Ubiquitination of Ebola virus VP35 at lysine 309 regulates viral transcription and assembly

Ebola virus (EBOV) VP35 is a polyfunctional protein involved in viral genome packaging, viral polymerase function, and host immune antagonism. The mechanisms regulating VP35's engagement in different functions are not well-understood. We previously showed that the host E3 ubiquitin ligase TRIM6 ubiquitinates VP35 at lysine 309 (K309) to facilitate virus replication. However, how K309 ubiquitination regulates the function of VP35 as the viral polymerase co-factor and the precise stage(s) of the EBOV replication cycle that require VP35 ubiquitination are not known. Here, 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 K309R mutant retains dsRNA binding and efficient type-I Interferon (IFN-I) antagonism due to the basic residue conservation. 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 disruption of the transcriptional gradient results in unbalanced viral protein production, including reduced synthesis of the viral transcription factor VP30. In addition, lack of ubiquitination on K309 results in enhanced interactions with the viral nucleoprotein and premature nucleocapsid packaging, leading to dysregulation of virus assembly. Overall, we identified a novel role of VP35 ubiquitination in coordinating viral transcription and assembly. Author summaryEbola is a pathogenic zoonotic virus that causes severe human disease. Advancing our understanding of the basic molecular mechanisms underlying this virus' replication strategy is critical to expanding the availability of virus-targeted therapeutics. Here, we identified the mechanism by which EBOV VP35 ubiquitination at lysine 309 provides an advantage to complete the viral replication cycle efficiently. We utilized two virus mutants to parse the contributions of ubiquitination and a basic residue at VP35/309 to the virus life cycle. Ubiquitination is critical for facilitating optimal viral transcriptional polymerase co-factor function without affecting transcriptional initiation. The loss of a basic charge at 309 further compromises VP35's function through diminished interaction with the viral matrix protein and type-I interferon antagonism. Overall, ubiquitination and retention of a basic residue at VP35/309 is critical for viral transcription and assembly.