Fig 1.
Changes in the relative abundance of HLA class I ligands in HCMV-infected fibroblasts.
MRC-5 cells infected with ΔUS2-6 or ΔUS2-6/US11 HCMV mutants at a multiplicity of infection (MOI) of 5 were collected at 48 h post-infection and MHC-I molecules were immunoprecipitated using the mAb W6/32. Peptide ligands were eluted and analyzed by mass spectrometry. (A) Volcano plots of changes in the relative abundances of HLA ligands in the two samples (Rep. #1 and #2). Each dot represents a specific HLA ligand. Log2-fold-changes of their abundance in US11+ compared to US11- infection are indicated on the x-axis, the corresponding significance levels after Benjamini-Hochberg correction are given on the y-axis. HLA ligands showing significant up- or downregulation (>4-fold change in abundance with p<0.01) are highlighted in red and blue, respectively. The numbers and percentages of significantly modulated ligands are specified in the corresponding quadrants. The reproducibility of HLA peptidome analysis was assessed by plotting HLA-I peptide abundances in biological replicates of HCMV infected MRC-5 cells (S2A Fig) (B) Distribution of HLA restrictions among peptides significantly down-regulated (blue) or up-regulated (red), as identified in A. (C) MRC-5 cells were mock-treated or infected with ΔUS2-6 or ΔUS2-6/US11 deletion mutants at an MOI of 5. At 48 h post-infection MHC-I cell surface expression was analyzed by flow cytometry with mAbs as indicated. (D) Quantification of fold downregulation by US11 shown in (C), as ratio of MFI measured in ΔUS2-6/US11-infected cells relative to ΔUS2-6. Error bars show SEM for at least three independent experiments. Statistical analyses were performed applying one-way analysis of variance (ANOVA) followed by a Tuckey’s multiple comparisons method for all pairwise differences of means. (E) HeLa cells stably expressing HA-tagged (~) HLA-A*02:01, A*03:01, B*07:02 or CD99 molecules were transduced with lentiviruses encoding US11 in front of an IRES-EGFP sequence. At 0, 24 and 48 h post-transduction the cells were analyzed by flow cytometry using an anti-HA mAb. The MFI in EGFP+ cells relative to MFI at 0 h post transduction is depicted. Two independent biological replicates of the experiment with similar outcomes were performed.
Fig 2.
HLA locus specific downregulation by US11.
HeLa cells were transiently co-transfected with US11 or a control pIRES-EGFP plasmid (CMV major IE promoter) together with the indicated HA-tagged (~) HLA molecules expressed from the pUC-IP vector (SFFV U3 promoter). (A) At 20 h post-transfection HLA-I cell surface expression of EGFP positive cells was measured by flow cytometry using anti-HA mAbs. (B) The HLA-I expression from (A) was defined as ratio of the MFI in US11 expressing cells compared to control cells, the value of which was normalized to the downregulation of a control molecule (HA-CD99). Bars represent normalized mean values ± SEM from three independent experiments. Statistical analyses were performed to compare HLA-A or -B alleles among themselves, applying one-way ANOVA followed by a Tuckey’s multiple comparisons method for all pairwise differences of means. Endogenous HLA-I expressed in MRC-5 or HeLa cells are indicated. (C) At 20 h post-transfection cells were labeled with [35S]-Met/Cys for 30 min and chased for 0 or 45 min and an immunoprecipitation was performed using anti-HA mAb. An uncropped autoradiography is depicted in S3 Fig. (D) Whole cell lysates were prepared and digested with EndoH prior to analysis by Western blot with antibodies as indicated. Equal loading of lysates was controlled by Ponceau S staining. (E) Cells were analyzed as described in A. In addition, the cells were incubated with LIR1-Fc. In the upper panel binding of LIR1-Fc to the CD99/ctrl transfected cells is shown in green. Representatives of two independent biological replicates with similar outcomes are shown.
Fig 3.
Analysis of factors that could affect HLA-B resistance against US11.
(A) HeLa cells were transiently co-transfected with US11 or a ctrl pIRES-EGFP plasmid together with indicated HA-tagged (~) HLA in pIRES-EGFP (CMV major IE promoter). At 20 h post-transfection cells were labeled with [35S]-Met/Cys for 30 min and chased for 0 or 45 min and a co-immunoprecipitation experiment was performed using anti-HA mAb or anti-US11 antiserum. A complete autoradiography is depicted in S5 Fig. A representative of two independent biological replicates with similar outcomes is shown. (B-C) HeLa cells were transiently co-transfected with US11 or a ctrl pIRES-EGFP plasmid together with indicated HA-tagged (~) MHC-I molecules and mutants encoded by the pUC-IP vector. At 20 h post-transfection flow cytometry and statistical analysis were performed as described in Fig 2A and 2B. (D) β2m-deficient FO-1 cells were co-transfected with US11 or a control pIRES-EGFP plasmid together with indicated HA-tagged (~) HLA alleles encoded by the pUC-IP vector. At 20 h post-transfection cell surface expression of EGFP positive cells was measured by flow cytometry using anti-HA mAbs. (E) FO-1 cells were transfected as described in (D). At 20 h post-transfection cells were labeled with [35S]-Met/Cys for 1 or 3 h and an immunoprecipitation was performed using anti-HA antibodies. A representative of two independent biological replicates with similar outcomes is shown. A complete autoradiography is depicted in S6 Fig.
Fig 4.
US11 co-immunoprecipitation with the PLC in HCMV-infected cells.
