FIG. 2.
Complementation of rSARS-CoVΔ6 with plasmid DNA encoding p6. Vero E6 cells were grown in 24- or 6-well plates and transfected for 16 h with 1.0 or 4.0 μg pCAGGS-ORF6-HA plasmid, using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. As controls, cells were transfected with the same amount of empty vector. (A) Transfection efficiency was determined by an immunofluorescence assay using mouse anti-hemagglutinin antibody (Covance, Berkeley, CA), followed by Cy3-conjugated donkey anti-mouse antibody (Jackson Immunoresearch, West Grove, PA) (red). Nuclei were stained with Topro-3 (blue). (B) Sixteen hours after transfection, cells were infected with rSARS-CoVΔ6 at an MOI of 0.01 in serum-free Dulbecco's modified Eagle's medium. Viral titers were determined at 8 h p.i. by plaque assay on Vero E6 cells. *, P < 0.05 by Student's t test.