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

EBOV VP35 is a polymerase co-factor to regulate viral RNA-dependent RNA polymerase function that is responsible for  replication of viral genome and transcription of mRNAs, and is also involved in viral genome packaging and host immmune antagonism. The host E3 ubiquitin ligase TRIM6 ubiquitinates VP35 at lysine 309 (K309) to facilitate viral replication. Two mutations K309G and K309R prohibited the VP35/K309 ubiquitination. Although the K309R mutant retained IFN-I antagonism, the K309G mutant lost the ability to antagonize the IFN-I response, indicating the basic residue in position 309 is critical for the IFN-I antagonism. Because these two mutants impaired an interaction of VP35 with viral polymerase, both mutant viruses poorly replicated in IFN-competent and-deficient cells. Loss of the K309 ubiqutination resulted in the dysregulated mRNA transcription gradient, leading to the disrupted balance of viral protein synthesis. Additionally, the lack of K309 ubiquitination resulted in enhanced interaction with viral nucleoprotein, causing a premature nucleocapsid packaging.