CD36 is a receptor for human sapovirus

Figures

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  Fig. 1.. CD36 is required for HuSaV infection. (A) Volcano plot of differentially expressed genes (DEG) between susceptible (Caco-2MC and ME) and unsusceptible (PG and PJ) cell lines; red and blue indicate up- and down-regulated genes in susceptible cells, respectively. (B) Immunofluorescence staining of CD36 in Caco-2MC, CD36 KO, and KO + CD36 cells. Cells were fixed with 4% paraformaldehyde and stained with anti-CD36 antibody (red) and Hoechst 33342 (blue). CD36-positive cells were quantified relative to total nuclei in three randomly selected fields per sample. Scale bar, 50 μm. (C) Surface CD36 expression analyzed by flow cytometry. The x axis indicates CD36 expression, and the y axis indicates number of cells. (D) CD36 KO inhibits HuSaV infection. Cells were infected with HuSaV GI.1 AH20 [multiplicity of infection (MOI) 1] for 1 hour, washed, and cultured with 1 mM GCA. Viral RNA in the cell lysates was quantified by quantitative polymerase chain reaction (qPCR) at 0 and 3 days postinfection. (E) TCID50 of progeny virus at 3 days postinfection. (F) Flow cytometric detection of HuSaV VP1 expression in infected cells with HuSaV GI.1 AH20 (MOI 10, at 3 days postinfection) following…

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  Fig. 2.. CD36 permits HuSaV infection in nonpermissive cells. (A) CD36 KI enables HuSaV infection in nonpermissive cells (293T, HuTu80, and HCT 116). Cells were infected with HuSaV GI.1 AH20 (MOI 1) for 1 hour, washed, and cultured with 1 mM GCA. Viral RNA in the cell lysates was quantified by qPCR at 0 and 5 days postinfection. (B) Flow cytometric detection of HuSaV VP1 in CD36 KI cells infected with HuSaV GI.1 AH20 (MOI 10, 1 day postinfection) following permeabilization and costaining for CD36 and VP1. Quantification was performed using three technical replicates, with the percentage of cells within the gated population determined by flow cytometry (table S2). (C) CD36 KI enables HuSaV infection in nonhuman-derived cells (CHO-K1, Vero E6, and NIH-3T3). Cells were infected as in (A), and viral RNA was quantified by qPCR at 0 and 3 days postinfection. (D) Flow cytometric detection of HuSaV VP1 in CD36 KI cells as in (B), with quantification based on gated population (table S2). (E) TCID50 of progeny virus at 3 days postinfection determined using Vero E6/CD36 clone#16 (VeroC16) cells. (F) Serial passage of multiple genotypes in VeroC16. VeroC16 cells were infected with multiple HuSaV…

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  Fig. 3.. CD36 is essential for HuSaV susceptibility in human intestinal organoids. (A) CD36 KO inhibits HuSaV infection in ileum-derived organoids (Ileum-1). Ileum-1 and CD36 KO organoids were infected with HuSaV GI.1 AH20 (MOI 1) for 1 hour, washed, and cultured with 1 mM GCA. Viral RNA in cell lysates was quantified by qPCR at 0 and 3 days postinfection. (B) TCID50 of progeny virus at 3 days postinfection in Ileum-1 and CD36 KO organoids using VeroC16. (C) CD36 KO does not affect susceptibility to HuNoV (GII. 4). Organoids were infected for 1 hour, washed, and cultured for 3 days. Viral RNA in supernatants was quantified by qPCR. (D) Immunofluorescence staining of human intestinal tissue section with anti-CD36 antibody (red), anti-villin antibody (green), and Hoechst 33342 (blue). The region enclosed by the dashed box is shown at higher magnification in the right panel. Images were acquired at ×4 (left) and ×20 (right). Scale bar, 200 μm (left) and 50 μm (right). (E) Viral replication in Ileum-1, CD36 KO, and KI organoids infected with HuSaV GI.1 AH20 (MOI 1). Viral RNA was measured at 0, 1, and 4 days postinfection by qPCR. (F) TCID50 of progeny virus at 4 days postinfection in…

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  Fig. 4.. CD36 directly binds HuSaV particles. (A and B) Interaction of recombinant CD36 (r-hCD36) with viral VLPs analyzed by BLI. r-hCD36 was immobilized and tested against HuSaV GII.3 VLP (A) or HuNoV GII.4 VLP (B). (C) Binding of recombinant CD36 to HuSaV virions by ELISA. Purified HuSaV GI.1 (AH20) virions (0.025 μg/ml) were immobilized on plates, fixed, and incubated with serially diluted recombinant human CD36-Fc (r-hCD36-Fc) or control human IgG1-Fc (r-Fc). Bound CD36 was detected using horseradish peroxidase–conjugated goat anti-human IgG antibody and TMB substrate. (D) Blocking assay with r-hCD36-Fc. HuSaV GI.1 AH20 (MOI 0.1) was preincubated with r-hCD36-Fc or r-Fc and used to infect to Caco-2MC cells. Viral RNA in the cell lysates was quantified by qPCR at 1 day postinfection. (E) Immunofluorescence analysis of infection in blocking assay. Cells were fixed and stained with anti-VP1 antibody (green) and Hoechst 33342 (blue). Images are representative of three independent experiments. VP1-positive cells were quantified relative to total nuclei in three randomly selected fields per sample. Scale bar, 50 μm. (F) Blocking assay with anti-CD36 antibody. Caco-2MC cells were…

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  Fig. 5.. Bile acids facilitate CD36-mediated viral entry. (A) Schematic diagram of the time-of-addition assay for GCA. Gray arrows indicate GCA treatment of virus in the inoculum; black arrows indicate GCA treatment of cells. The concentration of GCA was 1 mM [hr., hours]. (B) Viral RNA levels under indicated condition. Cells were infected for 1 hour with GCA pretreated virus (conditions 1 and 2) or untreated virus (conditions 3 to 6), washed, and cultured with or without 1 mM GCA. Viral RNA in cell lysates was quantified at 1 days postinfection by qPCR. (C) Effect of GCA on early infection. Caco-2MC and CD36 KI cells (293T, HuTu80, and VeroE6 cells) were infected with HuSaV GI.1 AH20 (MOI 1) in the presence or absence of 1 mM GCA for 1 hour. After washing, cell-associated viral RNA was quantified at 1 hour postinfection by qPCR. Vero E6/CD36 KI cells, not VeroC16 cells, were used. (D) TCID50 of progeny virus following RNA transfection. HuSaV (GI.1 AH20) genomic RNA was transfected into Caco-2MC and CD36 KO cells, and progeny virus at 3 days posttransfection was quantified using Caco-2MC (left) or VeroC16 (right) cells. For (B) to (D), dotted lines indicate detection limits. Data are…

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  Fig. 6.. CD36-mediated HuSaV infection model in intestine.