Use this URL to cite or link to this record in EThOS:
Title: Functional dissection of human cytomegalovirus immune evasion protein US6
Author: Dugan, Gillian Elizabeth
ISNI:       0000 0001 3434 175X
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2007
Availability of Full Text:
Access from EThOS:
Cytotoxic T lymphocytes (CTLs) are activated to kill virally infected cells by the recognition of viral peptides presented on the cell surface by major histocompatibility (MHC) class I molecules. Peptides are generated by proteasOIpal degradation of viral proteins in the cytosol, and then translocated into the lumen of the endoplasmic reticulum (ER) by the transporter associated with antigen processing (TAP). Here, peptides are loaded onto newly synthesised MHC class I molecules, before trafficking ') to the cell surface. Clearly it is advantageous' for viruses to inhibit cell- surface expression of MHC class I, as this enables them to avoid a CTL response. Human cytomegalovirus (HCMV) encodes US6, an ER resident glycoprotein that inhibits peptide translocation by TAP. HCMV US6 prevents ATP binding by TAP, effectively starving the transporter of its energy source, and cutting off the supply of peptides for loading onto MHC class I molecules. The mechanism through which HCMV US6 inhibits ATP binding by TAP is poorly understood, nor is it clear how HCMV US6 localises to the ER. Thus the aims of this study were three-fold.' Firstly, to further investigate HCMV US6 inhibition of MHC class I cell surface expression, in particular its interactions with TAP. Secondly, to characterise a chimpanzee cytomegalovirus (CCMV) homologue of HCMV US6, in order to identify conserved residues required for TAP inhibition. Thirdly, to ~nvestigate interactions that may mediate HCMV US6 localisation to the ER. A flow cytometric· assay for TAP activity was developed in which cell surface expression of the TAP-depe.ndent allele HLA-B2705 was measured. Using a series of HCMV US6 truncations and site directed mutants in this assay it was demonstrated that that residues 89-104 ofHCMV US6 are required for TAP inhibition. Additionally, two separate regions of HCMV US6 stabilise the interaction with TAP; one at the Nterminus comprising residues 81-89, and another at the C-terminus ofHCMV US6. In contrast CCMV US6 had no effect on HLA-B2705 expression and could not inhibit ATP binding by human TAP. Sequence alignments indicated that CCMV US6 differs from HCMV US6 in seven of the sixteen residues required for TAP inhibition, corresponding to residues 89-104 ofHCMV US6. Site directed mutagenesis was used to create a CCMV US6 molecule that was identical to HCMV US6 in fifteen of the sixteen residues required for TAP inhibition. Significantly this chimeric US6 protein reduced cell surface expression of HLA-B2705, and inhibited ATP binding by TAP. Overall, these data provide further insight into the molecular interactions between HCMV US6 and TAP, from which a possible mechanism ofinhibition can be proposed. Previously published work revealed that HCMV US6 binds both TAP and the ER chaperone calnexin. To investigate if either of these interactions was required for HCMV US6 localisation to the ER, TAP-deficient, and calnexin-deficient cell lines were utilised. In both of these cell types HCMV US6 localised to the ER, suggesting that neither TAP nor calnexin was, by itself, required to retain HCMV US6 in the ER. Additionally, the intracellular localisation of a HCMV US6 truncation series was assessed to map the sequence requirements for ER localisation; however this could not be conclusively defined.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available