Ebola virus VP35 interacts non-covalently with ubiquitin chains to promote viral replication

Abstract

Ebolavirus (EBOV) belongs to a family of highly pathogenic viruses that cause severe hemorrhagic fever in humans. EBOV replication requires the activity of the viral polymerase complex, which includes the cofactor and Interferon antagonist VP35. We previously showed that the covalent ubiquitination of VP35 promotes virus replication by regulating interactions with the polymerase complex. In addition, VP35 can also interact non-covalently with ubiquitin (Ub); however, the function of this interaction is unknown. Here, we report that VP35 interacts with free (unanchored) K63-linked polyUb chains. Ectopic expression of Isopeptidase T (USP5), which is known to degrade unanchored polyUb chains, reduced VP35 association with Ub and correlated with diminished polymerase activity in a minigenome assay. Using computational methods, we modeled the VP35-Ub non-covalent interacting complex, identified the VP35-Ub interacting surface, and tested mutations to validate the interface. Docking simulations identified chemical compounds that can block VP35-Ub interactions leading to reduced viral polymerase activity. Treatment with the compounds reduced replication of infectious EBOV in cells and in vivo in a mouse model. In conclusion, we identified a novel role of unanchored polyUb in regulating Ebola virus polymerase function and discovered compounds that have promising anti-Ebola virus activity.

Fig 1. EBOV VP35 protein interacts with Ub non-covalently.

(A) WCEs from HEK293T cells transfected with Flag-VP35 (VP35) and HA-Ub WT or HA-Ub ΔGG (cannot conjugate proteins) were used for HA IP under non-denaturing conditions (RIPA washes), followed by IB. (B) Purified recombinant K48 or K63 polyUb chains (mix of 2–7 Ub chains) were mixed in vitro with Flag-VP35, followed by Flag IP. Interacting proteins were eluted with Flag peptide. (C, D) Experiments performed as in (B) but using the C-terminal IID domain of VP35 (C), or the VP35 K309R or K309G mutants, which are not covalently ubiquitinated (D). * No ubiquitinated VP35, possibly phosphorylation. The data underlying the graphs shown in the figure can be found in S1 Data. EBOV, Ebolavirus; IB, immunoblot; IID, IFN-inhibitory domain; IP, immunoprecipitation; WCE, whole-cell extract; WT, wild type.

Fig 2. Unanchored ubiquitin interactions with VP35 promote viral polymerase activity.

(A) WCE from HEK293T cells transfected with His-IsoT WT, His-IsoT C335A, VP35 WT, and HA-Ub were used for IP with anti-HA beads. (B) Polymerase minigenome assay. HEK293T cells transfected with a monocistronic firefly luciferase-expressing minigenome, including VP30, L, and REN-Luc/pRL-TK, in the presence or absence of IsoT-WT or C335A mutant. Data are expressed as mean + SEM of 3 independent assays in triplicate. Tukey’s multiple comparisons tests. ** p < 0.001. The percent of activity from the luciferase and renilla (Luc/ren) ratio was calculated. The data underlying the graphs shown in the figure can be found in S1 Data. IP, immunoprecipitation; WCE, whole-cell extract; WT, wild type.

Fig 3. Model of VP35 interacting with Ubiquitin.

(A) The complex of VP35 (PDB ID 3JKE) and Ubiquitin (PDB ID 1UBQ) modeled using a combination of protein docking and molecular dynamics simulations. Within the complex, VP35 is shown on the left and Ubiquitin on the right. The K48 and K63 Ub residues are shown in cyan on the bottom left and C-terminal on the right within Ub. (B) One of the strongest interactions contributing to the stability of the complex is ARG225-GLU18. (C) Mutation of ARG225 to GLU affects interactions. PDB, Protein Data Bank.

Fig 4. An intact R225 residue on VP35 is required for optimal interaction with Ub and viral polymerase function.

(A) HEK293T cells were transfected with minigenome plasmids and VP35 WT, VP35 R225E, or VP35 R225K, followed by Luciferase assay. (B) HEK293T cells were transfected with plasmids encoding Flag-VP35 WT, VP35 R225E, or VP35 R225K. WCE were then used to isolate Flag-tagged proteins using anti-Flag beads. After washes, the beads containing VP35 were mixed with the WCE containing HA-Ub to test binding. (C) As in B, but instead of mixing with WCE, binding was performed using purified recombinant K63-linked polyUb chains, followed by Flag elution. Quantification by densitometry of 3 independent experiments is shown. The data underlying the graphs shown in the figure can be found in S1 Data. WCE, whole-cell extract; WT, wild type.

Fig 5. VP35-PolyUb-dsRNA predicted complex.

(A) The predicted structure of the VP35-Ub complex was used as a template to superpose the structure of VP35 bound to RNA (PDB ID 3KS8). (B) PolyUb was modeled using as a template the structure of K63 Di-Ubiquitin (PDB ID 2JF5). The residues K63, K48, and G76 of the central Ub bound to VP35 are labeled in magenta and contribute favorably to RNA binding in this model. (C) In vitro competition binding assay. Increasing amounts of purified recombinant Ub K63 [–12] were incubated with VP35 and Biotin-polyI:C, followed by IP with anti-flag beads. (D) The mixes from (C) containing VP35-Ub-PolyI:C were treated with or without Rnase III followed by IP. The data underlying the graphs shown in the figure can be found in S1 Data. IP, immunoprecipitation; PDB, Protein Data Bank.

Fig 6. Predicted poses of small molecules disrupting the VP35-Ub interaction.

(A) The cavity within the interface with Ub with predicted bound pCEBS (B) and SFC (C) in close proximity to a sulfate ion (D) observed experimentally (PDB ID 4IBG). PDB, Protein Data Bank.

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 [–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.