA) profile in the small intestine of infected piglets by untargeted metabolomic analysis. Using a newly established ex vivo model–the stem cell-derived porcine intestinal enteroid (PIE) culture–we demonstrated that certain BAs, cholic acid (CA) in particular, enhance SADS-CoV replication by acting on PIEs at the early phase of infection. We ruled out the possibility that CA exerts an augmenting effect on viral replication through classic farnesoid X receptor or Takeda G protein-coupled receptor 5 signaling, innate immune suppression or viral attachment. BA induced multiple cellular responses including rapid changes in caveolae-mediated endocytosis, endosomal acidification and dynamics of the endosomal/lysosomal system that are critical for SADS-CoV replication. Thus, our findings shed light on how SECoVs exploit microbiome-derived metabolite BAs to swiftly establish viral infection and accelerate replication within the intestinal microenvironment.

Bile acids (BAs), a commonly studied category of microbial metabolites, have long been acknowledged to have proviral or antiviral activities. Recent studies using different swine enteric coronaviruses (SECoVs) showed that BA play an important role in regulating viral replication in vitro. A mechanistic understanding of how BA regulates SECoV replication in small intestinal enterocytes is lacking. Herein, we utilized an emerging highly pathogenic SECoV, swine acute diarrhea syndrome (SADS)-CoV, which possesses the potential for zoonotic transmission, to investigate the crucial role of BA in modulating viral replication in porcine intestinal enteroids (PIEs). Our observations explain how BAs acts on epithelial cells to enhance SADS-CoV replication by inducing caveolae-mediated endocytosis and endosomal acidification, altering the dynamics of viral trafficking through the cellular endosomal/lysosomal system. Our results shed light on the role of BAs in the rapid establishment of SECoV infection within the intestinal microenvironment.

Funding: This research was funded by the National Key Research and Development Program of China (2021YFD1801103) to YWH, and supported by the National Natural Science Foundation of China (No. U21A20261 and No. 32172864) to SJZ and by the Laboratory of Lingnan Modern Agriculture Project (NG2022001) to YWH. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2022 Yang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

To confirm this hypothesis, we used untargeted metabolomics to profile small intestinal metabolites in SADS-CoV-infected piglets vs mock controls, discovering a series of BAs that were greatly enriched. Using porcine intestinal enteroids (PIEs) to mimic intestinal biology and physiology in vivo, we modeled the impact of BAs on SADS-CoV replication in intestinal enterocytes. We found that CA induces cellular changes that are of vital importance for SADS-CoV replication, including enhanced caveolae-mediated endocytosis (CavME), endosomal acidification and altered dynamics of the endo-lysosomal system. This novel role of BAs in promoting SECoV replication brings a new perspective on the establishment of viral infection in the intestinal microenvironment in vivo.

Primary BAs including cholic acid (CA) and chenodeoxycholic acid (CDCA) are synthesized from cholesterol-derived precursor molecules, conjugated to either taurine (mainly in rodents) or glycine (primarily in humans) within hepatocytes and excreted into the small intestines, where commensal bacteria deconjugate and convert them into secondary BAs, such as deoxycholic acid (DCA) and lithocholic acid (LCA) [ 23 ]. Approximately 95% of BAs are reabsorbed in the distal ileum and transported back to the liver to complete enterohepatic circulation. This makes the ileum the site of a large pool of various BAs with relatively high concentrations [ 24 ]. Thus, we hypothesized that SADS-CoV might take advantage of this BA-rich microenvironment to swiftly establish early infection and facilitate its spread in small intestinal epithelial cells.

A novel emerging pathogenic SECoV, swine acute diarrhea syndrome (SADS)-CoV, was first reported in suckling piglets with severe diarrhea in Guangdong, China in 2017 [ 16 , 17 ]. SADS-CoV preferentially infects the GI tract and causes clinical symptoms including acute vomiting and watery diarrhea [ 16 , 18 ]. This novel alphacoronavirus is most closely related to bat coronavirus HKU2 [ 16 , 19 ], and it is capable of infecting cell lines from several species including pigs, nonhuman primates and humans, raising the concern that it might possess the potential to jump to human beings [ 20 – 22 ]. The typical clinical presentation in the GI tract makes SADS-CoV a perfect model for the study of the critical role of BA in the regulation of SECoV replication.

