Analysis of E/N association. (A) E/N association is RNA independent. 293 T cells were co-transfected with the designated constructs. At 48 h post-transfection, equal amounts of the cell lysates were treated with or without 0.2 mg/ml DNase-free RNase A for 30 min at 25°C followed by mixing with glutathione-agarose beads. Complexes bound to beads were pelleted, washed, and subjected to Western immunoblotting. (B-D) Mapping of SARS-CoV N domains involved in E association. (B) Schematic representation of GST-N fusion constructs. PCR-amplified fragments containing various portions of SARS-CoV N coding sequences were fused to the carboxyl terminus of GST, directed by a mammalian elongation factor 1a promoter. (C) Association of GST-N fusion proteins with SARS-CoV E. 293 T cells were co-transfected with SARS-CoV HA-E and indicated GST-N fusion construct. Cells and supernatants were collected at 48 h post-transfection, prepared, and subjected to Western immunoblot analysis. (D) Co-precipitation of SARS-CoV E with GST-N fusion proteins. Equivalent amounts of cell lysates were analyzed by GST pull down assays as described above.