Mechanism of Adenovirus E4-ORF3-Mediated SUMO Modifications

ABSTRACT Regulation of a variety of different cellular processes, including posttranslational modifications, is critical for the ability of many viruses to replicate efficiently within host cells. The adenovirus (Ad) E4-ORF3 protein assembles into polymers and forms a unique nuclear scaffold that le...

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Autores principales:	Sook-Young Sohn, Patrick Hearing
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2019
Materias:	adenoviruses E3 ligase E4-ORF3 SUMO UBC9 Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/ca739257e324406f9509a9dafa96c7be
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spelling	oai:doaj.org-article:ca739257e324406f9509a9dafa96c7be2021-11-15T15:55:13ZMechanism of Adenovirus E4-ORF3-Mediated SUMO Modifications10.1128/mBio.00022-192150-7511https://doaj.org/article/ca739257e324406f9509a9dafa96c7be2019-02-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00022-19https://doaj.org/toc/2150-7511ABSTRACT Regulation of a variety of different cellular processes, including posttranslational modifications, is critical for the ability of many viruses to replicate efficiently within host cells. The adenovirus (Ad) E4-ORF3 protein assembles into polymers and forms a unique nuclear scaffold that leads to the relocalization and sequestration of cellular proteins, including small ubiquitin-like modifiers (SUMOs). Previously, we showed that E4-ORF3 functions as a SUMO E3 ligase of transcriptional intermediary factor-1 gamma (TIF-1γ) and promotes poly-SUMO chain formation. Here, we present cellular and biochemical data to further understand E4-ORF3 SUMO ligase activity. E4-ORF3 proteins from five different Ad species were found to possess SUMO E3 ligase activities in vitro. In infected cells, SUMO modifications of target proteins occurred only when the proteins were recruited into E4-ORF3 polymeric structures. By analyzing SUMO-deficient TIF-1γ, we demonstrated that SUMO conjugations are not required for E4-ORF3-mediated relocalization of target proteins in infected cells, implying that sequestration is followed by SUMO modification. In vitro SUMO conjugation assays revealed the Ad E1B-55K oncoprotein as a new viral target of E4-ORF3-mediated SUMOylation. We also verified a direct function of E4-ORF3 as a SUMO ligase for multiple cellular proteins, including transcription factor II-I (TFII-I), Nbs1, and Mre11. Moreover, we discovered that E4-ORF3 associates with SUMO-bound UBC9, and E4-ORF3 polymerization is crucial for this ternary interaction. Together, our findings characterize E4-ORF3 as a novel polymer-type SUMO E3 ligase and provide mechanistic insights into the role of E4-ORF3 in SUMO conjugation. IMPORTANCE Viruses interplay with the host SUMOylation system to manipulate diverse cellular responses. The Ad E4-ORF3 protein forms a dynamic nuclear network to interfere with and exploit different host processes, including the DNA damage and interferon responses. We previously reported that E4-ORF3 is a SUMO E3 ligase. Here, we demonstrate that this activity is a conserved function of evolutionarily diverse human Ad E4-ORF3 proteins and that E4-ORF3 functions directly to promote SUMO conjugations to multiple cellular proteins. Recruitment of cellular substrates into E4-ORF3 nuclear inclusions is required for SUMO conjugation to occur in vivo. We probed the mechanism by which E4-ORF3 functions as a SUMO E3 ligase. Only multimeric, but not dimeric, E4-ORF3 binds to the SUMO E2 conjugation enzyme UBC9 in vitro only in a trimeric complex with SUMO. These results reveal a novel mechanism by which a conserved viral protein usurps the cellular SUMO conjugation machinery.Sook-Young SohnPatrick HearingAmerican Society for MicrobiologyarticleadenovirusesE3 ligaseE4-ORF3SUMOUBC9MicrobiologyQR1-502ENmBio, Vol 10, Iss 1 (2019)
institution	DOAJ
collection	DOAJ
language	EN
