Human cytomegalovirus glycoprotein UL16 causes intracellular sequestration of NKG2D ligands, protecting against natural killer cell cytotoxicity

Abstract

The activating receptor, NKG2D, is expressed on a variety of immune effector cells and recognizes divergent families of major histocompatibility complex (MHC) class I-related ligands, including the MIC and ULBP proteins. Infection, stress, or transformation can induce NKG2D ligand expression, resulting in effector cell activation and killing of the ligand-expressing target cell. The human cytomegalovirus (HCMV) membrane glycoprotein, UL16, binds to three of the five known ligands for human NKG2D. UL16 is retained in the endoplasmic reticulum and cis-Golgi apparatus of cells and causes MICB to be similarly retained and stabilized within cells. Coexpression of UL16 markedly reduces cell surface levels of MICB, ULBP1, and ULBP2, and decreases susceptibility to natural killer cell-mediated cytotoxicity. Domain swapping experiments demonstrate that the transmembrane and cytoplasmic domains of UL16 are important for intracellular retention of UL16, whereas the ectodomain of UL16 participates in down-regulation of NKG2D ligands. The intracellular sequestration of NKG2D ligands by UL16 represents a novel HCMV immune evasion mechanism to add to the well-documented viral strategies directed against antigen presentation by classical MHC molecules.

Figure 1.

Soluble UL16 does not block ULBP-mediated cytotoxicity. Daudi cells expressing MHC class I and ULBP1 were used as targets in cytotoxicity assays using human NK cells, at the indicated effector/target ratios. Class I⁺/ULBP1⁺ Daudi targets were preincubated with the anti-ULBP1 Fab, M291, a control Fab, the UL16LZ protein, or a control LZ protein (RANK ligand-LZ), before the addition of effector cells. Individual data points are calculated from the averages of triplicate samples. The results shown are representative of three experiments using three separate donors.

Figure 2.

UL16 is a stable, highly glycosylated protein that localizes to the ER/cis-Golgi regions. (A) His16 cells were infected with AdtetUL16 and Adtet-trans (50 and 10 PFU/ml, respectively) for 20 h. Infected cells were labeled with [³⁵S]methionine, a 10-min pulse (labeled P), and chases for the indicated times (in minutes, labeled C). UL16 was immunoprecipitated from cell lysates with rabbit anti–UL16-C-terminal peptide sera. For endoH treatment, samples were divided in half and not treated (−) or treated (+) with endoH. Immunoprecipitates were analyzed by SDS-PAGE and autoradiography. Bands representing UL16 or UL16 with N-linked oligosaccharides removed (UL16-CHO) are indicated. Preimmune rabbit serum did not precipitate these bands (unpublished data). (B) His16 cells were infected with AdtetUL16 and Adtet-trans (25 and 5 PFU/cell, respectively). After 12 h the cells were fixed, permeabilized, and incubated simultaneously with rabbit anti–UL16-C (red) and mouse antibodies to the cellular markers calnexin (green) and gm130 (green) as indicated. Primary antibodies were visualized with secondary fluorescent antibodies using a scanning laser confocal microscope. No staining was seen with preimmune rabbit serum (unpublished data).

Figure 3.

UL16 reduces cell surface expression of NKG2D ligands. (A and B) His16 cells were infected with 50 PFU/cell AdtetMICB and coinfected with AdtetUL16 and Adtet-trans (30 and 6 PFU/cell, respectively) to induce UL16 expression, or with Adtet-trans alone as a negative control (36 PFU/cell) for 20 h. Cells were stained with anti-MICB M360 and analyzed by flow cytometry. In A: thin black line, AdtetUL16-infected cells; thick gray line, Adtet-trans infected cells; dotted line, secondary antibody control. B quantitates cell surface expression of MICB on cells infected with Adtet-trans versus AdtetUL16. (C) His16 cells were coinfected with AdtetMICA, AdtetMICB, or AdtetULBP2 and Adtet-trans (50 and 10 PFU/cell, respectively). The cells were also infected with AdtetUL16 and Adtet-trans (50 and 10 PFU/cell, respectively) or as negative control with AdtetUS9 and Adtet-trans (also 50 and 10 PFU/cell, respectively) for 20 h. Cells were stained with anti-MICA M673, anti-MICB M360, and anti-ULBP2 M311. The mean fluorescence in each sample was normalized to the values obtained when cells were transduced with each of the NKG2D ligands alone. These results are representative of three separate experiments.