Very few studies have focused on how BAs influence replication of swine enteric coronaviruses (SECoVs), though it was recently demonstrated that BAs increased infectivity of porcine epidemic diarrhea coronavirus (PEDV) strain icPC22A in Vero cells and the porcine small intestinal epithelial cell line IPEC-DQ [ 14 ]. However, a later study reported that BAs had antiviral activity against another SECoV, porcine deltacoronavirus (PDCoV), reducing its replication in LLC-PK1 and IPEC-J2 cells [ 15 ]. These seemingly contradictory outcomes suggest that BAs may modulate replication of distinct SECoVs very differently. Unfortunately, a mechanistic understanding of how BA regulates SECoVs replication in small intestinal enterocytes is still lacking.

Cumulative evidence supports the view that microbial metabolites can regulate enteric viral infection [ 3 – 5 ]. Among them, bile acids (BAs) have been shown to play crucial roles in enhancing replication of enteric viruses such as porcine sapoviruses (PoSaV), porcine enteric calicivirus (PEC) and noroviruses (NoVs) [ 6 – 8 ]. The replication of PEC in the porcine kidney cell line LLC-PK1 is dependent on the presence of BAs in the culture medium for at least two reasons: 1) BAs facilitate PEC escape from the endosome into cytoplasm for initiation of viral replication [ 8 ]; 2) BAs inhibit cellular innate immunity by downregulating phosphorylation of signal transducer and activator of transcription 1 (STAT1) upon PEC infection in LLC-PK1 [ 9 ]. Human NoV (HuNoV) subtype GII.3 replication in human intestinal enteroids (HIE) depends on the enhanced endosomal/lysosomal acidification and activation of sphinogomyelinase ASM caused by BAs [ 10 , 11 ]. The major capsid protein (VP1) of murine norovirus (MNV) binds to BAs, triggering a structural variation in the virion that enhances receptor binding and viral infectivity, as well as blocking antibody neutralization [ 12 , 13 ].

The mammalian gastrointestinal (GI) tract harbors an enormously diverse microbial community (termed ‘microbiota’) that develops a mutualistic relationship with its host, forming a complex ecosystem over millions of years of coevolution [ 1 ]. The intestinal microbiome generates immensely disparate metabolic products that can modulate host physiological activity and immune responses directly or indirectly [ 2 ]. Thus, infection by enteric viruses is not just a simple biological event between pathogen and host target cell, but rather a complicated process that takes place in the context of the intestinal microenvironment.

Results

SADS-CoV oral infection leads to a significant increase of BAs in the small intestine of piglets High viral titers and severe histopathological changes including diminishing capillaries and villous atrophy in the small intestines indicate that SADS-CoV infection is highly efficient and pathogenic in newborn suckling piglets [16,18,25]. Although previous studies did not profile the infection-related metabolites in the small intestine, we speculated that infection may result in perturbations to the gut microenvironment (i.e., redistribution of specific microbiota-derived metabolites) that would enhance SADS-CoV replication. Groups of 3-day-old SPF suckling piglets (n = 6) were orally inoculated with either vehicle or 3×10 5 PFU of SADS-CoV and monitored for viral shedding. At 7 days post-infection (dpi), coinciding with the peak of viral shedding (Fig 1A), animals were sacrificed, and proximal and distal segments of the small intestine were dissected and subjected to untargeted metabolomics analysis by liquid chromatography-mass spectrometry (LC-MS). As expected, the principal components analysis (PCA) revealed a distinct metabolomic profile in infected piglets from that of the negative controls (Fig 1B). Hierarchical clustering and KEGG analyses suggested that the increased differential metabolites in the small intestines of infected piglets were mainly enriched in lipid metabolism, more specifically primary bile acid biosynthesis (Fig 1C). Indeed, from a total of 364 metabolites that were remarkably upregulated (|log2 FC| >0.5, adjusted p < .05) in the small intestines of infected piglets, we discovered primary BAs such as CA, glycocholic acid (GCA), and secondary BAs like isohyodeoxycholic acid (isoHDCA) and tauroursodeoxycholic acid (TUDCA) (Fig 1D). We further compared the differentiated metabolites between the proximal and distal small intestine and discovered that SADS-CoV infection induced significantly higher level of CA, taurocholic acid (TCA) and isoHDCA in the distal, and to a lesser extent in the proximal small intestine (Fig 1E). On the contrary, the increase in GCA and TUDCA was less pronounced in the distal than proximal small intestine (Fig 1E). This finding suggests that SADS-CoV infection induces augmentation of BAs along the small intestine in a tissue-specific manner. To further confirm these results, a targeted metabolic profiling study was performed on BAs, finding a markedly higher absolute concentration of a series of BAs in the small intestine of infected compared to mock-infected piglets (Fig 1F). Collectively, the results from both metabolic profile analyses demonstrated that SADS-CoV infection greatly increased the concentration of BAs in the small intestine, which might be positively correlated with efficient viral replication and epithelial tissue damage. PPT PowerPoint slide