topic	adenoviruses E3 ligase E4-ORF3 SUMO UBC9 Microbiology QR1-502
spellingShingle	adenoviruses E3 ligase E4-ORF3 SUMO UBC9 Microbiology QR1-502 Sook-Young Sohn Patrick Hearing Mechanism of Adenovirus E4-ORF3-Mediated SUMO Modifications
description	ABSTRACT Regulation of a variety of different cellular processes, including posttranslational modifications, is critical for the ability of many viruses to replicate efficiently within host cells. The adenovirus (Ad) E4-ORF3 protein assembles into polymers and forms a unique nuclear scaffold that leads to the relocalization and sequestration of cellular proteins, including small ubiquitin-like modifiers (SUMOs). Previously, we showed that E4-ORF3 functions as a SUMO E3 ligase of transcriptional intermediary factor-1 gamma (TIF-1γ) and promotes poly-SUMO chain formation. Here, we present cellular and biochemical data to further understand E4-ORF3 SUMO ligase activity. E4-ORF3 proteins from five different Ad species were found to possess SUMO E3 ligase activities in vitro. In infected cells, SUMO modifications of target proteins occurred only when the proteins were recruited into E4-ORF3 polymeric structures. By analyzing SUMO-deficient TIF-1γ, we demonstrated that SUMO conjugations are not required for E4-ORF3-mediated relocalization of target proteins in infected cells, implying that sequestration is followed by SUMO modification. In vitro SUMO conjugation assays revealed the Ad E1B-55K oncoprotein as a new viral target of E4-ORF3-mediated SUMOylation. We also verified a direct function of E4-ORF3 as a SUMO ligase for multiple cellular proteins, including transcription factor II-I (TFII-I), Nbs1, and Mre11. Moreover, we discovered that E4-ORF3 associates with SUMO-bound UBC9, and E4-ORF3 polymerization is crucial for this ternary interaction. Together, our findings characterize E4-ORF3 as a novel polymer-type SUMO E3 ligase and provide mechanistic insights into the role of E4-ORF3 in SUMO conjugation. IMPORTANCE Viruses interplay with the host SUMOylation system to manipulate diverse cellular responses. The Ad E4-ORF3 protein forms a dynamic nuclear network to interfere with and exploit different host processes, including the DNA damage and interferon responses. We previously reported that E4-ORF3 is a SUMO E3 ligase. Here, we demonstrate that this activity is a conserved function of evolutionarily diverse human Ad E4-ORF3 proteins and that E4-ORF3 functions directly to promote SUMO conjugations to multiple cellular proteins. Recruitment of cellular substrates into E4-ORF3 nuclear inclusions is required for SUMO conjugation to occur in vivo. We probed the mechanism by which E4-ORF3 functions as a SUMO E3 ligase. Only multimeric, but not dimeric, E4-ORF3 binds to the SUMO E2 conjugation enzyme UBC9 in vitro only in a trimeric complex with SUMO. These results reveal a novel mechanism by which a conserved viral protein usurps the cellular SUMO conjugation machinery.
format	article
author	Sook-Young Sohn Patrick Hearing
author_facet	Sook-Young Sohn Patrick Hearing
author_sort	Sook-Young Sohn
title	Mechanism of Adenovirus E4-ORF3-Mediated SUMO Modifications
title_short	Mechanism of Adenovirus E4-ORF3-Mediated SUMO Modifications
title_full	Mechanism of Adenovirus E4-ORF3-Mediated SUMO Modifications
title_fullStr	Mechanism of Adenovirus E4-ORF3-Mediated SUMO Modifications
title_full_unstemmed	Mechanism of Adenovirus E4-ORF3-Mediated SUMO Modifications
title_sort	mechanism of adenovirus e4-orf3-mediated sumo modifications
publisher	American Society for Microbiology
publishDate	2019
url	https://doaj.org/article/ca739257e324406f9509a9dafa96c7be
work_keys_str_mv	AT sookyoungsohn mechanismofadenoviruse4orf3mediatedsumomodifications AT patrickhearing mechanismofadenoviruse4orf3mediatedsumomodifications
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Mechanism of Adenovirus E4-ORF3-Mediated SUMO Modifications

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