FIG. 3.

AIP1 is required for the incorporation of Mopeia virus NP into VLPs. (A) 293T cells were treated with siRNA for AIP1 or AllStar (nonspecific) siRNA or were left untreated. After 24 h, the treatment was repeated, and after another 24 h, the cells were cotransfected with pC-MopZ-HA and pC-MopNP-FLAG. VLP isolation and analysis with anti-HA and anti-FLAG antibodies by Western blotting were performed 48 h posttransfection with plasmids. The AIP1 silencing in the cell lysates was assessed with an anti-PDC6I (i.e., anti-AIP1) antibody, and an equal number of cells in each sample was confirmed with an anti-actin antibody. The VLP formation efficiencies were determined as described in the legend of Fig. 1C. The NP incorporation efficiencies were calculated as the amount of NP divided by that of Z in the VLP sample. The incorporation efficiencies for the NPs in the siRNA-treated samples are reported relative to the results for the untreated sample. The estimates of the VLP formation and NP incorporation efficiencies are averages of data from three independent experiments, and standard deviations are reported. (B) 293T cells were cotransfected with plasmids expressing Z and NP and increasing concentrations of DN AIP1. Forty-eight hours later, VLPs were subjected to ultracentrifugation, and their compositions were examined by Western blotting with anti-HA and anti-FLAG antibodies. The expression of dominant negative AIP1 was verified with an anti-PDC6I (i.e., anti-AIP1) antibody. The NP incorporation efficiencies were estimated as described above (A). (C) pC-MopZ-HA, pC-MopNP-FLAG, and increasing amounts of pC-FLAG-AIP1 were cotransfected into 293T cells. After 48 h, VLPs were pelleted and examined by Western blotting as described above (A).