NKG2D ligand MICA is retained in the cis-Golgi apparatus by human cytomegalovirus protein UL142

Abstract

Human cytomegalovirus (HCMV) evades T-cell recognition by down-regulating expression of major histocompatibility complex (MHC) class I and II molecules on the surfaces of infected cells. Contrary to the "missing-self" hypothesis, HCMV-infected cells are refractory to lysis by natural killer (NK) cells. Inhibition of NK cell function is mediated by a number of HCMV immune evasion molecules, which operate by delivering inhibitory signals to NK cells and preventing engagement of activating ligands. One such molecule is UL142, which is an MHC class I-related glycoprotein encoded by clinical isolates and low-passage-number strains of HCMV. UL142 is known to down-modulate surface expression of MHC class I-related chain A (MICA), which is a ligand of the activating NK receptor NKG2D. However, the mechanism by which UL142 interferes with MICA is unknown. Here, we show that UL142 localizes predominantly to the endoplasmic reticulum (ER) and cis-Golgi apparatus. The transmembrane domain of UL142 mediates its ER localization, while we propose that the UL142 luminal domain is involved in its cis-Golgi localization. We also confirm that UL142 down-modulates surface expression of full-length MICA alleles while having no effect on the truncated allele MICA*008. However, we demonstrate for the first time that UL142 retains full-length MICA alleles in the cis-Golgi apparatus. In addition, we propose that UL142 interacts with nascent MICA en route to the cell surface but not mature MICA at the cell surface. Our data also demonstrate that the UL142 luminal and transmembrane domains are involved in recognition and intracellular sequestration of full-length MICA alleles.

FIG. 1.

UL142 is localized predominantly to the ER and cis-Golgi apparatus. (A) Schematic of GFP-UL142, which is N-terminal GFP and Myc-tagged UL142. The amino acid sequences of the UL142 transmembrane domain (TMD) and cytoplasmic tail are shown. (B) HeLa cells transfected with GFP-UL142 (green) were fixed, permeabilized, and stained with antibodies against intracellular compartment markers (red) at 48 h posttransfection. Calreticulin served as a marker for the ER, GM130 was a cis-Golgi apparatus marker, and MHC class I was a marker for the cell surface. GFP-UL142 is localized predominantly to the ER and cis-Golgi apparatus. (C) Z-projection of high-resolution scans of GFP-UL142 (green)-expressing HeLa cells stained for the ER marker calreticulin (red) and cis-Golgi apparatus marker GM130 (blue). Magnified views of a small region showing the ER and cis-Golgi apparatus are also presented. The results show that GFP-UL142 is localized to the ER at 16 h posttransfection. (D) Mock-transfected HeLa cells and HeLa cells expressing GFP-UL142 or GFP-HLA-A2 were stained with rabbit anti-Myc antibody followed by Alexa Fluor 647-conjugated goat anti-rabbit IgG antibody. Surface expression of GFP-UL142 on a small proportion of transfected cells with very high levels of protein expression was observed. Control cell surface protein GFP-HLA-A2 was detected on all transfected cells regardless of the level of protein expression. As a control for the Myc staining, transfected cells were also stained with the Alexa Fluor 647-conjugated goat anti-rabbit IgG antibody alone. (E) HFFFs infected with HCMV for 72 h were transfected with GFP-UL142 (green) for the last 16 h of infection. Cells were stained for calreticulin as a marker of the ER (red) and immediate-early (IE) antigen as a marker of HCMV infection (gray). The results show that GFP-UL142 remains colocalized with the ER marker in virus-infected cells.

FIG. 2.

UL142 remains endo H sensitive following synthesis. (A, B, and C) HeLa cells transfected with GFP-UL142 or mock transfected were labeled with [³⁵S]methionine and [³⁵S]cysteine for 20 min and then chased in unlabeled methionine and cysteine for 0, 45, 180, or 360 min. GFP-UL142 was immunoprecipitated using either rabbit anti-UL142 sera (A) or monoclonal anti-GFP antibody (B). Fractions of the eluted proteins were digested with endo H and analyzed by gel electrophoresis. GFP-UL142 remained endo H sensitive and had disappeared by the 360-min chase time point. (D) The control cell surface protein MOG 25.1, which was analyzed in parallel, acquired endo H resistance after the 45-min chase. IP, immunoprecipitant; +, present; −, absent.

FIG. 3.

