Trophoblast class I major histocompatibility complex (MHC) products are resistant to rapid degradation imposed by the human cytomegalovirus (HCMV) gene products US2 and US11

Abstract

US11 and US2 encode gene products expressed early in the replicative cycle of human cytomegalovirus (HCMV), which cause dislocation of human and murine major histocompatibility complex (MHC) class I molecules from the lumen of the endoplasmic reticulum to the cytosol, where the class I heavy chains are rapidly degraded. Human histocompatibility leukocyte antigens (HLA)-C and HLA-G are uniquely resistant to the effects of both US11 and US2 in a human trophoblast cell line as well as in porcine endothelial cells stably transfected with human class I genes. Dislocation and degradation of MHC class I heavy chains do not appear to involve cell type-specific factors, as US11 and US2 are fully active in this xenogeneic model. Importantly, trophoblasts HLA-G and HLA-C possess unique characteristics that allow their escape from HCMV-associated MHC class I degradation. Trophoblast class I molecules could serve not only to block recognition by natural killer cells, but also to guide virus-specific HLA-C- and possibly HLA-G-restricted cytotoxic T-lymphocytes to their targets.

Figure 1

HLA-G and HLA-Cw*0401 from JEG 3 trophoblast-derived cells are not degraded in the presence of the HCMV gene product US11. Approximately 10⁷ JEG 3 cells were either infected with a vaccinia virus expressing the murine class I heavy chain, K^b (lacking its cytoplasmic tail, VVK^b, MOI = 5), or doubly infected with VVK^b (MOI = 5) and a vaccinia virus expressing the HCMV gene product US11 (VVUS11, MOI = 5). Cells were metabolically pulse labeled for 15 min with [³⁵S]methionine and chased with unlabeled media for 0 and 30 min. Lysates of these cells were sequentially immunoprecipitated with an antibody against mouse heavy chain (RafHC), with W6/32, and with αUS11.

Figure 2

In vitro–translated HLA-A2 associates with cotranslated US2 and β₂m; in vitro– translated trophoblast MHC class I heavy chains do not. (A) Human MHC class I heavy chains (HLA-A2, HLA-G, and HLA-C) were cotranslated in vitro with human β₂m and US2. Radioactively labeled translation products were either separated directly by SDS-PAGE, or were immunoprecipitated with W6/32 before electrophoretic analysis. As indicated, components of the cotranslation are present at the anticipated position in the direct load samples. (B) US2 fails to be coprecipitated with HLA-G and β₂m, despite optimization of the relative quantities of translated heavy chain, β₂m, and US2. Increasing amounts of transcribed US2 were added to the cotranslation mixture of HLA-G, β₂m, and US2 to allow relative quantities of class I heavy chain to US2 similar to those demonstrated for HLA-A2 in A. Lanes 1–4 represent separate translation mixtures in which the amounts of transcribed HLA-G and β₂m template were constant but transcribed US2 template was present at a ratio of 2:1, 5:1, 10:1, and 20:1, respectively. W6/32 immunoprecipitation and electrophoresis were as described above.

Figure 2

In vitro–translated HLA-A2 associates with cotranslated US2 and β₂m; in vitro– translated trophoblast MHC class I heavy chains do not. (A) Human MHC class I heavy chains (HLA-A2, HLA-G, and HLA-C) were cotranslated in vitro with human β₂m and US2. Radioactively labeled translation products were either separated directly by SDS-PAGE, or were immunoprecipitated with W6/32 before electrophoretic analysis. As indicated, components of the cotranslation are present at the anticipated position in the direct load samples. (B) US2 fails to be coprecipitated with HLA-G and β₂m, despite optimization of the relative quantities of translated heavy chain, β₂m, and US2. Increasing amounts of transcribed US2 were added to the cotranslation mixture of HLA-G, β₂m, and US2 to allow relative quantities of class I heavy chain to US2 similar to those demonstrated for HLA-A2 in A. Lanes 1–4 represent separate translation mixtures in which the amounts of transcribed HLA-G and β₂m template were constant but transcribed US2 template was present at a ratio of 2:1, 5:1, 10:1, and 20:1, respectively. W6/32 immunoprecipitation and electrophoresis were as described above.

