Figure 3. MVM production in cells expressing dominant-negative gelsolin mutants.

A9 cells and derivatives expressing dominant-negative gelsolin mutants (GlnY438A; GlnD565N) were infected with MVM (30 pfu/cell). (A) Infected cells were analyzed 48 h p.i. for the structure of their actin network by IF staining with rhodamine-phalloidin (upper panel) and by biochemical fractionation according to the solubility of actin filaments (lower panel). Cell extracts were treated with detergents in increasing amount and of increasing strength, and actin was quantified in the individual fractions by western blotting. LaminB served as a loading control. Fractions containing the most rigid filaments (i.e. insoluble components) are highlighted by dotted squares. Expression of the two gelsolin variants GlnY438A or GlnD565N, respectively, protect actin filaments from degradation through parvovirus MVM, i.e. exert a dominant-negative effect. (B) Cells and supernatants were collected separately at the indicated time p.i. Titers of cell-associated (cell) and released (medium) infectious virions were determined by standard plaque-assays on A9 cells and are expressed in plaque-forming units (PFU). Inactivation of gelsolin does not affect the production of infectious progeny particles, but inhibits egress of progeny virions into the medium supernatant.