Ebola virus VP35 interacts non-covalently with ubiquitin chains to promote viral replication
Fig 7
pCEBS and SFC compounds inhibit interactions between VP35 and K63-linked polyubiquitin chains and correlate with reduced viral polymerase activity and virus replication.
(A) Flag-VP35 bound to anti-Flag beads were incubated for 1 h at room temperature with different concentrations of pCEBS or SFC, followed by incubation with recombinant purified unanchored K63-linked polyUb chains [2–7]. VP35-Ub complexes were eluted with Flag-peptide and analyzed by Immunoblot. (B) 293T cells were transfected with minigenome components and 4 h post-transfection cells were treated with pCEBS and SFC compounds at different concentrations, and 50 h later cells were lysed for luciferase assay. (C) Cytotoxicity test (CyQUANT MTT Cell Viability Assay Thermo Fisher) using pCEBS and SFC at different dilutions (D) PR and (E) VYR assays, the cells were infected by 1 h and after 1 h the treatment was made with pCEBS, SFB compound, or DMSO: Dimethyl sulfoxide with the overlay. The number of plaques in each set of compound dilution were converted to a percentage relative to the untreated virus control. (F, G) 6-week-old BALB/c females uninfected and treated with PBS (Mock vehicle) (n = 5), uninfected treated with 100 mg/kg of SFC (n = 5) (Mock SFC), infected intraperitoneal (IP) with 100 PFU of maEBOV and treated with either vehicle (EBOV vehicle) (n = 10) or SFC (EBOV SFC) (n = 10). (F) Viral titers in serum of infected mice at days 2, 4, and 6 post-infection. No plaques were detected in the mock groups. (G) Clinical presentation of disease scored as 1: Healthy; 2: Ruffle fur and/or Lethargic; 3: scoring 2 + hunched posture; 4: Weight loss over 20% of initial weight or scoring 3 + unable to move when stimulated, unable to access food/water, or displaying a moribund appearance. The percent of activity from the ratio of luciferase and renilla (Luc/ren) was calculated. Data are depicted as mean + SEM of the 2 independent assays in triplicate. Tukey’s multiple comparisons tests. p < 0.001 **, p < 0.0001 ***, p < 0.00001 ****. The data underlying the graphs shown in the figure can be found in S1 Data. EBOV, Ebolavirus; PR, plaque reduction; VYR, virus yield reduction.