The UL142 transmembrane domain (TMD) mediates ER localization. (A) Schematic of CD8-WT and CD8-UL142 chimeras, which have an N-terminal Myc tag. (B) HeLa cells transfected with the CD8-WT, CD8-UL142Tail, CD8-UL142TMDTail, or CD8-UL142TMDonly construct were fixed and permeabilized and then stained with antibodies against the Myc tag (green) and MHC class I (red). MHC class I served as a marker for both the cell surface and the Golgi apparatus. CD8-UL142TMDTail and CD8-UL142TMDonly were localized predominantly intracellularly. (C) HeLa cells transfected with the CD8-UL142TMDTail or CD8-UL142TMDonly construct were fixed and permeabilized and then stained with antibodies against the Myc tag (green) and the ER marker calreticulin (red). CD8-UL142TMDTail and CD8-UL142TMDonly colocalized with calreticulin. (D) HeLa cells transfected with CD8-UL142TMDTail or CD8-UL142TMDonly were fixed and permeabilized and then stained with antibodies against the Myc tag (green) and the cis-Golgi apparatus marker GM130 (red). CD8-UL142TMDonly, but not CD8-UL142TMDTail, colocalized with GM130. (E) Western blot of mock-transfected HeLa cells and transfected HeLa cells expressing CD8-WT or a CD8-UL142 chimera. CD8 progresses through three maturation states as it traffics through the cell. CD8u is located in the ER, CD8i is located in the cis-Golgi apparatus, and CD8m is located at the cell surface. The chimeras CD8-UL142TMDTail and CD8-UL142TMDonly were predominantly in the CD8u state. Molecular mass differences among the maturation states of CD8-WT and those of the CD8-UL142 chimeras are due to the compositions of their domains.

FIG. 4.

UL142 down-modulates surface expression of full-length MICA alleles. (A) Sequence alignment of portions of the transmembrane domains (highlighted in gray) and cytoplasmic tails of full-length and truncated MICA alleles. (B) U373 cells express the full-length allele MICA*001, while HeLa cells express the truncated allele MICA*008. HeLa and U373 cells were transduced with RAd UL142 GFP, which expresses both UL142 and GFP, or RAd control GFP, which expresses GFP alone. Transduction efficiency of ∼100% was achieved. Ninety-six hours posttransduction, the cells were stained with anti-MICA, anti-ULBP2, or an isotype control antibody, followed by Alexa Fluor 647-conjugated secondary antibody. UL142 down-modulated expression of MICA*001 on the surfaces of U373 cells but not that of MICA*008 on the surfaces of HeLa cells. Surface expression of ULBP2 on cells of both lines was unaffected by UL142. Overlapping of histograms is depicted by cross-hatching.

FIG. 5.

UL142 retains full-length MICA alleles in the cis-Golgi apparatus. (A) HeLa cells transfected with either GFP-MICA*018 (GFP-tagged full-length MICA) or GFP-MICA*008 (GFP-tagged truncated MICA) were analyzed by confocal microscopy. Both alleles of MICA were localized to the cell surface and Golgi apparatus. (B) Immunofluorescence microscopy images of HeLa cells cotransfected with GFP-MICA*018 or GFP-MICA*008 (green) and FLAG-UL142 (red). Sixteen hours posttransfection, cells were fixed, permeabilized, and stained with antibodies against the FLAG tag and GM130 (blue), which served as a marker for the cis-Golgi apparatus. FLAG-UL142 prevents surface expression of GFP-MICA*018 by retaining the full-length MICA allele in the cis-Golgi apparatus. However, FLAG-UL142 has no effect on surface expression of the truncated allele GFP-MICA*008.

FIG. 6.

The UL142 luminal domain is required for intracellular retention of full-length MICA alleles. (A) HeLa cells cotransfected with GFP-MICA*018 (green) and CD8-UL142TMDTail (red) were fixed, permeabilized, and incubated with anti-UL142 rabbit sera. CD8-UL142TMDTail had no effect on surface expression of GFP-MICA*018. (B) Schematic of CD8-MICA*018TMDTail (a CD8 chimera with the transmembrane domain [TMD] and cytoplasmic tail of the full-length allele MICA*018), UL142Luminal-CD8TMDTail (a CD8 chimera with the luminal domain of UL142), and UL142LuminalTMD-CD8Tail (a CD8 chimera with the luminal and transmembrane domains of UL142). (C) HeLa cells cotransfected with CD8-MICA*018TMDTail and FLAG-UL142 were fixed and permeabilized and then stained with antibodies against the Myc (green) and FLAG (red) tags. FLAG-UL142 had no effect on the cell surface expression of CD8-MICA*018TMDTail. (D) HeLa cells cotransfected with UL142Luminal-CD8TMDTail or UL142LuminalTMD-CD8Tail (red) and untagged MICA*018 (green) were fixed and permeabilized and then stained with antibodies against the Myc tag (red) and MICA (green). Both chimeras were unable to intracellularly sequester MICA*018.