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. 2013 Nov;36(5):455-64.
doi: 10.1007/s10059-013-0221-8. Epub 2013 Nov 8.

Human cytomegalovirus (HCMV) US2 protein interacts with human CD1d (hCD1d) and down-regulates invariant NKT (iNKT) cell activity

Jihye Han  1 , Seung Bae Rho, Jae Yeon Lee, Joonbeom Bae, Se Ho Park, Suk Jun Lee, Sang Yeol Lee, Curie Ahn, Jae Young Kim, Taehoon Chun
Affiliations

Human cytomegalovirus (HCMV) US2 protein interacts with human CD1d (hCD1d) and down-regulates invariant NKT (iNKT) cell activity

Jihye Han et al. Mol Cells. 2013 Nov.

Abstract

To avoid host immune surveillance, human cytomegalovirus (HCMV) encoded endoplasmic reticulum (ER)-membrane glycoprotein US2, which interferes with antigen presenting mechanism of Major histocompatibility complex (MHC) class Ia and class II molecules. However, not many attempts have been made to study the effect of HCMV US2 on the expression of MHC class Ib molecules. In this study, we examined the effect of HCMV US2 on the expression and function of human CD1d (hCD1d), which presents glycolipid antigens to invariant NKT (iNKT) cells. Our results clearly showed that the physiological interaction between ER lumenal domain of HCMV US2 and α3 domain of hCD1d was observed within ER. Compared with mature form of hCD1d, immature form of hCD1d is more susceptible to ubiquitin-dependent proteasomal degradation mediated by HCMV US2. Moreover, the ectopic expression of HCMV US2 leads to the down-modulation of iNKT cell activity without significant change of hCD1d expression. These results will advance our understanding of the function of HCMV US2 in immune evasive mechanisms against anti-viral immunity of iNKT cells.

