Fig 7. Ubiquitination of VP35 at K309 impedes interaction with EBOV nucleoprotein.

(A) HA-NP (input) was added to beads bound with lysates from empty vector or FLAG-VP35 (wt, K309R, or K309G) transfected 293T cells, washed, and FLAG-eluted. Immunoblot quantification from two independent experiments. The binding ratio (HA-NP/FLAG-VP35) for each VP35 construct was divided by the ratio of wt VP35 to determine relative binding. (B) HA-NP and untagged wt VP35 were co-transfected into wt or T6-KO A549 cells and the WCE were immunoprecipitated with anti-HA beads. The blot quantifications are representative of two independent experiments. The binding ratio ((IP: VP35/HA-NP)/(WCE: (VP35/HA-NP)/Tubulin)) for lysates from wt and T6-KO cells was divided by the wt’s ratio to determine relative binding. (C) Diagram depicting the experiment set-up for the deubiquitinase experiment. When wild-type VP35 (pink rectangle) is expressed, several ubiquitin molecules will be ubiquitinated (white circle with ‘Ub’) at lysine 309. When co-expressed with catalytically active, wt ovarian tumor (OTU) deubiquitinase, the covalently linked ubiquitin will be cleaved from VP35. The catalytically inactive mutant, OTU-2A, has two key cysteine residues mutated to alanine and is not able to cleave ubiquitin from substrates. Lysates cells co-expressing VP35 and FLAG-OTU were added onto beads coated with either HA-NP (antibody molecule with green hexagon) or VP35-specific antibody (antibody with pink rectangle). (D) 293T cells were co-transfected with untagged VP35 (wt, K309R, or K-all-R) and empty vector or FLAG-OTU (wt or -2A). The WCE from VP35 FLAG-OTU co-transfected cells were incubated with the anti-HA (IP:HA), IgG-protein A, or anti-VP35-protein (IP: VP35) coated beads, bound with lysates from HA-NP or empty vector transfected cells to pulldown VP35 (IP:HA) or ubiquitin (IP: VP35). The western blot is representative of two independent experiments run in duplicate. (E) The area under the curve (AUC) for each protein from the western blots in panel D were calculated using ImageJ. The relative binding ratio (VP35 IP: (Ub/VP35)/WCE: (Ub/VP35)/tubulin) for VP35-associated ubiquitin was determined for each condition and divided by the ratio for wt VP35 without OTU treatment. (F) The area under the curve (AUC) for each protein from the western blots in panel D were calculated using ImageJ. The relative binding ratio (HA-NP IP: (VP35/HA-NP)/WCE: (VP35/tubulin) for VP35-NP binding was determined for each condition and divided by the ratio for wt VP35 without OTU treatment. The data analysis was done using a one-way ANOVA with Tukey’s post-test (A and B) or two-way ANOVA with Bonferroni’s post-test (E and F) for comparison between groups. P-value: *<0.05, **<0.01, ***<0.001, ****<0.0001; ns, not significant (p>0.05).