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TIFF original image Download: Fig 1. SADS-CoV oral infection leads to a significant increase of bile acids in the small intestine of piglets. (A) Kinetic of viral shedding in fecal swabs from sucking piglets orally infected with SADS-CoV (n = 6). (B) Principal components analysis (PCA) of the duodenum and ileum from SADS-CoV-infected and mock-infected piglets. (C) KEGG analysis of small intestinal metabolites in SADS-CoV-infected and mock-infected piglets. (D) Volcano plot of small intestinal metabolites in small intestine of SADS-CoV-infected and mock-infected animals. (E) Heatmap of small intestinal metabolites in the duodenum and ileum. (F) Small intestinal bile acid (BA) concentrations of SADS-CoV-infected and mock-infected piglets. P values were determined by unpaired two-tailed Student’s t test. *: p < .05; **: p < .01; ***: p < .001; ns, not significant. https://doi.org/10.1371/journal.ppat.1010620.g001

PIEs effectively support growth of SADS-CoV With the advantage of recapitulating the structural and functional features of natural intestinal epithelium in vivo, PIEs are useful as a novel ex vivo system to study the infections of SECoVs such as PEDV and PDCoV [26,27]. Therefore, we first generated three-dimensional (3D) enteroids from crypts harvested from the duodenum, jejunum or ileum of piglets and dissociate them into 2D enteroids monolayers (S1A and S1B Fig). As shown in S1C Fig, the 2D PIE cultures were stained positive with multiple cellular markers including villin (enterocytes), Ki-67 (proliferating cells), E-cadherin (epithelial tight junction) and chromogranin A (enteroendocrine cells). To evaluate whether 2D PIEs support SADS-CoV replication, we inoculated monolayers of duodenal, jejunal or ileal PIEs with a recombinant SADS-CoV expressing green fluorescent protein (GFP) under different Multiplicity of infections (MOIs) (0.01, 0.1 and 1). In ileal PIE, compared with 1 h post-inoculation (hpi), at 48 hpi the viral genome increased by 63-, 933- and 1175-fold and the virus titer reached 3.3, 5.14 and 6.02 log 10 , respectively, in an MOI-dependent manner (Fig 2A). A similar magnitude of replication was observed in duodenal and jejunal PIEs (S1D Fig). Cytopathic effect (CPE) such as cell rounding and syncytium formation were observed in SADS-CoV-inoculated ileal PIE cultures at 48 hpi (Fig 2B). Viral replication was further demonstrated by co-localization of the viral nucleocapsid N protein with nonstructural secreted GFP in infected cells by immunofluorescent assay (IFA) (Fig 2C). GFP signals were predominantly co-localized with cellular markers of E-cadherin, Ki-67 and villin, but not with chromogranin, indicating that SADS-CoV primarily infects and replicates in enterocytes instead of enteroendocrine cells (Fig 2D). Additionally, SADS-CoV infection exhibited comparable multi-step growth kinetics in all three PIE cultures, which showed a time course-dependent increase both in genomic RNA copies and infectious viral titers until a plateau was reached at 72 hpi (Fig 2E). Taken together, these results indicate that SADS-CoV infection of duodenal, jejunal or ileal PIEs results in an indiscriminately productive viral replication, and this novel infectious model can be used to investigate the relationship between microbiota-derived metabolites and SADS-CoV infection. PPT PowerPoint slide

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TIFF original image Download: Fig 2. Porcine intestinal enteroid (PIE) cultures support SADS-CoV replication. (A) Ileal PIE monolayers were incubated with medium or SADS-CoV-GFP at the indicated MOIs on a rotary shaker for 1 h at 37°C. The incubated monolayers were washed three times with PBS and harvested at 1 or 48 hpi. Supernatant RNA was extracted and the number of SADS-CoV-GFP genome copies was determined by RT-qPCR and viral titer was determined by a standard TCID 50 assay. (B) Cytopathic effect (CPE) was observed in ileal PIEs infected with SADS-CoV at 48 hpi (scale bar, 50 μm). (C) Immunofluorescence assay (IFA) was performed using rabbit anti-SADS-CoV-N polyclonal antibody and Alexa Fluor 594-conjugated anti-rabbit IgG as secondary antibody, and nuclei (blue) were visualized by DAPI (scale bar, 50 μm). (D) Colocalization of SADS-CoV-GFP and cellular markers (red) including E-cadherin (epithelial tight junction), Ki-67 (proliferating cells), villin (enterocytes) and chromogranin A (enteroendocrine cells), and nuclei (blue) stained by DAPI (scale bar, 50 μm). (E) Duodenal, jejunal and ileal PIE monolayers were incubated with SADS-CoV-GFP at MOI = 0.1 and the SADS-CoV-GFP replication kinetic was determined by RT-qPCR or TCID 50 . Data are from three independent experiments. P values were determined by unpaired two-tailed Student’s t test. *: p < .05; **: p < .01; ***: p < .001; ****: p < .0001; ns, not significant. https://doi.org/10.1371/journal.ppat.1010620.g002

