Proviral Roles of Ebola virus VP35 Ubiquitination

Journal Title
Journal ISSN
Volume Title

Ebola virus’ structural protein VP35 is polyfunctional and plays vital roles in Ebola’s life cycle from antagonizing the host’s type I interferon pathway to acting as the polymerase co-factor. The mechanisms that regulate which function any given VP35 molecule engages remains unknown. Previously, we observed the host E3 ubiquitin ligase TRIM6 conjugates ubiquitin onto VP35 at lysine (K) 309. This post-translational modification was found to be proviral, but we did not know which VP35 role(s) ubiquitination regulated. We generated recombinant EBOVs encoding glycine (G) or arginine (R) mutations at VP35/K309 (rEBOV-VP35/K309G/-R) and show that both mutations prohibit VP35/K309 ubiquitination. The rEBOV-VP35/K309G mutant loses the ability to efficiently antagonize the IFN-I response, while the rEBOV-VP35/K309R mutant’s suppression is enhanced. The replication of both mutants was significantly attenuated in both IFN-competent and -deficient cells due to impaired interactions with the viral polymerase. The lack of ubiquitination on VP35/K309 or TRIM6 deficiency disrupts viral transcription with increasing severity along the transcriptional gradient. This dysregulation of the transcriptional gradient results in unbalanced viral protein production, including reduced synthesis of the viral transcription factor VP30. Blocking VP35/K309 ubiquitination enhanced interaction with the viral nucleoprotein and may trigger premature nucleocapsid packaging. Prior work also showed that at least one lysine residue other than K309 is ubiquitinated and that VP35 non-covalently interacts with ubiquitin. We observed that multiple residues in VP35’s N-terminus can receive covalent ubiquitin, including K119, 126, and 141. Substitution of K119-, 126-, and/or 141-to-R significantly attenuates VP35’s polymerase co-factor activity, but their mutation does not alter interactions with the viral polymerase or nucleoprotein. When looking into VP35’s non-covalent ubiquitin interaction, we found that VP35 specifically binds K63-linked ubiquitin chains via its C-terminus. The specific cleavage of unanchored ubiquitin chains, using the deubiquitinase isopeptidase T, impedes VP35’s non-covalent binding to ubiquitin and stunts EBOV’s polymerase activity. Finally, we also found that TRIM25, TRIM6’s relative, facilitates ubiquitin ligation onto VP35 and enhances VP35’s non-covalent interaction with ubiquitin. EBOV replication is attenuated 100-10,000 fold in cells lacking TRIM25. Overall, our data support that TRIM6- and TRIM25-mediated VP35 ubiquitination and VP35’s non-covalent interaction with unanchored ubiquitin is proviral.

Biology, Virology, Biology, Microbiology