Western blot analysis of 3a protein. Human colonic adenocarcinoma Caco2 cells were infected with SCoV at a multiplicity of infection of 0.01, and cells were solubilized with sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample buffer (100 mM Tris-HCl [pH 6.8], 4% sodium dodecyl sulfate, 0.2% bromophenol blue, 20% glycerol, and 200 mM beta-mercaptoethanol) at 5 days p.i. Cell extracts were applied to a sodium dodecyl sulfate-15% polyacrylamide gel, and Western blot analysis was performed using anti-3a antibody. Lane 1, SCoV-infected Caco2 cells; lane 2, mock-infected Caco2 cells. Arrow; major 31-kDa 3a protein.

Confocal microscopic analysis of 3a protein in SCoV-infected cells. Vero E6 cells growing in eight-well chamber slides (Lab-Tek, Naperville, Ill.) were infected with SCoV at a multiplicity of infection of 1 (A) or mock infected (B). At 24 h p.i., cultures were incubated overnight with 4% paraformaldehyde and then treated with 0.25% Triton X-100 for 15 min. Subsequently, cells were incubated with anti-3a antibody and goat anti-rabbit secondary antibody conjugated with Alexa Fluor 488 dye (Molecular Probes, Eugene, Oreg.). Cells were observed under the Zeiss LSM 510 UV META laser scanning confocal microscope in the University of Texas Medical Branch Infectious Disease and Toxicology Optical Imaging Core.

Immunogold labeling of 3a protein in SCoV-infected cells. Caco2 cells were infected with SCoV at a multiplicity of infection of 0.5, fixed at 48 h p.i., and embedded in LR White resin. Ultrathin sections of the cells were incubated with anti-3a antibody and goat anti-rabbit immunoglobulin G (heavy plus light) conjugated to 15-nm colloidal gold particles (Amersham Biosciences). (A) In a SCoV-infected cell the label is clearly associated with intracellular virus (v) and with the virions at the cell surface (arrows). An uninfected cell in the upper left corner is devoid of label. n, nucleus. Bar = 1 μm. (B) 3a protein is associated with plasma membranes. V, SCoV particles associated with 3a protein. (C) Association of 3a protein with intracellular virus particles (arrows). (D) Portion of cytoplasm of a mock-infected Caco2 cell showing occasional staining of a few gold particles near the plasma membrane. Bars (B to D) = 0.5 μm.

Western blot analysis of N, M, and 3a proteins in purified SCoV. Caco2 cells were infected with SCoV at a multiplicity of infection of 1, and culture fluid was collected at 5 days p.i. Released SCoV was purified by sucrose gradient centrifugation as described in the text. (A) Ten fractions from a 20 to 60% sucrose gradient containing the virus particles were collected and numbered from bottom (B) to top (T) of the gradient. The top panel represents the density of each sucrose fraction. IC, intracellular proteins from SCoV-infected Caco2 cells. (B) Purified SCoV (lane 1), cell extracts from SCoV-infected Caco2 cells at 5 days p.i. (lane 2), and uninfected Caco2 cells at 5 days p.i. (lane 3) were analyzed for actin protein, N protein, 3a protein, and SCoV nsp1 protein.