(A) MRC-5 cells were treated with US11_1 (US11) or control (ctrl) siRNA (S7A Fig) 4 h prior to mock treatment (m) or infection with the ΔUS2-6 HCMV mutant at an MOI of 5. At 24 h post-infection cells were metabolically labeled with [35S]-Met/Cys for 2 h and an immunoprecipitation using anti-ERp57 or W6/32 mAbs was performed. (B) MRC-5 cells were mock treated or infected with ΔUS2-6 or ΔUS2-6/US11 HCMV mutants at an MOI of 5. At 24 h post-infection, cells were metabolically labeled with [35S]-Met/Cys for 2 h and an immunoprecipitation using W6/32 or anti-ERp57 antibodies was performed. In addition, anti-ERp57 recovered proteins were dissociated and re-immunoprecipitated using an anti-US11 antiserum. The gel image is displayed with various contrast and light conditions depending on the antibodies (marked by dotted lines) used. The image is displayed with same conditions for all parts in S7B Fig.
Fig 5.
The N-terminal LCR of US11 is required for MHC-I ER retention but not for degradation.
(A) Schematic presentation of HA-tagged US11 mutants stably transduced into HeLa cells. SP, signal peptide; HA, HA-tag, LCR, low-complexity region; N-glyc, N-glycosylation site; Ig-like, immunoglobulin-like domain; TM, transmembrane domain; asterisk, Q192A mutation. (B) Whole cell lysates of HeLa cells expressing HA-tagged US11 variants and US6-HA and US3 were analyzed by Western blot using HC10 for detection of MHC-I HC and anti-HA for detection of viral proteins. Equal loading of lysates was controlled by Ponceau S staining. (C) Analysis of MHC-I cell surface expression by flow cytometry using W6/32. (D) Stably transduced HeLa cells were labeled with [35S]-Met/Cys for 2 h and co-immunoprecipitation was performed using anti-HA, anti-tapasin, W6/32 or anti-ERp57 antibodies. W6/32 samples were subjected to EndoH (EH) digest. Black asterisk indicates US11 specific bands; the green asterisk indicates EndoH digested US11. The black vertical bar marks EndoH digested MHC-I HCs. The red asterisk marks co-immunoprecipitated endogenous HLA-A*68:02. The dotted vertical line marks separate gels. (E) HeLa cells stably transduced with US11 constructs as indicated were labeled with [35S]-Met/Cys for 2 h and immunoprecipitation was performed using anti-US11, W6/32 or HC10 antibodies. A long exposure of the gel is depicted in S9 Fig. A representative of at least two independent biological replicates with similar outcomes is shown in each panel.
Fig 6.
Resistant HLA-B in cells ectopically expressing US11.
(A) Stable HA-US11-HeLa cells (~US11) or control cells (-) were treated for 36 h with IFN-γ (500 U/ml) or left untreated. Cells were labeled with [35S]-Met/Cys for 2 h and immunoprecipitation was performed using indicated antibodies. Distinct MHC-I HCs are indicated with blue and red asterisks. A representative of two independent biological replicates with similar outcomes is shown. (B) Hela cells were transiently transfected with HA-tagged MHC-I encoded by the pUC-IP vector. At 20 h post-transfection an immunoprecipitation experiment as described in (A) was performed.
Fig 7.
Anchor residue usage of HLA-B ligands is modified by US11.
(A-B) Analysis of HLA class I ligands was performed in control HeLa cells or HeLa cells stably transduced with HA-tagged (~) US11Q/A, ΔLCRUS11Q/A or US3. Cells were collected and MHC-I molecules were isolated using the mAb W6/32. Peptide ligands were eluted and analyzed by mass spectrometry. (A) The relative distribution of MHC-I specific 9-mer ligands between HLA-A*68:02 and B*15:03 is shown. (B) The frequency of P2 peptide anchor residues of HLA-B*15:03 9-mer ligands was determined and depicted as percentage of total pool at that specific position. Two independent biological replicates of the experiment are shown (#1 and #2). (C-D) Pooled #1 and #2 ligands from Fig 1A predicted by NetMHC3.4 to bind to HLA-B*07:02 and B*44:02 with an affinity of <500 and <1000 nM, respectively, were divided into common and unique ΔUS2-6 and ΔUS2-6/US11 ligands respectively (S13A Fig). From these pools the frequency of specific amino acids (x-axis) at positions P1 and P3 of HLA-B*07:02 (C) and positions P3 and P4 of HLA-B*44:02 (D) was determined and depicted as percentage of total pool at that specific position.
Table 1.
Number of 9-mer MHC-I ligands isolated from HeLa cell lines.
Fig 8.
Model for US11 regulation of HLA-A and HLA-B.
The HC of MHC-I dimerizes with β2m and is recruited to the PLC, where peptide (green) loading takes place. The stabilized MHC-I/peptide complex leaves the PLC for cell surface expression through the secretory pathway. In the presence of US11 (yellow), MHC-I molecules are redirected for proteasomal degradation via an ERAD pathway. This function can be executed by US11 also without the LCR. HLA-B HCs comprise an intrinsic resistance against US11-mediated degradation and dimerize more efficiently with β2m in the presence of US11. In an LCR-dependent manner US11 modifies peptide (orange) loading. The relative effect of US11 on HLA-A and HLA-B is depicted with arrows, the thickness of which is indicative of the efficacy of degradation (red) and surface expression (black).
Table 2.
Primer sequences used for cloning.