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) Plasmids expressing SBP-tagged LCMV Z WT, Z containing lysine to arginine mutations at each of the six lysine residues, Z-NoK, or an empty vector were transfected into HEK293T cells along with a plasmid expressing HA-tagged ubiquitin (HA-Ub) and then two days later Z was affinity purified and was detected with ubiquitin by two color western blotting. (B) Plasmids expressing HA-Ub and LCMV Z or an empty vector were transfected into HEK293T cells, affinity purified, and detected via western blotting as in (A), except the lysine mutant Z constructs that were used each contained only the one intact lysine residue indicated, or no intact lysines (NoK). (C) Plasmids expressing HA-Ub and an empty vector or LCMV Z WT, LCMV Z mutant with both lysines 10 and 77 mutated to arginine, LCMV Z-NoK, or a LCMV Z PPXY late domain mutant (PPXY/AAAA) were transfected into HEK293T cells, affinity purified, and detected as in (A-B). Each panel is representative of at least 3 independent experiments.