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 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 ...... 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 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. ...... 100 4.2. Experimental procedures...... 100 4.2.1. Plasmid construction...... 100 4.2.2. Reconstitution and propagation of HCMV...... 102 4.2.3. Cell culture methods...... 103 4.2.4. analysis...... 105

References...... 109

Annex...... 123 Delayed maturation of MHC I is not caused by pp71 or UL18...... 123 Tested group I and group II alleles were classified to the A2 or A3 lineages...... 125 Testing of 0811 specific polyclonal antibodies...... 126

Table of figures...... 127

Danksagung...... 130

Curriculum vitae...... 131

Erklärung...... 134