Figure 4.

UL16 causes intracellular retention and stabilization of MICB. (A) MICB-transfected MBN15 cells (top) or His16 cells expressing MICA after infection with AdtetMICA and Adtet-trans (50 and 10 PFU/cell, respectively) were coinfected with AdtetUL16 (100 PFU/cell) or AdtetUS9 (100 PFU/cell) and Adtet-trans (20 PFU/cell) for 20 h or were left uninfected. Infected cells were labeled for 10 min with [³⁵S]methionine and the label was chased for 60 min. MICB and MICA were immunoprecipitated from cell extracts with mouse monoclonal antibodies M360 and 3H5, respectively, treated with endoH where indicated, and analyzed by SDS-PAGE and phosphorimager. The positions of molecular weight markers of 45 and 31 kD and of several species representing mature MICB and MICA, as well as MICB and MICA with N-linked oligosaccharides removed (MICB-CHO, MICA-CHO), are indicated. MICA- and MICB-specific bands were identified by comparison with immunoprecipitates using isotype control antibodies and untransfected or uninfected cells (unpublished data). (B) MBN15 cells infected with AdtetUS9 or AdtetUL16 as described above were labeled for 10 min with [³⁵S]methionine (labeled P) and the label was chased for the indicated times (in min, labeled C). MICB was immunoprecipitated as described above.

Figure 5.

MICB colocalizes with UL16 and is retained/stabilized by the interaction. MBN15 cells expressing MICB were infected with either AdtetUL16 (25 PFU/cell) in A, B, C, G, H, and I or AdtetUS9 (25 PFU/cell) in D, E, F, J, K, and L and Adtet-trans (5 PFU/cell; in all cases) for 12 h. The infected cells were fixed immediately (A–F) or incubated for 4 h with 100 μg/ml cycloheximide and then fixed (G–L). The cells were permeabilized and incubated simultaneously with anti-MICB M360 (green) and rabbit anti–UL16-C sera (red), followed by secondary fluorescent antibodies and visualized by confocal microscopy. No staining was seen with preimmune rabbit serum or isotype control monoclonal antibody (unpublished data).

Figure 6.

UL16 decreases cell surface expression of ULBP1, ULBP2, and MICB, but not ULBP3, in EL4 cells. (A) EL4 cells expressing the ULBPs or MICB were further transduced with retroviruses encoding both UL16 and GFP. Transduced cells were enriched by one round of fluorescence-activated cell sorting for cells expressing GFP, stained with specific monoclonal antibodies, and analyzed by flow cytometry. (B) EL4 cells transduced with ULBP2 and UL16 (ULBP2⁺/UL16 EL4) were further sorted by flow cytometry to obtain populations expressing high (BP2^hi/UL16 EL4) or low (BP2^low/UL16 EL4) cell surface levels of ULBP2. (C) Cell lysates were prepared from ULBP2⁺ EL4 cells, ULBP2⁺/UL16 EL4 cells, ULBP2^hi/UL16 EL4 cells, ULBP2^low/UL16 EL4 cells, or from CV-1 cells transiently transfected with an expression vector alone (vector) or with an expression vector containing the gene for UL16. Cell lysates were treated with N-glycanase and analyzed by immunoblotting with anti-UL16 M230. Blots were stripped and reprobed with anti-STAT5 to demonstrate equal protein loading.

Figure 6.

