Institut für Virologie Departement für Medizinische Mikrobiologie und Hygiene Albert-Ludwigs-Universität Freiburg im Breisgau Functional and structural dissection of the human cytomegalovirus encoded immunoevasin US11 reveals co-adaptation to HLA-A INAUGURAL-DISSERTATION zur Erlangung der Doktorwürde der Fakultät für Biologie und der Fakultät für Medizin der Albert-Ludwigs-Universität Freiburg im Breisgau vorgelegt von Cosima Zimmermann geboren in Raeren, Belgien Freiburg im Breisgau Juni 2016 Table of contents Abbreviations..................................................................................................................... 1 Summary.............................................................................................................................4 Zusammenfassung............................................................................................................. 6 1. Introduction.................................................................................................................... 8 1.1. The MHC class I antigen presentation pathway............................................................8 1.1.1. Structure of MHC 1.........................................................................................................8 1.1.2. The MHC I peptide loading complex and antigen presentation...................................9 1.1.3. MHC I polymorphism.................................................................................................. 11 1.1.4. Antigen recognition and immune surveillance..........................................................12 1.2. The human cytomegalovirus.........................................................................................13 1.2.1. Epidemiology and health impact................................................................................13 1.2.2. Genome organization and structure.......................................................................... 15 1.2.3. Replication and species-specificity.............................................................................16 1.2.4. Immunobiology of HCMV infection............................................................................ 17 1.3. MHC class I inhibitors.................................................................................................... 19 1.3.1. US2...............................................................................................................................20 1.3.2. US3...............................................................................................................................21 1.3.3. US6...............................................................................................................................22 1.3.4. US11............................................................................................................................ 22 1.3.5. Cooperation and other immunoevasins.....................................................................25 1.4. Aim of the study..............................................................................................................27 2. Results............................................................................................................................28 2.1. Early modifications of the PLC in HCMV infected cells coincide with US3 and US11 binding to MHC 1........................................................................................................................28 2.1.1. Disturbed MHC I/PLC interaction and MHC I maturation early in infection.............28 2.1.2. The immunoevasins US3 and US11 are co-immunoprecipitated ..........with 29MHC I 2.2. The immunoevasin US11 inhibits efficient recruitment of MHC I to the....... PLC33 2.2.1. Factors interfering with MHC I/PLC interaction are encoded in the gene region US2-11 .....................................................................................................................................33 2.2.2. Inefficient recruitment of MHC I to the PLCUS2-6 in A infected cells is rescued by additional deletionUS11 of and US11 interacts with the ................................................PLC 35 2.3. Distinct allele-specificity of US11 in HCMV infected fibroblasts.............................38 2.3.1. Changes in the relative abundance of HLA class .........................................1 ligands 38 2.3.2. Allotype specific MHC class I regulation by US11......................................................41 2.4. Distinct domains of US11 targets different lineages of HLA-A allotypes................44 2.4.1. The N-terminus of 11811 is required for HLA-A*02:01 down-regulation................. 44 2.4.2. The N-terminus of US11 specifically recognizes group I HLA-A.................... alleles 46 2.5. US11 uses different sub-ectodomains to target different domains on HLA-A*02:01 and HLA-A*03:01 molecules for down-regulation................................................................54 2.5.1. Residues 184-207 of HLA-A*03:01 confer US11 sensitivity..................................... 54 2.5.2. US11 N-terminus recognizes the al-2 domains of HLA-A*02:01............................. 57 2.6. Targeting of MHC I by the US11 N-terminus specifically evolved with A2 lineage allotypes......................................................................................................................................61 2.6.1. The CCMV US11 homologue is notable to down-regulate HLA-A*02:01................. 61 2.6.2. The proline at position 184 is a key residue for USll-mediated down-regulation of HLA-A*03:01.............................................................................................................................63 2.6.3. Appearance of alanine at position 184 was an early event during evolution of the A2 lineage ..................................................................................................................................... 65 2.7. A novel MHC class I allotype-dependent function of the HCMV immunoevasin US11 manipulates MHC I quality control...............................................................................67 2.7.1. Study of 11811 interaction partners............................................................................ 67 2.7.2. The N-terminus of US11 is required for MHC I retention........................................ 70 2.7.3. 11811 modifies the usage of HLA-B*15:03 anchor residues.......................................73 3. Discussion......................................................................................................................76 3.1. US11 is highly MHC I allele specific: HLA-A is strongly down-regulated, whereas HLA-B is not affected................................................................................................................. 76 3.2. US11 adapted to the evolution of HLA-A by targeting different A lineages with distinct luminal domains..........................................................................................................78 3.3. The US11 N-terminus attacks HLA-A*02:01 independently of the cytosolic CysLysVal motif.......................................................................................................................... 79 3.4. Did US11 contribute to the split of HLA-A lineages?................................................... 81 3.5. A novel MHC I allele-dependent function of US11 manipulates the ligandome of HLA-B*15:03.............................................................................................................................. 83 3.6. The role of the US11 N-terminus...................................................................................86 3.7. Perspectives.....................................................................................................................87 4. Material and Methods.................................................................................................. 89 4.1. Material..............................................................................................................................89 4.1.1. Biochemicals and Chemicals.......................................................................................89 4.1.2. Enzymes.......................................................................................................................90 4.1.3. Buffers and solutions..................................................................................................90 4.1.4. Kits.............................................................................................................................. 93 4.1.5. Oligonucleotides......................................................................................................... 94 4.1.6. Plasmids......................................................................................................................96 4.1.7. Antibodies................................................................................... 97 4.1.8. Small interfering RNA................................................................................................. 98 4.1.9. Cells.............................................................................................................................99 4.1.10. Viruses.....................................................................................................................100 4.2. Experimental procedures............................................................................................100