The initial findings described in the previous section were the basis for this thesis, during which we focused on further delineating the roles MDV USP played in viral replication and/or tumourigenesis. The first aim of this study was to identify MDV USP interacting proteins to better understand what makes this fragment of pUL36 so important for replication and/or tumourigenesis. Essentially, our goal was to determine whether MDV USP interacts with the MDV-encoded oncoprotein Meq or cell cycle regulators. For this purpose, tandem affinity purification (TAP) and immunofluorescence (IF) experiments where performed. Unfortunately, development of these systems was difficult and therefore shall only be briefly mentioned in the results section. In a near future, yeast two hybrid (Y2H) assays in collaboration with Professor Jürgen C. Haas and Clark Russell from the University of Edinburgh will hopefully contribute to the discovery of MDV USP substrates.
The second aim was to study the role of MDV USP in replication and tumourigenesis during virus infection. For this part of the project, several recombinants (r) MDV were generated using two-step Red recombination and the reconstituted viruses (v) were compared regarding their plaque sizes and growth kinetics. More specifically, we studied the replication ability of MDV mutants in the absence of the putative USP sequence or its cysteine active site, Cys box, and also whether defective viral phenotypes (both by cysteine to alanine mutation or complete removal of the USP portion) can be restored by ectopic expression of USP during virus infection.
Finally, rabbit anti-MDV USP and anti-MDV pUL36 antibodies developed, following peptide immunization, were analysed by western blot (WB) and IF analyses of infected and transfected cell lysates.
THE UL36-ENCODED UBIQUITIN-SPECIFIC PROTEASE
IN MAREK’S DISEASE VIRUS REPLICATION AND TUMOURIGENESIS
Publication date: 15/05/2013
El vendedor asume toda la responsabilidad de esta entrada.