Figure 1. Tsg101 knockdown in MARV-infected cells results in reduced particle release.

MARV-infected Huh-7 cells (MOI of 1) were transfected twice with Tsg101-specific siRNA or control siRNA (1 h and 18 h p.i.). Cells and virus particles were harvested at 48 h p.i. (A) Quantification of Tsg101 protein level was performed by Western Blot in cells transfected with Tsg101-specific siRNA and control siRNA. Tsg101 levels in cells transfected with control siRNA (ctr.) were set to 100%. (B) Lysates and supernatants of MARV infected cells transfected with Tsg101-specific or control siRNA were subjected to Western Blot and analyzed for the presence of viral proteins NP and VP40 and Tsg101. (C) The 65 kDa form of Tsg101 is ubiquitinated. HEK293 cells were transfected with Tsg101-Flag and HA-Ubiquitin expression plasmids. At 48 h p.tr., cell lysates were subjected to immunoprecipitation with anti-HA-agarose. Cell lysates and precipitates were separated by SDS-PAGE and analyzed by Western Blot using HA- and Tsg101-specific antibodies. The position of the ubiquitinated Tsg101 (Ub-Tsg101) band is indicated by an arrow between 55 and 70 kDa. (D) Virus titers in the supernatants of MARV infected cells transfected with Tsg101-specific or control siRNA were determined by TCID₅₀ titration, p-value (_*, P≤0.05). (E) Phenotype of Tsg101 knockdown in MARV-infected cells. Huh-7 cells were infected with MARV and treated with Tsg101 specific or control siRNA and subjected to immunofluorescence analysis using a guinea pig anti-NP and secondary goat anti-guinea pig FITC-conjugated antibody. Grey boxes indicate marginal region of cells. Lower panels show higher magnification of boxed area, arrows indicate accumulation of nucleocapsids upon Tsg101 knockdown. Bars, 10 µm. (F) Western blot analysis of Tsg101 knockdown. Cells transfected with Tsg101-specific or control siRNA were analyzed by Western Blot using Tsg101- and tubulin-specific antibodies.