FIG 8.

Infectious SDeV particles are formed when I/1Ki-Δ cells are transfected with viral glycoproteins. (A) I/1Ki-Δ cells transfected with HISV-1 GPC (top row), Puumala virus glycoproteins (PUUV Gn&Gc, second row), UGV-1 GPC (third row), UGV-1 ZP and GPC (fourth row), HBV S-Ag (fifth row), and empty pCAGGS-MCS plasmid (bottom row) were stained for HA tag (anti-HA [1:4,000] and Alexa Fluor 594-labeled donkey anti-mouse immunoglobulin [1:1,000], left panels, red) and SDAg (α-SDAg-AF488, middle panels, green). Hoechst 33342 was used to visualize the nuclei. The panels on the right show an overlay of the two images. A 30-μm bar is shown in the bottom right corner. All images were taken at ×400 magnification using a Zeiss Axioplan 2 microscope. (B) Supernatants collected from I/1Ki-Δ cells transfected with empty pCAGGS-MCS plasmid, UGV-1 ZP, UGV-1 GPC and ZP, UGV-1 GPC, HISV-1 GPC, LCMV GPC, JUNV GPC, PUUV glycoproteins, and HBV S-Ag were pelleted by ultracentrifugation and analyzed by Western blotting. The left panel shows anti-SDAg staining, and the right panel shows anti-SDAg and anti-HA staining. (C) Supernatants collected from I/1Ki-Δ cells transfected with empty pCAGGS-MCS plasmid, UGV-1 ZP, UGV-1 GPC and ZP, UGV-1 GPC, HISV-1 GPC, LCMV GPC, JUNV GPC, PUUV glycoproteins, and HBV S-Ag were titrated on clean I/1Ki cells. The plasmid used for transfection is shown in the left column, and the corresponding SDeV titer (in fluorescent focus-forming units [fffus] per milliliter) is shown in the right column.