Human Norovirus Triggers Primary B Cell Immune ActivationIn Vitro

Figures

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  FIG 1. HNoV replicates in primary B cells, and replication is restricted by interferon. (A) Primary B cells were extracted from whole blood and infected with GII.4- and GII.6-positive stool samples in 1.5-mL tubes. One hour after adsorption, cells were washed and incubated for 3 days at 37°C in coculture with hCD40L-3T3 cells. The graph represents the fold increases in viral genome copies obtained by RT-qPCR at 3 dpi versus 0 dpi. Each data point represents a biological replicate (different blood donor). (B) Primary B cells were infected with GII.4- and GII.6-positive stool samples in the absence or presence of 2′-C-methylcytidine (2′CMC). The graph represents fold increases in viral genome copies. Each dot is a single biological replicate. (C) Primary B cells were isolated from spleen and lymph nodes of patients’ biopsy specimens. The graph represents the fold increase in viral genome copies. Each dot is a single biological replicate (single donor), which is the average from three technical replicates. (D) Primary B cells from spleen and BJAB cells were treated with IFN-β prior to infection with HNoV-positive stool samples and viral loads determined as before. (E) Primary B cells from…

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  FIG 2. HNoV infection is more efficient in freshly isolated B cells. (A) Whole-blood-derived B cells were infected with GII.6 HNoV-positive stool samples at the moment of isolation or after 2 or 5 days of coculture with hCD40L-3T3 cells. The graph represents the fold increases in viral genome copies obtained by RT-qPCR at 3 dpi versus 0 dpi. Cell viability was determined on uninfected cells by flow cytometry as a percentage of live/dead cells after staining with Live/Dead fixable aqua dead cell stain. Each data point in the graph indicates a biological replicate from one donor (day 0, n = 11; day 2 and day 5, n = 8). Statistical tests were performed using GraphPad Prism, and the day 5 group is statistically different from the day 0 group by a Mann-Whitney test (P = 0.035). (B) Primary B cells from various donors were subjected to flow cytometry analysis after isolation (day 0) (n = 10) and at day 2 (n = 5) and day 5 (n = 5) of coculture with hCD40L-3T3 cells. The percentages of CD27− IgD+ (naive), CD27+ IgD− (memory-switched), CD27+ IgD+ (memory-unswitched), and CD27− IgD− (double-negative) B cells were calculated with FlowJo software and represent averages and standard deviations (SD)…

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  FIG 3. HNoV tropism is not restricted to a specific B cell subset. (A) Freshly isolated primary B cells were infected or mock infected with GII.6-positive stool samples and harvested at 3 dpi for flow cytometry analysis after staining with Live/Dead fixable aqua dead cell stain, PerCP/Cy5.5 anti-human CD20, PE-CF594 mouse anti-human CD27, Pacific Blue anti-human IgD, and biotinylated dsRNA (J2) for the detection of infected cells. B cells were also infected in parallel to determine the fold increase (FI) in viral replication by RT-qPCR. The graph shows the percentage of dsRNA-positive (dsRNA+) cells of infected B cells. Each data point is an independent biological replicate from a different donor. Permissive donors were defined according to a 3-FI or higher as defined by RT-qPCR. (B) Representative flow plots of infected and mock-infected B cells from permissive and nonpermissive donors. (C) Percentages of dsRNA+ cells in the different functional B cell subsets. The dsRNA+ gate was arbitrarily defined on the HNoV-infected B cells from nonpermissive donors to include ≤1% events. Percentages are calculated in the infected gate (4 to 10% of total B cells). Each data point on the graph…

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  FIG 4. HNoV infection or treatment with virus-like particles (VLPs) induces changes in B cell functional subset distributions. (A) Changes in B cell subsets expressed as a ratio of the percentages in HNoV-infected to noninfected (Mock) B cells. Permissive (n = 13) and nonpermissive (n = 13) donors were defined according to FIs in viral replication and/or <2% of dsRNA+ cells. Values close to 1 represent no or few changes, and values of >1 or <1 represent increases and decreases in the percentages of cells for each subpopulation, respectively. Statistical tests were performed using GraphPad Prism as Student’s t test on the ratio between permissive and nonpermissive donors. (B) Changes in B cell subsets upon treatment with IL-4 (20 ng/mL) for 3 days, expressed as a ratio of treated to nontreated (Mock) B cells. (C and D) Changes in B cell subpopulations 3 days after treatment with HNoV VLPs (0.1, 1, and 10 μg/mL) (C) or poly(I·C) (1 μg) (D), expressed as a ratio of treated to nontreated (Mock) B cells. Violin plots depict means and interquartile ranges, and each dot represents an independent biological replicate (single donor). Statistical tests were performed using GraphPad Prism as…

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  FIG 5. NS1 protein from genogroup GII is cleaved in vitro by caspase 7. (A) Fifteen percent SDS-PAGE gel of NT*HNoV NS1/2-TM cleaved with caspase 3 or 7. For the caspase 7 (Casp-7)-treated sample, the uncleaved protein is shown as band 1, and the cleavage products generated by caspase 7 treatment are shown as bands 2 to 5. The identities of protein bands 1 to 5 produced by caspase 7 treatment were confirmed by mass spectrometry and are shown schematically. The protein band schematics for the caspase 7-generated fragments (bands 2 to 5) include the molecular weight (MW) estimated by gel migration and by sequence prediction (gel estimated/predicted MW in kilodaltons). Using mass spectrometry, the C-terminal 4 amino acids of band 4 were determined to be SSSD, and the N-terminal 4 amino acids of band 5 were identified as GVSG, corresponding to two predicted caspase 7 cleavage sites in the full-length protein (SSSDGVLS and SAKGVSG), with the cleavage point indicated by the vertical arrows. The signal intensity of peptides detected by mass spectrometry plotted along the protein sequence supports the expected termini of bands 2 and 3. (B) Fifteen percent SDS-PAGE gel of NT*MNV NS1/2-TM cleaved…

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  FIG 6. NS1 treatment enhances B cell activation and metabolism. (A) Changes in B cell subpopulations 3 days after HNoV NS1 treatment (1 and 10 μg). (B) Primary B cells were collected and treated with HNoV NS1 (10 μg). Cells were harvested at 3 dpt and prepared for flow cytometry analysis to quantify the surface expression of the activation marker CD68. Cells were analyzed with a BD Fortessa instrument and FlowJo software. Each data point represents a technical replicate from 4 independent biological experiments. (C) Supernatants from primary B cells treated with HNoV NS1 (10 μg) were analyzed by ELISA. (D) A total of 106 primary B cells from three different donors were treated with HNoV NS1 protein (10 μg) for 16 h. Cells were harvested, and metabolites were collected in 80% ice-cold ethanol, followed by snapshot metabolomics analysis. Data are visualized by Volcano plot produced in GraphPad Prism, with the x axis representing the log2 fold change of selected metabolites under NS1-treated versus nontreated conditions and the y axis representing the −log10 P value for each metabolite. (E) Heat map of metabolites significantly upregulated (red) and downregulated (blue) under NS1…