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Marek´s disease (MD) is a poultry disease with a worldwide distribution.
The disease is caused by MD virus (MDV), an alphaherpesvirus in the Mardivirus genus.
MDV causes neurological symptoms and CD4+ T cell visceral lymphomas, which eventually lead to the death of the affected animals. First described in 1907 by Jozef Marek as a slowly progressing chronic polyneuritis, MD became a major worry for poultry producers due to the severity of symptoms and the lack of effective control measures until vaccines were developed in the 1960s. The various vaccines have resulted in a reduction of disease incidence, but not in sterilizing immunity, which has led to constantly increasing virulence of circulating field strains, ultimately leading to the selection of highly virulent viruses. These strains continue to threaten chickens and it is, therefore, important to study MDV genes involved in viral pathogenesis. In addition to the MDV-encoded oncoprotein Meq and the viral telomerase, the UL36 gene plays an essential role in MDV pathogenesis. This gene was recently found to encode a deubiquitinase (DUB). The ubiquitin-specific protease (USP) is embedded in the N-terminal part of the large tegument protein (pUL36). The USP is highly conserved among herpesviruses and replacement of its putative active site cysteine by an alanine had a profound effect on in vitro replication and in vivo tumourigenesis. However, in these previous studies it was not possible to determine which proteins interact with MDV USP, and also if the observed effects were due to structural significance of the N-terminus of the UL36-encoded large tegument protein or solely to its enzymatic activity.
In this dissertation, USP-Meq interaction studies were performed and several mutant viruses based on the very virulent RB-1B MDV strain were constructed by two-step Red mediated recombination. The mutants were designed to allow differentiation between USP enzymatic and structural function. Even if the interaction between MDV USP and Meq could not be confirmed, it was shown that both the N-terminal UL36 region and its highly-conserved USP cysteine box are essential for viral replication. Also, ectopical expression of a functional, synthetic USP (SynUSP) in several locations of the MDV genome could not compensate for the absence of a (functional) USP, suggesting that the large tegument protein can only exert its functions if USP is expressed in frame within the UL36 protein. We concluded from our studies that MDV USP has a structural role beyond its enzymatic activity, which implies that the decreased tumourigenesis observed in the absence of an active USP is partly due to MDV replication impairment and not to a direct activity of USP in tumourigenesis.
Additionally, specific antibodies for MDV USP and for pUL36 were produced. Two 40 kDa bands were detected with one of the antibodies in MDV-infected cell lysates, which could correspond to cleaved MDV USP, similar to what was previously identified in other herpesviruses.