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

Abstract

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.

Inhibitors of the Ubiquitin-Mediated Signaling Pathway Exhibit Broad-Spectrum Antiviral Activities against New World Alphaviruses

“…Targeting the E3 ligase Cullin 2 exerts an inhibitory effect on dengue virus, adding support to the potential of these enzymes to be targeted to achieve viral inhibitory outcomes [ 64 ]. The Ebola virus protein VP35 is known to be ubiquitinated, which is required for the regulation of viral transcription and assembly [ 65 ]. The ubiquitination of VP40 has been demonstrated to be important for filovirus budding and egress [ 65 , 66 , 67 , 68 ].…”

“…The Ebola virus protein VP35 is known to be ubiquitinated, which is required for the regulation of viral transcription and assembly [ 65 ]. The ubiquitination of VP40 has been demonstrated to be important for filovirus budding and egress [ 65 , 66 , 67 , 68 ]. In the context of New World alphaviruses, it has been demonstrated that inhibition of the proteasome function using the FDA-approved small molecule, bortezomib, exerted robust viral inhibitory activity against VEEV, by potentially impacting the ubiquitination status of the capsid protein [ 25 ].…”

Ebola virus sequesters IRF3 in viral inclusion bodies to evade host antiviral immunity

“…In IBs, the EBOV genome is replicated and transcribed by viral polymerase complexes ( Misasi and Sullivan, 2014 ). VP35 serves as a cofactor of RNA-dependent RNA polymerase and contributes to viral replication by homo-oligomerization through a coiled-coil domain ( Reid et al, 2005 ) as well as through its phosphorylation and ubiquitination, which was recently discovered ( van Tol et al, 2022 ; Zhu et al, 2020 ).…”

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

“…The RDRP is composed of the catalytic subunit of the polymerase L and the polymerase cofactor protein VP35, which interact with the viral envelope through the VP40 and minor VP24 matrix proteins to form the virion [1][2][3]. In addition to VP35's critical role as a cofactor of the viral polymerase, it has been extensively studied for its function in the inhibition of innate signaling pathways and antagonism of antiviral type-I Interferon (IFN-I) [4][5][6][7][8][9][10][11][12][13][14][15].…”