Figure 3

HLA-A2 is degraded in porcine endothelial cells in the presence of either US2 or US11. Approximately 10⁷ 2A2-A2/8.3, HLA-A2–expressing porcine endothelial cells were either infected with a vaccinia virus driving the expression of US2 (VVUS2, MOI = 5), with VVUS11 (MOI = 5), or left uninfected (Uninf). Cells were pretreated with the proteasome inhibitor, ZL₃H, for 45 min, pulse labeled for 15 min with [³⁵S]methionine, and then chased with unlabeled media for 0 and 30 min (all in the presence of ZL₃H). Lysed cells were sequentially immunoprecipitated with a polyclonal antibody against class I heavy chain, RαHC, and with αUS11 and αUS2. 10 × 10⁶ parental 2A2 cells were included as a specificity control for the RαHC antibody and were pretreated, pulse labeled, and lysed as above. These lysates were immunoprecipitated with RαHC only. (A) Immunoprecipitations with the polyclonal anti–human MHC class I heavy chain antibody, RαHC. (B) Immunoprecipitations with polyclonal antibodies raised against US2 and US11 (αUS2 and αUS11, respectively).

Figure 3

HLA-A2 is degraded in porcine endothelial cells in the presence of either US2 or US11. Approximately 10⁷ 2A2-A2/8.3, HLA-A2–expressing porcine endothelial cells were either infected with a vaccinia virus driving the expression of US2 (VVUS2, MOI = 5), with VVUS11 (MOI = 5), or left uninfected (Uninf). Cells were pretreated with the proteasome inhibitor, ZL₃H, for 45 min, pulse labeled for 15 min with [³⁵S]methionine, and then chased with unlabeled media for 0 and 30 min (all in the presence of ZL₃H). Lysed cells were sequentially immunoprecipitated with a polyclonal antibody against class I heavy chain, RαHC, and with αUS11 and αUS2. 10 × 10⁶ parental 2A2 cells were included as a specificity control for the RαHC antibody and were pretreated, pulse labeled, and lysed as above. These lysates were immunoprecipitated with RαHC only. (A) Immunoprecipitations with the polyclonal anti–human MHC class I heavy chain antibody, RαHC. (B) Immunoprecipitations with polyclonal antibodies raised against US2 and US11 (αUS2 and αUS11, respectively).

Figure 4

HLA-G stably expressed in porcine endothelial cells is resistant to class I heavy chain degradation associated with US2 and US11. Identical experiments to those described for Fig. 3 were performed using 2A2-HLA-G/1.2 cells, a porcine endothelial cell line stably transfected with HLA-G. For these experiments, primary RαHC immunoprecipitates were reimmunoprecipitated with RαHC before SDS-PAGE. (A) Immunoprecipitations with the polyclonal anti–human MHC class I heavy chain antibody, RαHC. (B) Immunoprecipitations with polyclonal antibodies raised against US2 and US11 (αUS2 and αUS11, respectively). Uninf, Uninfected.

Figure 4

HLA-G stably expressed in porcine endothelial cells is resistant to class I heavy chain degradation associated with US2 and US11. Identical experiments to those described for Fig. 3 were performed using 2A2-HLA-G/1.2 cells, a porcine endothelial cell line stably transfected with HLA-G. For these experiments, primary RαHC immunoprecipitates were reimmunoprecipitated with RαHC before SDS-PAGE. (A) Immunoprecipitations with the polyclonal anti–human MHC class I heavy chain antibody, RαHC. (B) Immunoprecipitations with polyclonal antibodies raised against US2 and US11 (αUS2 and αUS11, respectively). Uninf, Uninfected.

Figure 5

HLA-Cw3 stably expressed in porcine endothelial cells is resistant to class I heavy chain degradation associated with US2 and US11. Identical experiments to those described for Figs. 3 and 4 were performed using 2A2-HLA-Cw3/7 cells, a porcine endothelial cell line stably transfected with HLA-Cw3. (A) Immunoprecipitations with the polyclonal anti–human MHC class I heavy chain antibody, RαHC. (B) Immunoprecipitations with polyclonal antibodies raised against US2 and US11 (αUS2 and αUS11, respectively). Uninf, Uninfected.

Figure 5

HLA-Cw3 stably expressed in porcine endothelial cells is resistant to class I heavy chain degradation associated with US2 and US11. Identical experiments to those described for Figs. 3 and 4 were performed using 2A2-HLA-Cw3/7 cells, a porcine endothelial cell line stably transfected with HLA-Cw3. (A) Immunoprecipitations with the polyclonal anti–human MHC class I heavy chain antibody, RαHC. (B) Immunoprecipitations with polyclonal antibodies raised against US2 and US11 (αUS2 and αUS11, respectively). Uninf, Uninfected.