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-B) Reverse genetics was used to generate recombinant (r)LCMV containing mutations at the two ubiquitination sites (K10 and K77) as well as a virus with a lysine-free (NoK) or a late domain-free (PPPY>AAAA) Z protein. The growth kinetics of these viruses were then measured by infecting A549 cells with these viruses and measuring the infectious titers by plaque assay (A) and DI particle titers by plaque interfering assay (B) at 1, 16, 24, 40, 48 and 72 hours after infection. (C) Summary of a two-way ANOVA with Holm-Sidak’s test for multiple comparisons used to compare the log-transformed mean titer values from the growth curve in (A-B). (D-E) Viral RNA was isolated from clarified cell culture media from the 72 hours post-infection time point in (A-B) and the quantities of genomic S-segment (D) or L-segment (E) vRNA were determined by quantitative real time RT-PCR. (F) The release of virus-like particles (VLPs) from HEK293T cells transfected with plasmids expressing SBP-tagged LCMV Z protein with the indicated mutations was measured. The graphs in (A-B and D-F) represent the mean values ± SEM of three independent experiments with two technical replicates each. A one-way ANOVA with Holm-Sidak’s test for multiple comparisons was used to compare the mean values to the WT control in (D-F). *p< 0.05, **p< 0.01, ***p< 0.001, ****p< 0.0001.