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Tsg101/ESCRT-I recruitment regulated by the dual binding modes of K63-linked diubiquitin.

ABSTRACT: The ESCRT-I protein Tsg101 plays a critical role in viral budding and endocytic sorting. Although Tsg101 is known to recognize monoubiquitin (Ub₁), here we show that it can also bind several diubiquitins (K48-Ub₂, N-Ub₂, and K63-Ub₂), with a preference for K63-linked Ub₂. The NMR structure of the Tsg101:K63-Ub₂ complex showed that while the Ub₁-binding site accommodates the distal domain of Ub₂, the proximal domain alternatively binds two different sites, the vestigial active site and an N-terminal helix. Mutation of each site results in distinct phenotypes regarding the recruitment of Tsg101 partners. Mutation in the vestigial active site abrogates interaction between Tsg101 and the HIV-1 protein Gag but not Hrs, a cellular protein. Mutation at the N-terminal helix alters Gag but not Hrs-Tsg101 localization. Given the broad involvement of Tsg101 in diverse cellular functions, this discovery advances our understanding of how the ESCRT protein recognizes binding partners and sorts endocytic cargo.

SUBMITTER: Strickland M

PROVIDER: S-EPMC10015442 | biostudies-literature | 2022 Feb

REPOSITORIES: biostudies-literature

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Tsg101/ESCRT-I recruitment regulated by the dual binding modes of K63-linked diubiquitin.

Strickland Madeleine M Watanabe Susan S Bonn Steven M SM Camara Christina M CM Starich Mary R MR Fushman David D Carter Carol A CA Tjandra Nico N

Structure (London, England : 1993) 20220201 2

The ESCRT-I protein Tsg101 plays a critical role in viral budding and endocytic sorting. Although Tsg101 is known to recognize monoubiquitin (Ub1), here we show that it can also bind several diubiquitins (K48-Ub2, N-Ub2, and K63-Ub2), with a preference for K63-linked Ub2. The NMR structure of the Tsg101:K63-Ub2 complex showed that while the Ub1-binding site accommodates the distal domain of Ub2, the proximal ...[more]

PMID: 35120596

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Project description:Classical swine fever (CSF), caused by classical swine fever virus (CSFV), is a highly contagious disease of swine with high morbidity and mortality that negatively affects the pig industry worldwide, in particular in China. Soon after the endocytosis of CSFV, the virus makes full use of the components of host cells to complete its life cycle. The endocytosis sorting complex required for transport (ESCRT) system is a central molecular machine for membrane protein sorting and scission in eukaryotic cells that plays an essential role in many physiological metabolic processes, including invasion and egress of envelope viruses. However, the molecular mechanism that ESCRT uses to regulate the replication of CSFV is unknown. In this study, we demonstrated that the ESCRT-I complex Tsg101 protein participates in clathrin-mediated endocytosis of CSFV and is also involved in CSFV trafficking. Tsg101 assists the virus in entering the host cell through the late endosome (Rab7 and Rab9) and finally reaching the lysosome (Lamp-1). Interestingly, Tsg101 is also involved in the viral replication process by interacting with nonstructural proteins 4B and 5B of CSFV. Finally, confocal microscopy showed that the replication complex of Tsg101 and double-stranded RNA (dsRNA) or NS4B and NS5B protein was close to the endoplasmic reticulum (ER), not the Golgi, in the cytoplasm. Collectively, our finding highlights that Tsg101 regulates the process of CSFV entry and replication, indicating that the ESCRT plays an important role in the life cycle of CSFV. Thus, ESCRT molecules could serve as therapeutic targets to combat CSFV infection.IMPORTANCE CSF, caused by CSFV, is a World Organization for Animal Health (OIE) notifiable disease and causes significant financial losses to the pig industry globally. The ESCRT machinery plays an important regulatory role in several members of the genera Flavivirus and Hepacivirus within the family Flaviviridae, such as hepatitis C virus, Japanese encephalitis virus, and dengue virus. Previous reports have shown that assembling and budding of these viruses require ESCRT. However, the role of ESCRT in Pestivirus infection remains to be elucidated. We determined the molecular mechanisms of the regulation of CSFV infection by the major subunit Tsg101 of ESCRT-I. Interestingly, Tsg101 plays an essential regulatory role in both clathrin-mediated endocytosis and genome replication of CSFV. Overall, the results of this study provide further insights into the molecular function of ESCRT-I complex protein Tsg101 during CSFV infection, which may serve as a molecular target for pestivirus inhibitors.

Dataset Information

Tsg101/ESCRT-I recruitment regulated by the dual binding modes of K63-linked diubiquitin.

Publications

Tsg101/ESCRT-I recruitment regulated by the dual binding modes of K63-linked diubiquitin.

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