Differential susceptibility of RAE-1 isoforms to mouse cytomegalovirus

Abstract

The NKG2D receptor is one of the most potent activating natural killer cell receptors involved in antiviral responses. The mouse NKG2D ligands MULT-1, RAE-1, and H60 are regulated by murine cytomegalovirus (MCMV) proteins m145, m152, and m155, respectively. In addition, the m138 protein interferes with the expression of both MULT-1 and H60. We show here that one of five RAE-1 isoforms, RAE-1delta, is resistant to downregulation by MCMV and that this escape has functional importance in vivo. Although m152 retained newly synthesized RAE-1delta and RAE-1gamma in the endoplasmic reticulum, no viral regulator was able to affect the mature RAE-1delta form which remains expressed on the surfaces of infected cells. This differential susceptibility to downregulation by MCMV is not a consequence of faster maturation of RAE-1delta compared to RAE-1gamma but rather an intrinsic property of the mature surface-resident protein. This difference can be attributed to the absence of a PLWY motif from RAE-1delta. Altogether, these findings provide evidence for a novel mechanism of host escape from viral immunoevasion of NKG2D-dependent control.

FIG. 1.

NKG2D-dependent virus control in vivo. (A) CBA/J, C3H/J, and BALB/c mice were injected i.v. with 3 × 10⁵ PFU of WT MCMV (MW97.01). C57BL/6 mice received the same dose of Δm157 MCMV. Mice were injected i.p. with PBS or with blocking anti-NKG2D MAbs. Virus titers were determined 3 days p.i. There were significant differences in virus titers in spleen between the groups of untreated mice and groups treated with anti-NKG2D MAbs: for CBA/J mice (P = 0.008) and for C57BL/6 mice (P = 0.038). Because of small number of animals per group (n = 3) for C3H/J mice, although the virus titer differences are indicative, there were no statistically significant differences. (B) C57BL/6 mice, injected i.p. with either PBS or blocking NKG2D MAbs either alone or in combination with cytolytic anti-CD4 and anti-CD8 MAbs, were injected i.v. with 2 × 10⁵ PFU of Δm157 virus. Virus titers were determined 7 days p.i. There were significant differences between the untreated group and the T-cell-depleted group (P = 0.029), as well as between the untreated group and the group treated with anti-NKG2D, anti-CD4, and anti-CD8 MAbs (P = 0.049). (C) DBA/2 mice were injected i.p. with PBS or blocking NKG2D MAbs or, in addition, with cytolytic anti-CD4 and anti-CD8 MAbs. The mice were also injected i.p. with 5 × 10⁴ PFU of SGV-WT MCMV. Virus titers were determined 11 days p.i. There were significant differences between the untreated group and the T-cell-depleted group (P = 0.049), as well as the untreated group and the group treated with anti-NKG2D, anti-CD4, and anti-CD8 MAbs (P = 0.029). Titers for individual mice (circles) and median values (horizontal bars) are shown.

FIG. 2.

Different susceptibilities of NKG2D ligands to MCMV. (A) NIH 3T3 and SVEC4-10 cells were infected for 12 h with one PFU of WT-gfp MCMV/cell or left uninfected. Cells were stained with PE-labeled NKG2D tetramer (filled histograms). AV-PE was used as a control (open histograms). (B) Expression of NKG2D ligands on SVEC4-10 cells was tested by specific MAbs: rat anti-MULT-1, mouse anti-RAE-1δ, rat anti-RAE-1ɛ, rat anti-RAE-1αβγ, and rat anti-H60, followed by biotinylated-goat anti-rat IgG or biotinylated-goat anti-mouse IgG and PE-labeled streptavidin (filled histograms). Isotype-matched rat IgG2a and mouse IgG1 MAbs were used as a negative control (open histograms). (C) SVEC4-10 and (D) C3H/J derived MEFs were infected for 12 h with one PFU of WT-gfp MCMV/cell or left uninfected. Cells were stained with PE-labeled NKG2D tetramer or with MAbs to RAE-1δ, RAE-1ɛ, and MULT-1 (filled histograms). Isotype-matched irrelevant MAbs and AV-PE were used as controls for staining with specific antibodies and tetramer, respectively (open histograms).

