Figure 2.

Intracellular Retention of Orf7a

Vero cells were either mock infected ([A], [C], and [E]) or infected with SARS-CoV ([B], [D], and [F]) for 18 hr at an moi of 5. As expected, the spike protein was clearly present at the surface of the SARS-CoV-infected but not mock-infected cells ([A] and [B]). Incubation with the anti-orf7a monoclonal antibody 2E11 demonstrated only limited cell-surface expression of orf7a. An intracellular pool of orf7a was clearly evident after saponin permeabilization ([E] and [F]). The anti-SARS antibody is a mixture of hybridoma supernatants specific for SARS proteins and found to primarily recognize the S protein by Western blotting (a gift of Larry Anderson, CDC). Intracellular retention of orf7a does not require other viral proteins. When an orf7a cDNA is used to transiently transfect 293T cells, very little orf7a can be detected at the cell surface using 2E11 (G). Again, saponin pretreatment allowed staining of the intracellular orf7a (H). Addition of a C-terminal GFP tag did not significantly alter this intracellular distribution. A good colocalization was observed between orf7a staining (red) and GFP fluorescence (green) in this transfectant ([I] and [J]). The orf7a-GFP distribution was distinct from that seen for GFP alone (K).