UL16 decreases cell surface expression of ULBP1, ULBP2, and MICB, but not ULBP3, in EL4 cells. (A) EL4 cells expressing the ULBPs or MICB were further transduced with retroviruses encoding both UL16 and GFP. Transduced cells were enriched by one round of fluorescence-activated cell sorting for cells expressing GFP, stained with specific monoclonal antibodies, and analyzed by flow cytometry. (B) EL4 cells transduced with ULBP2 and UL16 (ULBP2⁺/UL16 EL4) were further sorted by flow cytometry to obtain populations expressing high (BP2^hi/UL16 EL4) or low (BP2^low/UL16 EL4) cell surface levels of ULBP2. (C) Cell lysates were prepared from ULBP2⁺ EL4 cells, ULBP2⁺/UL16 EL4 cells, ULBP2^hi/UL16 EL4 cells, ULBP2^low/UL16 EL4 cells, or from CV-1 cells transiently transfected with an expression vector alone (vector) or with an expression vector containing the gene for UL16. Cell lysates were treated with N-glycanase and analyzed by immunoblotting with anti-UL16 M230. Blots were stripped and reprobed with anti-STAT5 to demonstrate equal protein loading.

Figure 7.

The UL16 transmembrane and cytoplasmic domains are involved in the intracellular retention of UL16, whereas the UL16 ectodomain is required for intracellular retention of ULBP2. (A) EL4 cells or EL4 cells transduced with a VSVG-pseudotyped retrovirus encoding UL16 and GFP were incubated in the presence or absence of 0.1% saponin to permeabilize cells. Cells were stained with anti-UL16 M230 or an isotype control antibody, fixed with 2% paraformaldehyde, and analyzed via flow cytometry. The histograms depict the mean fluorescence intensity (MFI) of cells stained with M230 minus the MFI of the cells stained with the control antibody. The results shown are representative of three separate experiments. (B) EL4 cells or EL4 cells transduced with a VSVG-pseudotyped retrovirus encoding the IL-4R/UL16 chimeric protein and GFP were stained as described above with anti-murine IL-4R M2 or an isotype control antibody. The histograms shown depict the MFI of cells stained with M2 minus the MFI of the cells stained with the isotype-matched control antibody. The results shown are representative of three separate experiments. (C) ULBP2⁺ EL4 cells were transduced with VSVG-pseudotyped retroviruses encoding UL16 and GFP, the UL16/IL-4R chimera and GFP, and the IL-4R/UL16 chimera and GFP. Transduced cells were enriched by fluorescence-activated cell sorting for cells expressing GFP, stained with anti-UL16 or with anti–IL-4R M2, and analyzed by flow cytometry. (D) ULBP2⁺ EL4 cells were transduced with VSVG-pseudotyped retroviruses encoding UL16 and GFP, the UL16/IL-4R chimera and GFP, and the IL-4R/UL16 chimera and GFP. Transduced cells were enriched by fluorescence-activated cell sorting of cells expressing GFP, stained with anti-ULBP2 M311, and analyzed by flow cytometry.

Figure 8.

Enhanced cytotoxicity of human and mouse NK cells against EL4 cells transduced with ULBPs or MICB. (A) EL4 cells expressing ULBP1, ULBP2, ULBP3, or MICB were tested as targets in cytotoxicity assays using mouse NK cells as effectors. Individual data points are calculated from the averages of triplicate samples. The results shown are representative of three separate experiments. (B) EL4 cells expressing ULBP2, ULBP3, or MICB were tested as targets in cytotoxicity assays using human NK cells as effectors. The results shown are from a single donor and are representative of three experiments using three separate donors.

Figure 9.

Cell surface expression of ULBP1, ULBP2, or MICB correlates with sensitivity to NK lysis. EL4 cells were transduced with amphotropic retroviruses encoding ULBP1 (A), ULBP2 (B), ULBP3 (C), or MICB (D). The ULBP- or MICB-expressing EL4 populations were further transduced with VSVG-pseudotyped retroviruses encoding both UL16 and GFP. Transduced cells were separated into two cell populations: those expressing GFP and UL16 (UL16⁺) and those not expressing GFP and UL16 (UL16⁻). Cell populations were used as targets in cytotoxicity assays using mouse NK cells (A–C) or human NK cells (D). The results shown are representative of three separate experiments.