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Figures

Fig. 1
Fig. 1
Mapping of the critical interaction region between hCD1d and HCMV US2 using the yeast two-hybrid system. The cDNA constructs were co-transformed into EGY48 yeast cells to test a protein-protein interaction within the yeast two-hybrid system. The results are representative of three separate experiments. Data are shown as means ± SEM. (A) Upper panel shows the schematic representation of cDNA constructs for each hCD1d (CD1d) deletion mutant and full-length HCMV US2 (US2) fusion proteins in the yeast two-hybrid system. Lower panel shows the result of protein-protein interaction determined in the yeast two-hybrid system. The values of β-galactosidase activity (Unit) measured by σNPG assays are indicated below the corresponding lanes. (B) Upper panel shows the schematic representation of cDNA constructs for each HCMV US2 (US2) deletion mutant and full-length hCD1d (CD1d) fusion proteins in the yeast two-hybrid system. Lower panel shows the result of protein-protein interaction determined in the yeast two-hybrid system. The values of β-galactosidase activity (Unit) measured by σNPG assays are indicated below the corresponding lanes. (C) Interaction between cDNA constructs for an hCD1d (CD1d) deletion mutant (Val203-Trp295) and three HCMV US2 (US2) deletion (Met1-Leu21, Pro22-Thr160 and His161-Cys199) fusion proteins in the two-hybrid system. “No” means empty vector only.
Fig. 2
Fig. 2
HCMV US2 and hCD1d interact in the ER. Overexpression of the HCMV US2 in C1R.CD1d cells and Jurkat cells (A, B, and C). (A) Detection of HCMV US2 (US2) mRNA transcripts in C1R.CD1d cells and Jurkat cells by RT-PCR. The Gapdh mRNA transcripts were used as a loading control. Mock means empty vector transfectants and US2 means HCMV US2 transfectants. (B) Flow cytometry analyses of the EGFP fusion HCMV US2 protein (EGFP-US2) expression in C1R.CD1d cells and Jurkat cells. Mock means empty EGFP vector transfectants and US2 means EGFP-US2 transfectants. Dotted line, isotype control; solid line, EGFP expression. (C) To measure transfection efficiency, cells were visualized under confocal microscopy after transfection. Control means untransfectants, Mock means empty EGFP vector transfectants and US2 means EGFP-US2 transfectants. The results are representative of three independent experiments. (D) Confocal microscopy analyses shows co-localization of HCMV US2 and hCD1d in the ER. The top panel shows hCD1d (CD1d) is expressed in intracellular compartment and cell surface, and a portion of hCD1d molecules is merged with calreticulin, ER specific marker. The middle panel shows HCMV US2 (US2) is existed in ER. The bottom panel indicates HCMV US2 (US2) and hCD1d (CD1d) proteins are partially co-localized in the ER. The results are representative of three independent experiments.
Fig. 3
Fig. 3
Destabilization of hCD1d expression by HCMV US2. Analyses on the stability hCD1d proteins in empty vector transfectants (Mock) and HCMV US2 transfectants (US2) (A, B). (A) hCD1d protein level in CD1d. C1R cells at 0 h, 2 h, 4 h, and 8 h after cycloheximide treatment were determined by immunoblotting. Anti-β actin was used as an internal control. Mock means empty vector transfectants and US2 means HCMV US2 transfectants. The results are representative of three independent experiments. (B) hCD1d protein level in Jurkat cells at 0 h, 2 h, 4 h, and 8 h after cycloheximide treatment were determined by immunoblotting. Anti-β actin was used as an internal control. Mock means empty vector transfectants and US2 means HCMV US2 transfectants. The results are representative of three independent experiments. (C) Ubiquitination assays of hCD1d proteins. Immunoblot analysis of ubiquitin conjugation level of CD1d.C1R cells with or without HCMV US2. Cells were treated with MG-132, proteasome inhibitor, harvested, and resuspended with NP-40 lysis buffer. Upper panel shows the immunoprecipitation (IP) of cell lysates using 51.1 antibody (51.1) and Western blot analyses (WB) using anti-ubiquitin antibody. The 51.1 antibody (51.1) detects only mature form of hCD1d. Lower panel shows the immunoprecipitation (IP) of cell lysates using NOR3.2 antibody and Western blot analyses (WB) using anti-ubiquitin antibody. The NOR3.2 antibody (NOR3.2) detects both immature and mature form of hCD1d. The equal amounts of immunoprecipitated hCD1d proteins are confirmed by Western blot analyses using C3D5 antibody and the β-actin was used as the loading control. The results are representative of three independent experiments.
Fig. 4
Fig. 4
The ectopic expression of HCMV US2 does not significantly change the expression of hCD1d. (A) Flow cytometry analyses on the empty vector transfected C1R.CD1d cells (Mock) and HCMV US2 transfected C1R.CD1d cells (US2). Cells were harvested and stained with PE conjugated anti-hCD1d antibody (CD1d42 clone, mouse IgG1κ) and PE conjugated isotype control (mouse IgG1κ). (B) Flow cytometry analyses on the empty vector transfected Jurkat cells (Mock) and HCMV US2 transfected jurkat cells (US2). Cells were harvested and stained with PE conjugated anti-hCD1d antibody (CD1d42 clone, mouse IgG1κ), PE conjugated anti-HLA class Ia (HLA-A, B, C; G46-2.6 clone, mouse IgG1κ) antibody and PE conjugated isotype control (mouse IgG1κ).
Fig. 5
Fig. 5
Down-regulation of iNKT cell activity by HCMV US2. (A) The empty vector transfected Jurkat cells (Mock) and US2 transfected Jurkat cells (US2) were treated with various concentration of the synthetic KRN7000 and inactivated. Then, inactivated cells were co-cultured with the iNKT cells (DN32.D3). After three days, IL-2 concentrations in culture supernatants were determined by ELISA. Data are shown are means ± SEM. (B) The empty vector transfected C1R.CD1d cells (Mock) and US2 transfected C1R.CD1d cells (US2) were treated with 0.5 μg/ml of the synthetic KRN7000 and inactivated. Then, inactivated cells were co-cultured with the iNKT cells (DN32.D3). After three days, IL-2 and IFN-γ concentrations in culture supernatants were determined by ELISA. Data are shown are means ± SEM.
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