Multimodal HLA-I genotype regulation by human cytomegalovirus US10 and resulting surface patterning
- Institute of Virology, Medical Center University of Freiburg, Germany
- Faculty of Medicine, University of Freiburg, Germany
- Spemann Graduate School of Biology and Medicine (SGBM), University of Freiburg, Germany
- Faculty of Biology, University of Freiburg, Germany
- Rudolf Virchow Center, Center for Integrative and Translational Bioimaging, University of Würzburg, Germany
- Department of Medicine II (Gastroenterology, Hepatology, Endocrinology and Infectious Diseases), Medical Center University of Freiburg, Germany
- Institute for Virology and Immunobiology, University of Würzburg, Germany
- Department of Immunopathology, Sanquin Research, Netherlands
- Landsteiner Laboratory, Amsterdam UMC, University of Amsterdam, Netherlands
- Institute for Transfusion Medicine and Gene Therapy, Medical Center University of Freiburg, Germany
- Center for Chronic Immunodeficiency, Medical Center University of Freiburg, Germany
- Institute for Virology, University Hospital Essen, University of Duisburg-Essen, Germany
- Institute for the Research on HIV and AIDS-associated Diseases, University Hospital Essen, Germany
- Institute of Virology, Hannover Medical School, Germany
- St. Anna Children’s Cancer Research Institute (CCRI), Austria
- Clinical Cooperation Unit Applied Tumor Immunity, German Cancer Research Center, National Center for Tumor Diseases (NCT), Heidelberg University Hospital, Germany
Figures
Figure 1
Geno- and allotype-specific regulation of HLA-I by US10.
(A) HeLa cells were transiently co-transfected with plasmids encoding HA-tagged (~) molecules or non-tagged HLA-A2E*01:01 (HLA-E was expressed with an HLA-A*02 signal peptide, a natural HLA-E …
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Figure 1—source data 1
Immunoprecipitation in Figure 1C.
- https://cdn.elifesciences.org/articles/85560/elife-85560-fig1-data1-v1.zip
Figure 2 with 1 supplement
US10 blocks human leucocyte antigen class I (HLA-I) interaction with the peptide loading complex (PLC).
(A) Control HeLa cells or cells stably expressing US10 were induced by IFNγ overnight and subsequently metabolically labeled for 30 min and chased as indicated. After immunoprecipitation by W6/32, …
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Figure 2—source data 1
Immunoprecipitation in Figure 2A.
- https://cdn.elifesciences.org/articles/85560/elife-85560-fig2-data1-v1.zip
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Figure 2—source data 2
Immunoprecipitation in Figure 2C.
- https://cdn.elifesciences.org/articles/85560/elife-85560-fig2-data2-v1.zip
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Figure 2—source data 3
Immunoprecipitation in Figure 2E.
- https://cdn.elifesciences.org/articles/85560/elife-85560-fig2-data3-v1.zip
Figure 2—figure supplement 1
US10 blocks human leucocyte antigen class I (HLA-I) interaction with the peptide loading complex (PLC).
(A) Experiment from Figure 2C showing the full size of the gel (Figure 2—figure supplement 1—source data 1). (B) Alignment of the sequences of HeLa HLA-I (HLA-A*68:02, -B*15:03, -C*12:03 and -E). …
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Figure 2—figure supplement 1—source data 1
Immunoprecipitation in Figure 2—figure supplement 1A.
- https://cdn.elifesciences.org/articles/85560/elife-85560-fig2-figsupp1-data1-v1.zip
Figure 3 with 1 supplement
Higher tapasin dependency in HLA-B correlates with increased sensitivity to US10.
(A) Wild-type or tapasin knockout HeLa cells were transiently co-transfected as indicated and treated with IFNγ overnight. Cell surface expression of the HA-tagged molecules or non-tagged HLA-A2E*01:…
Figure 3—figure supplement 1
HLA-B regulation by US10 correlates with HLA-B tapasin dependency.
(A) Wild-type and two clones of tapasin KO HeLa cells (31-I and 102-I) were metabolically labeled for 2 hr. Immunoprecipitations using anti-tapasin and anti-ERp57 were performed. Red asterisk: …
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Figure 3—figure supplement 1—source data 1
Immunoprecipitation in Figure 3—figure supplement 1A.