BA-associated enhancement of SADS-CoV replication is not dependent on BA receptor signaling, innate immune pathways or viral binding One mechanism by which microbiota-derived metabolites regulate enteric virus infection is to skew the antiviral innate immune response. BAs are known to act on two major receptors: the membrane receptor, Takeda G protein-coupled receptor 5 (TGR5); or nuclear farnesoid X receptor (FXR) [28]. Cumulative evidence has demonstrated that BA signaling via TGR5 or FXR is linked to an anti-inflammatory response involving suppression of NF-κB activity and results in attenuated induction of proinflammatory cytokines in macrophages and monocytes [29,30]. To answer the question of whether CA-treated PIEs have an impaired capacity to limit SADS-CoV replication due to diminished antiviral innate immune responses via TGR5 or FXR signaling pathways, we first treated PIEs with different doses of TGR5 or FXR agonists (INT-777 or INT-747, respectively). No altered viral genomic RNA copies or increase in infectious viral progeny was observed upon SADS-CoV inoculation. Additionally, experiments were carried out treating PIEs with different doses of TGR5 antagonist triamterene or FXR antagonist guggulsterone, then subsequently treated with CA and infected with SADS-CoV. Comparable viral titers were seen between receptor antagonist-treated and untreated PIEs in the absence or presence of CA, implying that TGR5 or FXR signaling is dispensable for SADS-CoV replication or CA-mediated augmentation of viral replication (Figs 4A–4D and S3A and S3B). Next, we evaluated the expression of genes associated with innate immunity antiviral responses in SADS-CoV-infected PIEs, with or without CA treatment at 6 and 24 hpi. However, no significant reduction in the expression of these genes was observed in the CA-treated PIEs compared to NT controls following SADS-CoV infection (Fig 4E), indicating that CA-driven enhancement of SADS-CoV replication is likely unrelated to innate immunity suppression. PPT PowerPoint slide

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TIFF original image Download: Fig 4. Bile acid (BA)-associated enhancement of SADS-CoV replication is not dependent on BA receptor signaling, innate immune regulation or viral binding. Porcine intestinal enteroid (PIE) monolayers were infected with SADS-CoV-GFP at an MOI = 0.1 for 48 h. (A) TGR5 agonist INT-777 or (B) FXR agonist INT-747 was added to medium during and post-infection at the indicated concentrations. PIE monolayers were pretreated with (C) TGR5 antagonist triamterene or (D) FXR antagonist guggulsterone for 2 h, and then antagonists and cholic acid (CA) were added to the medium at the indicated concentrations during and after SADS-CoV-GFP infection for 48 h. (E) Expression of innate immune-associated antiviral response genes in PIE monolayers infected with SADS-CoV-GFP in the presence or absence of CA at 6 and 24 hpi. Gene expression was determined by qRT-PCR. (F) PIE monolayers were incubated with SADS-CoV-GFP or simultaneously with CA for the indicated time points at 4°C. Viral genome copies were quantified from unwashed cells to determine the amount of input virus and from washed cells to determine the amount of cell-attached virus. Data are reported as the percent of viral genomes remaining cell-associated compared with input. Data are from three independent experiments. https://doi.org/10.1371/journal.ppat.1010620.g004 As Nelson et al. reported that BAs are cofactors that enhance MNV cell-binding and infectivity [13], we next tested whether CA-mediated stimulation could be attributed to enhanced viral attachment to PIEs. No elevated percentage of viral attachment was observed at any of the time points in CA-treated PIEs compared to NT controls (Fig 4F). In summary, despite acting on PIEs at an early phase of SADS-CoV infection, it is unlikely for BAs to promote viral replication by skewing antiviral innate immune responses through TGR5 or FXR signaling or altering viral attachment.

