FIG. 5.
Budding of VLPs with protein omissions. VLPs were prepared by transfecting 293T cells as described in Materials and Methods. Where indicated, plasmids encoding individual proteins were omitted from the transfection, the plasmids encoding the three polymerase proteins (PB1, PB2, and PA) were omitted (Pol−), or exogenous bacterial NA (exo NA) was added to the replacement media following transfection. The culture medium was harvested at 48 h p.t. and pelleted through a 30% sucrose cushion, and cells were prepared as described in Materials and Methods. Samples were analyzed by SDS-PAGE followed by immunoblotting to detect viral proteins. M1 protein in the 30% sucrose pellet was quantified by using the Odyssey infrared imaging system and normalized to the amount of M1 protein found in the cell lysate. The amount of M1 released from cells expressing all VLP proteins (WT, lane 1) was set to 1.0. Error bars represent standard deviations from the averages for three experiments.