- https://cdn.elifesciences.org/articles/85560/elife-85560-fig3-figsupp1-data1-v1.zip
Figure 4 with 1 supplement
US10 binding to β2m/HC heterodimers correlates with human leucocyte antigen class I (HLA-I) endoplasmic reticulum (ER) retention.
(A) HLA-I KO HeLa cells were transiently co-transfected with indicated HA-HLA-I-expressing plasmids comprising a mutated gRNA binding site together with a US10- or a control-pIRES-EGFP plasmid. To …
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Figure 4—source data 1
Immunoprecipitations with W6/32 and anti-HA in Figure 4A.
- https://cdn.elifesciences.org/articles/85560/elife-85560-fig4-data1-v1.zip
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Figure 4—source data 2
Immunoprecipitation with anti-US10 in Figure 4A.
- https://cdn.elifesciences.org/articles/85560/elife-85560-fig4-data2-v1.zip
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Figure 4—source data 3
Immunoprecipitation in Figure 4C.
- https://cdn.elifesciences.org/articles/85560/elife-85560-fig4-data3-v1.zip
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Figure 4—source data 4
Immunoprecipitation in Figure 4D.
- https://cdn.elifesciences.org/articles/85560/elife-85560-fig4-data4-v1.zip
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Figure 4—source data 5
Immunoprecipitation in Figure 4F.
- https://cdn.elifesciences.org/articles/85560/elife-85560-fig4-data5-v1.zip
Figure 4—figure supplement 1
US10 forms stable complexes with assembled HLA-C and -G, but not with HLA-A and -B.
(A) Immunoprecipitations were performed as described in Figure 4A using the antibodies anti-β2m and anti-US10 (Figure 4—figure supplement 1—source data 1). (B) The ratio (US10/control) of single …
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Figure 4—figure supplement 1—source data 1
Immunoprecipitations in Figure 4—figure supplement 1A.
- https://cdn.elifesciences.org/articles/85560/elife-85560-fig4-figsupp1-data1-v1.zip
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Figure 4—figure supplement 1—source data 2
Immunoprecipitations in Figure 4—figure supplement 1E.
- https://cdn.elifesciences.org/articles/85560/elife-85560-fig4-figsupp1-data2-v1.zip
Figure 5 with 1 supplement
Quantitative human leucocyte antigen class I (HLA-I) ligandome analysis confirms genotype-dependent effects by US10.
(A) Wild-type HeLa cells or TagBFP-T2A-US10i clone #5 were treated with doxycycline (0.33 µg/mL) or DMSO for 24 hr. Subsequently. cells were stained by anti-CD71, anti-Bw6, or anti-HLA-C antibodies …
Figure 5—figure supplement 1
Quantitative human leucocyte antigen class I (HLA-I) ligandome analysis confirms genotype-dependent effects by US10.
(A, B) TagBFP-T2A-US10i HeLa cell pool and cell clones and wild-type HeLa cells were treated with doxycycline (0.33 µg/mL) for 24 hr. In (A), TagBFP expression was analyzed by flow cytometry (dot …
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Figure 5—figure supplement 1—source data 1
Immunoprecipitations in Figure 5—figure supplement 1B.
- https://cdn.elifesciences.org/articles/85560/elife-85560-fig5-figsupp1-data1-v1.zip
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Figure 5—figure supplement 1—source data 2
Immunoprecipitations in Figure 5—figure supplement 1C.
- https://cdn.elifesciences.org/articles/85560/elife-85560-fig5-figsupp1-data2-v1.zip
Figure 6 with 2 supplements
Downregulation of overlapping US10 and US11 transcripts in human cytomegalovirus (HCMV)-infected cells rescues human leucocyte antigen class I (HLA-I) interaction with the peptide loading complex (PLC).
(A) MRC-5 fibroblasts were nucleofected with US10-specific or non-targeting siRNA 24 hr prior to mock treatment or infection with HCMV ΔUS2-6 mutant BAC2 at an MOI (multiplicity of infection) of 5. …
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Figure 6—source data 1
Immunoprecipitation in Figure 6B.
