NEDD4 family ubiquitin ligases associate with LCMV Z’s PPXY domain and are required for virus budding, but not via direct ubiquitination of Z
(A-C) Human lung carcinoma (A549) cells were reverse transfected with small interfering (si)RNAs to the indicated targets or a non-targeting control siRNA then two days later were infected with LCMV and cells and virus-containing cell culture media were collected after an additional two days. (A) Expression of siRNA-targeted proteins, the viral nucleoprotein (NP), and actin was measured in cells by western blot. Infectious titers were determined by plaque assay (B) and LCMV DI particle titers were measured by plaque interfering assay (C). (D-E) HEK293T cells were reverse transfected with siRNAs to the indicated targets or non-targeting control siRNA then two days later were transfected with a plasmid expressing SBP-tagged WT LCMV Z. (D) Expression of siRNA-targeted proteins was measured in cells by western blot and (E) the quantity of Z in cells and VLPs was determined by quantitative western blotting from which the percent VLP release was determined. (F-I) HEK293T cells were infected with LCMV and then treated after 1 hour with compound #5 (F-G) or #4 (H-I). Infectious titers and DI particle titers were measured from the cell culture supernatant collected two days after infection. (J-K) Release of VLPs from LCMV (J) or LASV (K) Z-transfected HEK293T cells treated with compound #4 was measured by quantitative western blotting. A one-way ANOVA with Holm-Sidak’s test for multiple comparisons was used to compare the mean values to the control siRNA (B, C and E) or the DMSO control (F-K). Data in (B-C and E-K) represent the mean ± SEM from four (B-C) or three (E-K) independent experiments. *p< 0.05, **p< 0.01, ***p< 0.001, ****p< 0.0001.