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ARS Home » Southeast Area » Athens, Georgia » U.S. National Poultry Research Center » Endemic Poultry Viral Diseases Research » Research » Publications at this Location » Publication #371984

Research Project: Enhancing Genetic Resistance to Marek’s Disease in Poultry

Location: Endemic Poultry Viral Diseases Research

Title: UL36 encoded by Marek’s Disease Virus exhibits linkage-specific deubiquitinase activity

item LIN, JUNYAN - Jilin University
item AI, YONGXING - Jilin University
item ZHOU, HONGDA - Jilin University
item LV, YAN - Jilin University
item WANG, MENGHAN - Jilin University
item XU, JIACUI - Jilin University
item YU, CONG - Changchun Institute Of Applied Chemistry
item Zhang, Huanmin
item WANG, MENGYUN - Jilin University

Submitted to: International Journal of Molecular Sciences
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/2/2020
Publication Date: 3/5/2020
Citation: Lin, J., Ai, Y., Zhou, H., Lv, Y., Wang, M., Xu, J., Yu, C., Zhang, H., Wang, M. 2020. UL36 encoded by Marek’s Disease Virus exhibits linkage-specific deubiquitinase activity. International Journal of Molecular Sciences. 21(5).

Interpretive Summary: Protein metabolism is essential for normal cellular function that involves both synthesis and degradation of proteins. Ubiquitination is a post-translational modification that plays important roles in cellular processes. Deubiquitination refers to cleavage of ubiquitin from proteins. Deubiquitinases or deubiquitinating enzymes (DUB) are any enzymes that catalyze a deubiquitination reaction. This study investigated a Marek’s disease virus-encoded giant protein, known as UL36, which contains a putative DUB. The findings came from this study indicated that this Marek’s disease virus UL36-DUB possesses very interesting properties and could potentially be used to identify inhibitors to battle Marek’s disease virus replication and spreading, thereby, to prevent or reduce Marek’s disease incidence in chicken.

Technical Abstract: Deubiquitinase (DUB) regulates various important cellular processes via reversing the protein ubiquitination. The N-terminal fragment of Marek’s disease (MD) virus (MDV) encodes a giant tegument protein, UL36, which encompasses a putative DUB (UL36-DUB) and shares no homology with any known DUBs. The N-terminus 75 kDa fragment of UL36 exists in MD T lymphoma cells at a high level and participates in MDV pathogenicity. To characterize the deubiquitinating activity and substrate specificity of UL36-DUB, the UL36 N-terminal fragments, UL36(323), UL36(480) and mutants were prepared using BAC-to-BAC system. The deubiquitinating activity and substrate specificity of these recombinant UL36-DUBs were analyzed using various ubiquitin (Ub) or ubiquitin-like (UbL) substrates and activity-based deubiquitinating enzyme probes. Results from this study indicated that wild type UL36-DUBs showed a different hydrolysis ability against varied types of ubiquitin chains, which presented the highest activity to K11, K48 and K63 linkage Ub chains, weak activity to K6, K29 and K33 Ub chains, and no activity to K27 linkage Ub chain. The UL36 had higher cleavage efficiency for K48 and K63 poly-ubiquitin than linear ubiquitin chain (M1-Ub4), but no activity on various ubiquitin-like modifiers. The mutation of C98 and H234 residues eliminated the deubiquitinating activity of UL36-DUB. D232A mutation impacted, but not eliminated UL36(480) activity. Ub-Br probe can bind to wild type of UL36-DUB, and mutants UL36(480)H234A and UL36(480)D232A, but not C98 mutants. These results suggested that the C98 and H234 in vitro are essential catalytic residues of UL36-DUB. UL36-DUB exhibited a strict substrate specificity. Inhibition assay revealed that UL36-DUB exhibited a resistance to the Roche protease inhibitor (CPIC) and Serine protease inhibitor, but not to Solarbio protease inhibitor. Taking together, UL36-DUB was demonstrated to exhibit the strict substrate preference, and the protocol developed in this study for obtaining active UL36-DUB protein should promote the high-throughput screening of UL36 inhibitors and the study on the function of MDV-encoded UL36.