Intracellular Sequestration of the NKG2D Ligand MIC B by Species F Adenovirus

Abstract

The enteric human adenoviruses of species F (HAdVs-F), which comprise HAdV-F40 and HAdV-F41, are significant pathogens that cause acute gastroenteritis in children worldwide. The early transcription unit 3 (E3) of HAdVs-F is markedly different from that of all other HAdV species. To date, the E3 proteins unique to HAdVs-F have not been characterized and the mechanism by which HAdVs-F evade immune defenses in the gastrointestinal (GI) tract is poorly understood. Here, we show that HAdV-F41 infection of human intestinal HCT116 cells upregulated the expression of MHC class I-related chain A (MIC A) and MIC B relative to uninfected cells. Our results also showed that, for MIC B, this response did not however result in a significant increase of MIC B on the cell surface. Instead, MIC B was largely sequestered intracellularly. Thus, although HAdV-F41 infection of HCT116 cells upregulated MIC B expression, the ligand remained inside infected cells. A similar observation could not be made for MIC A in these cells. Our preliminary findings represent a novel function of HAdVs-F that may enable these viruses to evade immune surveillance by natural killer (NK) cells in the infected gut, thereby paving the way for the future investigation of their unique E3 proteins.

Keywords: MIC A and MIC B; NK cells; adenovirus species F; adenoviruses; enteric viruses; gut immune system; immune evasion; viral tropism.

Figure 1

Sequence alignment showing the coding potential of E3 regions of the most common HAdVs-A, -B, -C, -D, -E, and -F. The expected molecular mass of each gene product is indicated. Proteins with amino acid sequence homology, generally ~35%, have the same shade coding: 19.4K and 31.6K are unique to HAdV-F.

Figure 2

Immunofluorescence staining of HAdV pVIII protein in HAdV-F41-infected HCT116 cells. Cells infected with HAdV-F41 (MOI 0.5) at day 2 post-infection showing nuclear localization of the viral structural pVIII protein (red). Actin fibers and cell chromatin are presented in green and blue, respectively. Samples were analyzed under an Olympus BX51 IF microscope coupled with a CCD camera Acquired channels were merged using ImageJ software v1.53a. Uninfected cells or secondary Ab alone yielded no relevant signals.

Figure 3

Expression of MIC ligands in uninfected HCT116 cells. (a) Flow cytometry histograms showing levels of MIC A and MIC B on the surface and in the intracellular environment of uninfected HCT116 cells. Cells were harvested at day 2 and 4 in culture. Isotype Abs recommended by the manufacturer were used as negative controls. Sample were analyzed on a Gallios (Beckman & Coulter, Brea, CA, USA) flow instrument and analysis was done offline using FlowJo software v10. Histograms are gated on live cells, after exclusion of cell debris and aggregates. (b) Immunofluorescence staining of MIC A and MIC B in uninfected HCT116 cells at day 2 in culture, with DAPI in blue and MICs in green. Data are representative of three independent experiments.

Figure 4

Expression of MIC ligands in HAdV-F41-infected HCT116 cells. HCT116 cells were infected with HAdV-F41 (MOI 0.5) and levels of MIC A and MIC B were assessed on the cell surface and intracellularly by flow cytometry, on day 2 and 4 post-infection. (a) Flow cytometry histograms showing staining of HAd41-infected cells (Hexon⁺) using a 2Hx-2 monoclonal anti-hexon Ab [34] with further detection with a secondary anti-mouse-FITC Ab. (b) Flow cytometry histogram showing expression levels of MIC A and MIC B on the cell surface and intracellularly. Hexon^- populations were gated from uninfected samples. Dashed lines represent the MFI levels of MIC A or MIC B on uninfected samples. (c) Fold increases in expression of MIC A and MIC B upon HAdV-F41 infection were calculated as MFI^hexon+ / MFI^hexon-. ** p < 0.01 defined by t student test.

Figure 5

Immunofluorescence assay of MIC B in HAdV-F41-infected HCT116 cells. IF assay showing (a) uninfected and (b) HAdV-F41-infected HCT116 cells (MOI 0.5; day 2). HAdV-F41 was traced using a rabbit polyclonal anti-pVIII Ab and a secondary goat anti-rabbit-Rhodamine. MIC B detection was performed using a mouse anti-MIC B Ab followed by incubation with a goat anti-mouse-FITC. Cell nuclei were counterstained with DAPI. Arrows show HAdV-F41-infected cells exhibiting stronger signals of MIC B compared to uninfected cells.