Marek’s Disease Virus Regulates the Ubiquitylome of Chicken CD4+ T Cells to Promote Tumorigenesis

Abstract

Ubiquitination and deubiquitination of cellular proteins are reciprocal reactions catalyzed by ubiquitination-related enzymes and deubiquitinase (DUB) which regulate almost all cellular processes. Marek’s disease virus (MDV) encodes a viral DUB that plays an important role in the MDV pathogenicity. Chicken CD4+ T-cell lymphoma induced by MDV is a key contributor to multiple visceral tumors and immunosuppression of chickens with Marek’s disease (MD). However, alterations in the ubiquitylome of MDV-induced T lymphoma cells are still unclear. In this study, a specific antibody against K-ε-GG was used to isolate ubiquitinated peptides from CD4+ T cells and MD T lymphoma cells. Mass spectrometry was used to compare and analyze alterations in the ubiquitylome. Our results showed that the ubiquitination of 717 and 778 proteins was significantly up- and downregulated, respectively, in T lymphoma cells. MDV up- and downregulated ubiquitination of a similar percentage of proteins. The ubiquitination of transferases, especially serine/threonine kinases, was the main regulatory target of MDV. Compared with CD4+ T cells of the control group, MDV mainly altered the ubiquitylome associated with the signal transduction, immune system, cancer, and infectious disease pathways in T lymphoma cells. In these pathways, the ubiquitination of CDK1, IL-18, PRKCB, ETV6, and EST1 proteins was significantly up- or downregulated as shown by immunoblotting. The current study revealed that the MDV infection could exert a significant influence on the ubiquitylome of CD4+ T cells.

UL36 Encoded by Marek’s Disease Virus Exhibits Linkage-Specific Deubiquitinase Activity

“…These observations indicated that UL36-DUB may have strong anti-antagonism and anti-proteolysis characteristics, and could be kept intact in host cells. It has been observed that UL36 was susceptible to proteolytic degradation during the lysis of MDV-induced T lymphoma cells in our previous study [28]. Thus, PMSF and Roche CPIC could be used in cell lysis during the survey of the function of UL36 to protect UL36 from degradation, as well as to avoid interference with UL36 activity.…”

“…The K11 or K48 type Ub chain modification is mainly involved in the proteasome degradation of the modified protein, while the K63 type Ub chain modification regulates the function of the modified protein [38]. In addition, MDV can reverse the ubiquitination that relates to the infection, inflammation, tumor, and immune-related proteins in T lymphocyte tumors [28]. These observations indicated that the cellular proteins modified by these three types of Ub chains should be the targets of MDV-encoded UL36.…”

“…For example, PMSF and Roche CPIC did not inhibit the activity of UL36 from a working concentration up to 5 × concentration, but the Solarbio inhibitor cocktail did. In addition, the UL36-DUB exists in MDV-induced T lymphoma cells for a long term [28], and chicken cells did not wipe off this foreign enzyme that seriously affects the intracellular environment promoting MDV activity. These observations indicated that UL36-DUB may have strong anti-antagonism and anti-proteolysis characteristics, and could be kept intact in host cells.…”

“…The mechanism of MDV pathogenesis and virulence enhancement has not been fully elucidated, although specific MDV-encoded genes have been revealed to be important to the MDV pathogenicity [21][22][23][24][25][26][27]. Our previous study found MDV could reverse the ubiquitylome of chicken T-cell lymphoma by comparison with normal CD4 + T cells, and the N-terminal 75 kDa fragment of UL36, which contains the viral DUB catalytic domain (UL36-DUB), exists in MDV-induced T lymphoma cells in high level [28]. The amino acid sequence of UL36-DUB is highly consistent throughout all virulent strains of MDV, but not in that of other virus species.…”

Morphological and Immunohistochemical Examination of Lymphoproliferative Lesions Caused by Marek’s Disease Virus in Breeder Chickens

“…Slides were treated with 1% bovine serum albumin (BSA) for 30 min and incubated for 1 h at room temperature in the presence of 0.1% BSA with an antibody against CD3 (Polyclonal Rabbit, 1:50, A0452 Dako-Agilent, Santa Clara, CA, USA) and an antibody against CD20 (Polyclonal Rabbit, 1:800, PA5-16701, ThermoFisher Scientific, Waltham, MA, USA) that identify T-lymphocytes (cytoplasmic region of the CD3ε-chain) and B-lymphocytes (C-terminus of CD20 protein). These antibodies were tested by several authors [19][20][21][22][23] that proved cross-reactivity with chicken T-and B-lymphocytes. After two rinses with phosphate-buffered saline (PBS), tissue sections were treated for 30 min with universal polymer labeled with horseradish peroxidase (MACH-1, Bio Optica, Milan, Italy), followed by the chromogen 3-3 diamminobenzidine tetrahydrochloride (DAB) for 3 min and counterstained with Mayer's hematoxylin.…”