BA-mediated enhancement of SADS-CoV replication is dependent on lipid rafts Having excluded a role for CA in the regulation of SADS-CoV attachment (Fig 4F), we next determined whether BAs affect viral entry in some other way. Since previous studies demonstrated that the entry of the avian CoV infectious bronchitis virus (IBV) is lipid raft-associated [31], we hypothesized that BA enhances the interactions between SADS-CoV and lipid rafts in order to facilitate viral entry. Indeed, pre-infection supplementation with methyl-β-cyclodextrin (MβCD), which impairs lipid rafts in the plasma membrane that are essential for membrane invagination and endocytosis, led to a significant decrease of SADS-CoV titers at 48 hpi in a dose-dependent manner. Additionally, the CA stimulatory effect was abrogated by MβCD supplementation, also in a dose-dependent manner (Figs 5A and S3C). To test whether this inhibitory effect was attributed to cholesterol depletion from lipid rafts, PIE monolayers were pretreated with MβCD, supplemented with exogenous cholesterol and subsequently infected with SADS-CoV in the presence or absence of CA. Impairment of the CA stimulatory effect by MβCD pretreatment was restored by cholesterol replenishment (Fig 5B), suggesting that BA-associated virus entry depends on intact lipid rafts and membrane cholesterol-mediated endocytosis. PPT PowerPoint slide

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TIFF original image Download: Fig 5. Bile acid (BA) promotion of SADS-CoV replication is dependent on lipid rafts. (A) Porcine intestinal enteroid (PIE) monolayers were pretreated with MβCD for 1 h, and then MβCD and cholic acid (CA) were added to the medium at the indicated concentration during and after SADS-CoV-GFP infection for 48 h. (B) PIE monolayers were pretreated with 1 mM MβCD for 1 h and supplemented with 1 mM cholesterol for 1 h, and then infected with SADS-CoV-GFP, CA and cholesterol were present during and post SADS-CoV-GFP infection. MβCD and CA-treated monolayers without cholesterol replenishment were set up as control. (C) PIE monolayers were treated with either medium alone, 100 μM CA, 100 μM UDCA or 1 mM propionate. Endocytic vesicles (red) were labeled by FM1-43FX and nuclei (blue) were visualized by DAPI (scale bar, 10 μm). Images were acquired by an LSM880 confocal laser-scanning microscope (Zeiss). Data are from three independent experiments; P values were determined by unpaired two-tailed Student’s t test. *: p < .05; **: p < .01; ns, not significant. https://doi.org/10.1371/journal.ppat.1010620.g005 Next, we further confirmed the role of BAs in modulating endocytosis in PIEs using the lipophilic dye FM1-43FX, which incorporates into the cellular membrane and stains endocytic vesicles migrating from the apical brush border. FM1-43FX staining exhibited a remarkable increase of labeled endocytic vesicles in the presence of CA, whereas no phenotype was observed with UDCA or propionate (Fig 5C), consistent with the finding that UDCA did not promote SADS-CoV replication in PIEs (Fig 3B). Together, these data indicate that CA enhances SADS-CoV replication and is associate with lipid raft and membrane cholesterol-mediated endocytosis.

BA enhances SADS-CoV entry via caveolae-mediated endocytosis In mammalian cells, multiple mechanisms are available for the endocytic internalization of virus particles including macropinocytosis, clathrin-mediated endocytosis (CME), caveolae-mediate endocytosis (CavME), and endocytic pathways independent on either clathrin or caveolae [32]. To determine the endocytic pathway on which BAs act to facilitate SADS-CoV replication, we employed diverse pharmacological drugs to block specific endocytic pathways in PIEs and assessed their effects on viral replication. The cytotoxicity of each drug was carefully evaluated by CCK8 assay (S3D Fig). Chlorpromazine (inhibits formation of clathrin-coated pits) or amiloride (specific inhibitor of Na+/H+ exchanger activity which is fundamental for macropinosome formation) did not hinder SADS-CoV replication or attenuate the stimulatory effect of CA in infected PIEs at 48 hpi (Fig 6A and 6B). The effects of these inhibitors were confirmed with respective controls (transferrin for chlorpromazine and 70kDa dextran for amiloride) (S3E and S3F Fig). PPT PowerPoint slide