- https://cdn.elifesciences.org/articles/85560/elife-85560-fig6-data1-v1.zip
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Figure 6—source data 2
Immunoprecipitations in Figure 6C and D.
- https://cdn.elifesciences.org/articles/85560/elife-85560-fig6-data2-v1.zip
Figure 6—figure supplement 1
Overlapping US10 and US11 transcripts in human cytomegalovirus (HCMV)-infected cells.
(A) Genome browser showing the US10/11 locus. The tracks show (from top to bottom) the genomic position, identified sequence motifs, the known open-reading frames (ORFs) encoding US10 and US11, the …
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Figure 6—figure supplement 1—source data 1
Western blots in Figure 6—figure supplement 2.
- https://cdn.elifesciences.org/articles/85560/elife-85560-fig6-figsupp1-data1-v1.zip
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Figure 6—figure supplement 1—source data 2
Immunoprecipitation in Figure 6—figure supplement 2.
- https://cdn.elifesciences.org/articles/85560/elife-85560-fig6-figsupp1-data2-v1.zip
Figure 6—figure supplement 2
Downregulation of overlapping US10 and US11 transcripts in human cytomegalovirus (HCMV)-infected cells rescues human leucocyte antigen class I (HLA-I) interaction with the peptide loading complex (PLC).
(A) Depiction of the coding sequences (CDS) for US11 and US10 and binding sites for the primers used for RT-PCR in Figure 6A and of US10-targeting siRNA #1. (B) Stable HeLa-US10HA cells were …
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Figure 6—figure supplement 2—source data 1
Uncropped blots from Figure 6—figure supplement 2B.
- https://cdn.elifesciences.org/articles/85560/elife-85560-fig6-figsupp2-data1-v1.zip
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Figure 6—figure supplement 2—source data 2
Uncropped version of Figure 6—figure supplement 2E.
- https://cdn.elifesciences.org/articles/85560/elife-85560-fig6-figsupp2-data2-v1.zip
Figure 7 with 2 supplements
US10 siRNA treatment of human cytomegalovirus (HCMV)-infected cells has little effect on HLA-A, but induce HLA-B1027 antigen presentation.
(A, B) MRC-5 (A) or HF99/7 (B) fibroblasts were nucleofected with US10-specific or non-targeting siRNA 24 hr prior to mock treatment or infection at an MOI of 5 with HCMV ΔUS2-6 mutant BAC2 or with …
Figure 7—figure supplement 1
Human leucocyte antigen class I (HLA-I) analysis of US10 siRNA-treated human cytomegalovirus (HCMV)-infected fibroblasts.
Figure 7—figure supplement 2
CD8+ T-cell activation after co-culture with US10 siRNA-treated human cytomegalovirus (HCMV)-infected fibroblasts.
Figure 8
Model of human leucocyte antigen class I (HLA-I) geno- and allotype-dependent targeting by US10.
US10 (red) is able to bind to all HLA-I heavy chains (HCs) early after their synthesis and prior to dimerization with β2m. HLA-A (blue) and HLA-B (green colors) molecules can escape from US10 by …
Tables
Table 1
Primer sequences for molecular cloning.
| HA-HLA-A*24:02 |
|
| HA-HLA-C*04:01 |
|
| HA-HLA-C*05:01 |
|
| HA-HLA-G*01:01 |
|
| HA-H-2Kb |
|
| HA-MICA*004 |
|
| HLA-A2E*01:01 |
|
| ΔCRISPR2-HA-HLA-A*02:01 |
|
| ΔCRISPR2-HA-HLA-B*07:02 |
|
| ΔCRISPR2-HA-HLA-B*44:02 |
|
| ΔCRISPR2-HA-HLA-C*05:01 |
|
| ΔCRISPR2-HA-HLA-C*07:02 |
|
| ΔCRISPR2-HA-HLA-E*01:01 |
|
| ΔCRISPR2-HA-HLA-G*01:01 |
|
| RL8 |
|
| US10 |
|
| US10HA |
|
| US2 |
|
| US2HA |
|
| US3HA |
|
| US9HA |
|
| UL40 |
|
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*
Primers written in italics are identical.
