Figure 6.

Validation of the role of CHMP1A in viral, but not virus-like MLV particle release. (A) CHMP1A is dispensable for the release of FLAG-tagged MLV.Gag^FLAG/YFP. HuH-7 cells were treated with control siRNA or siRNA pools targeting CHMP1A. Cells were transfected with an YFP-tagged MLV.Gag construct carrying a FLAG tag within the p12 domain. MLV.Gag^FLAG/YFP expression (Cell) and release (SPN) were assayed by FLAG-specific WB prior to GFP-specific immunoblotting of the same blot. Non-specific bands stained by the antisera served as controls for gel loading (Load). The degree of VLP release was quantified by densitometric analysis and demonstrated in percent amount relative to control cells (n = 2); (B) CHMP1A is essential for MLV virion release in HEK 293T cells. Following treatment of HEK 293T cells with control siRNA or siRNA pools against CHMP1A or CHMP1B, they were transfected with MLV.WT^FLAG/HA. Intra- and extracellular Env and Gag levels were determined by anti-HA and anti-FLAG WB on separate blots. The anti-ß-Actin blot served as a loading control. For quantification of extracellular virions, Env- and Gag-specific signals were measured by densitometry, summed, and demonstrated in percent amount relative to control cells (n = 2).