Norovirus VLP Binding to Host Cells

The binding of norovirus VLPs to cells in vitro was assessed using HT-29 (human colorectal adenocarcinoma cells), a cell line with well-characterized H antigen and A antigen expression. In addition, Chinese hamster ovary (CHO) cells transfected with rat α1,2-fucosyltransferase B cDNA, with or without cotransfection with the rat A enzyme cDNA or B enzyme cDNA, were utilized. The CHO cells were generated as part of a previous study (11 (link)). A total of 2.5 × 10⁵ viable cells were incubated with 10 μg/ml VLPs in PBS-0.1% BSA for 1 h at 4°C. After 3 washes with the same buffer, a 30-min incubation was performed with anti-VLP antibody. After washes, a third incubation was performed with biotinylated anti-rat secondary antibody under the same conditions. The final incubation step used streptavidin-phycoerythrin (BD Pharmingen). After final washes in PBS alone, fluorescence analysis was performed on a FACSCalibur (Becton, Dickinson, Rungis, France) using the CELLQuest program. Blocking of VLP binding with synthetic oligosaccharides was achieved by preincubating 10 μg/ml VLPs with 400 μg/ml oligosaccharides for 1 h at 37°C. Fucosidase treatment of cells was achieved by incubation at 37°C with 10 μg fucosidase in 100 mmol/liter sodium phosphate buffer, pH 6.5, for 1 h.