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TIFF original image Download: Fig 6. Bile acid (BA) enhances SADS-CoV entry via caveolae-mediated endocytosis. Porcine intestinal enteroid (PIE) monolayers infected with SADS-CoV-GFP in the presence of (A) chlorpromazine (CME inhibitor), (B) amiloride (macropinocytosis inhibitor), (C) nystatin (CavME inhibitor) or (D) dynasore (dynamin 2 inhibitor) at the indicated concentration and harvested at 48 hpi. Chlorpromazine, amiloride, nystatin and dynasore were added during and post-infection; data are from three independent experiments. (E) Caveolin-1 immunogold electron microscopy of SADS-CoV entry. Caveolin-1 was labeled with 10-nm immunogold, as indicated by the green arrow; SADS-CoV is indicated by the blue arrow (scale bar, 100 nm). P values were determined by unpaired two-tailed Student’s t test. *: p < .05; **: p < .01; ***: p < .001; ns, not significant. https://doi.org/10.1371/journal.ppat.1010620.g006 Next, we treated PIEs with a CavME inhibitor, nystatin, and discovered that it blunted both SADS-CoV replication and the CA stimulatory effect in a dose-dependent manner, suggestive of a vital role of CavME in SADS-CoV entry and BA-associated SADS-CoV replication enhancement (Fig 6C). As previously documented, IBV entry is dependent on dynamin 2, which is a GTPase that facilitates membrane fission to generate endocytic vesicles in CME and CavME [31]. To delineate whether dynamin 2 is involved in CA-enhanced endocytosis, we used the specific inhibitor dynasore to block the formation of coated vesicles. Indeed, addition of dynasore diminished both SADS-CoV replication and the CA stimulatory effect in a dose-dependent manner (Fig 6D). Caveolin-1 immunogold electron microscopy (EM) further confirmed the involvement of CavME in SADS-CoV entry in PIEs. Caveolae regions were present on the cell membrane during SADS-CoV invagination (Fig 6E, left panel) and caveolin-1 colocalized with SADS-CoV virions (Fig 6E, right panel). Overall, these results demonstrate BAs may facilitate SADS-CoV entry by influencing CavME, and dynamin 2 is required for this effect.

BA enhances SADS-CoV replication through endosomal acidification Viruses depend on the decreasing pH of endocytic organelles as a cue to activate uncoating and penetration into the cytoplasm. Thus, it was necessary to test whether the effect of BA on SADS-CoV entry is dependent on low pH. As shown in Fig 7A, the significant effect of CA treatment on viral genomic RNA and viral titers was abrogated in the presence of the endosome acidification inhibitor NH 4 Cl in a dose-dependent way. We repeated the experiments with bafilomycin A1, a specific inhibitor of vacuolar H+-ATPase (V-ATPase) which inhibits endosomal acidification. Consistent with previous studies, viral genomic RNAs and viral titers were greatly reduced in bafilomycin A1-treated PIEs compared to NT controls with or without CA. The effect of bafilomycin A1 was dose dependent (Figs 7B and S3G). Further, we used LysoTracker, a fluorescent dye for labeling and tracking acidic organelles, to determine whether CA treatment significantly augments acidic endo-lysosomal compartments in PIEs. Unsurprisingly, in PIE monolayers only treated with CA, numerous acidic vesicles (LysoTracker-positive red staining) were distributed throughout the cytoplasm. In contrast, CA-treated PIE cultures supplemented with NH 4 Cl or bafilomycin A1 showed very few LysoTracker-positive signals (Fig 7C). These concordant results indicate an important role for endosomal acidification in CA-stimulated viral internalization at the early phase of infection. PPT PowerPoint slide

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TIFF original image Download: Fig 7. Bile acid (BA) enhances SADS-CoV replication through endosomal acidification. Porcine intestinal enteroid (PIE) monolayers were pretreated with (A) NH 4 Cl and (B) bafilomycin A1 at the indicated concentrations for 1 h before infection, and harvested at 48 hpi. NH 4 Cl and bafilomycin A1 were supplemented during and post-infection. (C) PIE monolayers were treated with either medium alone, 100 μM CA, 100 μM cholic acid (CA) plus 30 mM NH 4 Cl or 100 μM CA plus 200 nM bafilomycin A1 for 1 h, washed with PBS three times and incubated with 200 nM LysoTracker for 30 min at 37°C (scale bar, 10 μm). Images were collected on an LSM880 confocal laser-scanning microscope (Zeiss). Data are from three independent experiments. P values were determined by unpaired two-tailed Student’s t test; *: p < .05; **: p < .01; ***: p < .001; ns, not significant. https://doi.org/10.1371/journal.ppat.1010620.g007

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