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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 ubiq...

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Autores principales: van Tol, Sarah, Kalveram, Birte, Ilinykh, Philipp A., Ronk, Adam, Huang, Kai, Aguilera-Aguirre, Leopoldo, Bharaj, Preeti, Hage, Adam, Atkins, Colm, Giraldo, Maria I., Wakamiya, Maki, Gonzalez-Orozco, Maria, Warren, Abbey N., Bukreyev, Alexander, Freiberg, Alexander N., Rajsbaum, Ricardo
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9119628/
https://www.ncbi.nlm.nih.gov/pubmed/35533195
http://dx.doi.org/10.1371/journal.ppat.1010532
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author van Tol, Sarah
Kalveram, Birte
Ilinykh, Philipp A.
Ronk, Adam
Huang, Kai
Aguilera-Aguirre, Leopoldo
Bharaj, Preeti
Hage, Adam
Atkins, Colm
Giraldo, Maria I.
Wakamiya, Maki
Gonzalez-Orozco, Maria
Warren, Abbey N.
Bukreyev, Alexander
Freiberg, Alexander N.
Rajsbaum, Ricardo
author_facet van Tol, Sarah
Kalveram, Birte
Ilinykh, Philipp A.
Ronk, Adam
Huang, Kai
Aguilera-Aguirre, Leopoldo
Bharaj, Preeti
Hage, Adam
Atkins, Colm
Giraldo, Maria I.
Wakamiya, Maki
Gonzalez-Orozco, Maria
Warren, Abbey N.
Bukreyev, Alexander
Freiberg, Alexander N.
Rajsbaum, Ricardo
author_sort van Tol, Sarah
collection PubMed
description 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.
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spelling pubmed-91196282022-05-20 Ubiquitination of Ebola virus VP35 at lysine 309 regulates viral transcription and assembly van Tol, Sarah Kalveram, Birte Ilinykh, Philipp A. Ronk, Adam Huang, Kai Aguilera-Aguirre, Leopoldo Bharaj, Preeti Hage, Adam Atkins, Colm Giraldo, Maria I. Wakamiya, Maki Gonzalez-Orozco, Maria Warren, Abbey N. Bukreyev, Alexander Freiberg, Alexander N. Rajsbaum, Ricardo PLoS Pathog Research Article 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. Public Library of Science 2022-05-09 /pmc/articles/PMC9119628/ /pubmed/35533195 http://dx.doi.org/10.1371/journal.ppat.1010532 Text en © 2022 van Tol et al https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
van Tol, Sarah
Kalveram, Birte
Ilinykh, Philipp A.
Ronk, Adam
Huang, Kai
Aguilera-Aguirre, Leopoldo
Bharaj, Preeti
Hage, Adam
Atkins, Colm
Giraldo, Maria I.
Wakamiya, Maki
Gonzalez-Orozco, Maria
Warren, Abbey N.
Bukreyev, Alexander
Freiberg, Alexander N.
Rajsbaum, Ricardo
Ubiquitination of Ebola virus VP35 at lysine 309 regulates viral transcription and assembly
title Ubiquitination of Ebola virus VP35 at lysine 309 regulates viral transcription and assembly
title_full Ubiquitination of Ebola virus VP35 at lysine 309 regulates viral transcription and assembly
title_fullStr Ubiquitination of Ebola virus VP35 at lysine 309 regulates viral transcription and assembly
title_full_unstemmed Ubiquitination of Ebola virus VP35 at lysine 309 regulates viral transcription and assembly
title_short Ubiquitination of Ebola virus VP35 at lysine 309 regulates viral transcription and assembly
title_sort ubiquitination of ebola virus vp35 at lysine 309 regulates viral transcription and assembly
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9119628/
https://www.ncbi.nlm.nih.gov/pubmed/35533195
http://dx.doi.org/10.1371/journal.ppat.1010532
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