Structural Mechanism of Orthopoxvirus Sabotage of MHCI Antigen Presentation

Immunomodulatory proteins that subvert major histocompatibility complex class I: MHCI) antigen presentation can help viruses to evade cytotoxic T-lymphocyte: CTL) detection and clearance. Cowpox virus, like other orthopoxvirus family members, is a large DNA virus, whose genomic termini encode numerous immunomodulatory genes, including two ER-resident MHCI saboteurs: CPXV012 and CPXV203). CPXV012 inhibits peptide loading of MHCI within the peptide-loading complex: PLC), while CPXV203 inhibits surface expression of both murine and human MHCI through the use of a C-terminal ER-retention sequence: KTEL). An association: direct or indirect) between CPXV203 and MHCI was first demonstrated in pull-down experiments. This doctoral work explores and dissects the molecular details of this interaction. In order to elucidate the nature of CPXV203/MHCI association, biophysical studies were pursued using recombinant CPXV203 and MHCI. Binding studies demonstrated that CPXV203 directly binds mature MHCI heterodimers, thus bridging MHCI to the KDEL receptor: KDELR) rescue pathway. Unlike other ER-resident immune evasion proteins, CPXV203 did not display distinct allelic preferences, but instead proved to be an extremely promiscuous MHCI-binder: murine and primate, Ia and Ib). Similar to KDELR/KDEL binding, CPXV203/MHCI binding was found to be pH-dependent, insuring high-affinity association of KDELR/CPXV203/MHCI in the Golgi and rapid dissociation within the ER. Molecular characterization of the pH-dependent nature of CPXV203/MHCI binding required knowledge of the interface contacts. X-ray crystallography was used to examine the CPXV203/MHCI complex at low pH revealing that CPXV203 interacts with MHCI by binding conserved surfaces required for tapasin association/function. CPXV203 is an 11-stranded β-sandwich consisting of two β-sheets stabilized by five disulfide bonds. CPXV203 topology is reminiscent of the poxvirus chemokine-binding family of proteins, though MHCI contacts largely originate from structural regions unique to CPXV203. Structure-based mutations introduced into CPXV203 and MHCI validated the crystallographically-defined binding geometry and identified several binding and functional hotspots. These studies support that CPXV203 binds mature MHCI in the Golgi and releases it in the ER where it accumulates and is eventually degraded via non-viral mechanisms. These results suggest that CPXV203 could be used as a viral tool to probe natural ERAD pathways that dispose of properly folded proteins that accumulate in the ER