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Thesis Details
TitleCharacterization of an Orf virus RING-H2 protein, B5L : a mimic of cellular anaphase promoting complex subunit 11
AuthorMo, Min
InstitutionUniversity of Otago
Date2009
AbstractThe anaphase promoting complex (APC/C) is an ubiquitin ligase that is an essential regulator of multiple steps in the cell cycle. The complex consists of at least 12 subunits with a catalytic core formed by a scaffold protein, APC2, and a RING-H2 protein, APC11. The Parapoxvirus, Orf virus (OV), encodes a RING-H2 protein, B5L, with clear sequence similarities to APC11. The disruption of APC/C function leads to pre-mature entry into S phase and a delayed M phase exit and, potentially, apoptosis. This investigation explored the functional significance of the similarity between B5L and APC11 and specifically sought to determine if B5L manipulates cell cycle regulation by targeting APC/C function. Co-immunoprecipitation experiments from lysates of cells expressing a range of constructs revealed an interaction between B5L and APC2 in the same manner as seen with APC11. Furthermore, B5L was found to associate with endogenous APC/C. However, although APC11 promoted the formation of polyubiquitin chains in substrate-independent in vitro assays, B5L was inactive in this assay. Bioinformatics comparisons of APC11 and other known RING ubiquitin ligases with B5L and its poxviral homologues revealed some subtle differences. In particular a domain of APC11 (amino acids 61-74), that is essential for its ubiquitin ligase activity is not conserved in B5L or its homologues. When this APC11 domain was incorporated in place of the corresponding region of B5L (amino acids 59-67), the mutated B5L acquired ubiquitin ligase activity. On the other hand, APC11 protein in which the domain was replaced with that of B5L lost ubiquitin ligase activity. Stable cell lines expressing B5L showed an increased number of cells in G2/M phase (30±4%) compared with cell lines expressing APC11 (11±2%, n=3, p<0.05, ANOVA, Tukey’s), consistent with impaired APC/C function. APC/C substrates such as cyclin A, cyclin B and the thymidine kinase were stablized in B5L-expressing cells compared with control cells. Furthermore, transient hyper-expression of B5L induced apoptosis in 25±2% (n=3, p<0.05) of the cell population compared with only 6±1% apoptotic cells when APC11 was hyper-expressed. Analysis of the DNA content of OV-infected cells revealed enhanced DNA synthesis compared with cells infected with a B5L knockout OV. These observations indicate that B5L is a non-functional mimic of APC11. It associates with APC/C, but lacks ubiquitin ligase activity, and hence disrupts APC/C function. These abilities may enable OV to induce a cellular environment that enhances viral replication.
Thesis E-Thesis currently not available.