MHC class I expression in intestinal cells is reduced by rotavirus infection and increased in bystander cells lacking rotavirus antigen

Figures

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  Figure 1. Levels of cell-surface and total MHCI following rotavirus infection of HT-29 cells. Cells were mock-infected or infected with RRV at a multiplicity of infection (m.o.i.) of 1. After 16 h cells were fixed, stained with antibodies to MHCI (Surface) then permeabilized and stained with antibodies to rotavirus and analysed by flow cytometry. Representative dot plots of rotavirus-exposed cells with the Bystander and Infected gates indicated with a red line (a) and mock-infected cells with the Mock gate indicated similarly (b) are shown. The fluorescence intensity dot-plots were set so the mock-infected cell population fell centrally on the plot, to facilitate detection of both increased and decreased intensities in virus-exposed cells. (c) Representative histograms of rotavirus antigen levels on the combined (Bystander + infected) and gated (Bystander, Infected) populations of rotavirus-exposed cells, and the gated mock-infected cells (Mock). (d) The histograms of MHCI levels on the same cell populations analysed for (c). In (e), cells were infected and stained for cell surface MHCI as above (Cell surface), or fixed and permeabilized, stained for MHCI and rotavirus (Total), and…

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  Figure 2. Rotavirus NSP1 reduces MHCI expression in uninfected bystander cells. Cells were mock-infected or infected with SA11-4F, SA11-5S or A5-16 rotavirus at a m.o.i. of 1. After 16 h cells were fixed and permeabilized, stained for rotavirus and total MHCI, and analysed by flow cytometry as described in the legend to Fig. 1. The mean ± S.D. of the MHCI MFI from three biological replicates is shown.

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  Figure 3. Influence of rotavirus replicative ability on total MHCI expression in infected cells and bystander uninfected cells. Cells were mock-infected, infected with SA11-5S at a m.o.i. of 1, or exposed to a corresponding inoculum of inactivated SA11-5S (I-SA11-5S). After 16 h cells were fixed and permeabilized, stained for rotavirus (a) and total MHCI (b), and analysed by flow cytometry as described in the legend to Fig. 1. (c) The mean ± S.D. of the MHCI MFI from three biological replicates is shown.

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  Figure 4. Effect of rotavirus m.o.i. on total MHCI expression in infected cells and bystander uninfected cells. Cells were mock-infected or infected with SA11-4F at m.o.i. of 0.05, 0.25 or 1 for 16 h. Cells were fixed and permeabilized, stained for rotavirus and total MHCI, and analysed by flow cytometry as described in the legend to Fig. 1. The mean ± S.D. of the MHCI MFI from three biological replicates is shown.

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  Figure 5. Involvement of IFN receptor signalling in modulation of MHCI expression by rotavirus. (a) Cells left untreated (Untr.) or treated with IFN-α, IFN-γ or IFN-λ for 16 h were fixed and stained for total MHCI and analysed by flow cytometry. (b) Cells were mock-infected (Mock) or infected with SA11-5S at a m.o.i of 1 for 16 h, or left untreated or treated with IFNs as for (a), and analysed by Western blot for levels of STAT1 or phosphorylated STAT1 (pSTAT1). Full-length blots are presented in Supplementary Figure 3. (c) Cells were mock-infected or infected with SA11-5S at a m.o.i. of 1 in the presence of IFN-α and/or blocking antibodies to IFNAR1, or control antibodies. After 16 h cells were fixed and permeabilized, stained for MHCI and rotavirus, and analysed by flow cytometry. For (a,c), the mean ± S.D. of the MHCI MFI from three biological replicates is shown.

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  Figure 6. Transcriptional regulation of MHCI in rotavirus-infected and bystander cells. HT-29 cells were mock-infected or infected with RRV at a m.o.i. of 0.5 for 16 h, fixed with methanol and stained for rotavirus antigen and MHCI. Infected (MHCIlo) and uninfected (MHCIhi) cell populations in the RRV-infected cell cultures were collected by cell sorting (a). Total cellular RNA was extracted from the sorted cell populations and mock-infected cells. Relative levels of mRNA were determined by qPCR for HLA-A, HLA-B and NLRC5 (b), and IL-8 (c). The mean ± S.D. of triplicate analyses is shown.

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  Figure 7. Localization of activated STAT1 and IRF1 in rotavirus-infected and bystander cells. HT-29 cells were infected with RRV at a m.o.i. of 1 or mock-infected. After 7 h cells were fixed, permeabilised and stained with antibodies to rotavirus VP6 and phosphorylated STAT1 (pSTAT1; a,b) or VP6 and IRF1 (c,d). Nuclei were stained with DAPI and representative images obtained by confocal microscopy. The VP6-positive (infected) cells in the merged images are indicated with white asterisks. Nuclear to cytoplasmic fluorescence ratios (Fn/c) of pSTAT1 (b) and IRF1 (d) were calculated for the uninfected and infected cell populations, analysing 7 to 14 randomly selected cells of each population type from 2 independent experiments. Images were obtained at ×1000 magnification. ***P < 0.0001.