FIG. 3.

m152 affects the maturation of RAE-1 proteins. RAE-1γ- and RAE-1δ-transfected NIH 3T3 cells were infected with four PFU of WT MCMV or Δm152 MCMV/cell or left uninfected. Cells were analyzed with specific anti-RAE-1 MAbs by flow cytometry (A), by immunoblotting (IB) after immunoprecipitation (IP) with anti-FLAG M2 Sepharose (B), and by confocal microscopy (C).

FIG. 4.

The PLWY motif of RAE-1 protein modulates sensitivity to MCMV. (A) Comparison of the amino acid sequences of RAE-1 isoforms using the Vector NT AlignX program (NCBI Pub-Med database search). The PLWY motif (amino acids 49 to 52) is marked. (B) RAE-1γ-, RAE-1δ-, and RAE-1δ-PLWY-transfected NIH 3T3 cells were infected for 12 h with the indicated viruses and analyzed for the expression of RAE-1 isoforms using the anti-FLAG M2 MAb. (C and D) RAE-1δ- and RAE-1δ-PLWY-transfected NIH 3T3 cells were infected for 12 h with four PFU of indicated viruses/cell and analyzed either by immunoblotting (IB) using the anti-RAE-1 MAbs after the immunoprecipitation (IP) with anti-FLAG-M2 Sepharose (C) or by confocal microscopy (D).

FIG. 5.

Differential stability of RAE-1δ and RAE-1γ mature forms. (A) RAE-1δ-transfected NIH 3T3 cells were infected with four PFU of WT MCMV or Δm152 MCMV/cell or left uninfected. RAE-1δ was immunoblotted from EndoH-treated or untreated lysates with anti-RAE-1δ MAbs. (B) RAE-1δ- or empty vector-transfected NIH 3T3 cells were infected with four PFU of WT MCMV or left uninfected. Cells were metabolically labeled with 300 μCi of [³⁵S]methionine/ml 6 h to 8 h after infection and chased for the indicated periods of time, and immunoprecipitation was performed using anti-RAE-1δ MAbs, followed by protein G-Sepharose. (C) RAE-1δ- or RAE-1γ-transfected NIH 3T3 cells were labeled with 500 μCi of [³⁵S]methionine/ml for 1 h and chased for the indicated periods of time. After immunoprecipitation with either anti-RAE-1δ or anti-RAE-1γ MAbs followed by protein G-Sepharose, eluted proteins were treated with EndoH or left untreated. (D) RAE-1γ-, RAE-1δ-, and RAE-1δ-PLWY-transfected NIH 3T3 cells were untreated or treated for 12 h with tunicamycin (2 μg/ml). Cell lysates were immunoblotted using specific anti-RAE-1 antibodies. (E) RAE-1δ-, RAE-1γ-, or empty vector-transfected NIH 3T3 cells were metabolically labeled with 300 μCi of [³⁵S]methionine/ml for 2 h before infection with four PFU of WT MCMV. Cells were chased for indicated periods of time and immunoprecipitation was performed with either anti-RAE-1δ MAbs or anti-RAE-1γ antibodies followed by protein G-Sepharose. (F) RAE-1δ-, RAE-1γ-, and RAE-1δ-PLWY-transfected NIH 3T3 cells were surface biotinylated. After the indicated periods of time, RAE-1 molecules were immunoprecipitated using anti-FLAG M2-Sepharose, followed by immunoblotting with SA-POD. Arrows indicate different maturation forms of RAE-1 proteins: R, resistant to EndoH; S, sensitive to EndoH; D, digested with EndoH. RAE-1 Tun.R indicates the matured form of RAE-1 which is resistant to tunicamycin, whereas RAE-1 Tun.S indicates the deglycosylated form of RAE-1 proteins by tunicamycin.