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. 2005 Jan 17;201(2):211-20.
doi: 10.1084/jem.20041617. Epub 2005 Jan 10.

NK cell activation through the NKG2D ligand MULT-1 is selectively prevented by the glycoprotein encoded by mouse cytomegalovirus gene m145

Astrid Krmpotic  1 , Milena Hasan, Andrea Loewendorf, Tanja Saulig, Anne Halenius, Tihana Lenac, Bojan Polic, Ivan Bubic, Anja Kriegeskorte, Ester Pernjak-Pugel, Martin Messerle, Hartmut Hengel, Dirk H Busch, Ulrich H Koszinowski, Stipan Jonjic
Affiliations

NK cell activation through the NKG2D ligand MULT-1 is selectively prevented by the glycoprotein encoded by mouse cytomegalovirus gene m145

Astrid Krmpotic et al. J Exp Med. .

Abstract

The NK cell-activating receptor NKG2D interacts with three different cellular ligands, all of which are regulated by mouse cytomegalovirus (MCMV). We set out to define the viral gene product regulating murine UL16-binding protein-like transcript (MULT)-1, a newly described NKG2D ligand. We show that MCMV infection strongly induces MULT-1 gene expression, but surface expression of this glycoprotein is nevertheless completely abolished by the virus. Screening a panel of MCMV deletion mutants defined the gene m145 as the viral regulator of MULT-1. The MCMV m145-encoded glycoprotein turned out to be necessary and sufficient to regulate MULT-1 by preventing plasma membrane residence of MULT-1. The importance of MULT-1 in NK cell regulation in vivo was confirmed by the attenuating effect of the m145 deletion that was lifted after NK cell depletion. Our findings underline the significance of escaping MULT-1/NKG2D signaling for viral survival and maintenance.

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Figures

Figure 1.
Figure 1.
NKG2D ligands are down-regulated by protein products of MCMV genes located between m144- m158 . (A) The HindIII cleavage map of the MCMV genome is shown (top) and the expanded region below indicates the positions of the m145 gene family members (shaded boxes) in the WT (w.t.) virus as well as the deletions (dashed lines) of the recombinant viruses. (B) NIH/3T3 cells were analyzed for expression of surface NKG2D ligands by staining with PE-NKG2D tetramer upon 12-h infection with indicated GFP-expressing viruses. Cells stained with streptavidin-PE were used as negative controls (dotted line). (C) NIH/3T3 and B12 cells were infected with indicated viruses and, after 12 h, stained with anti-H60 and anti–RAE-1α,β,γ mAbs, respectively. Cells stained with second antibody only were used as negative control (dotted line). (B and C) Each histogram represents 104 gated propidium iodide-negative, GFP-positive (infected) or GFP-negative (uninfected) cells.
Figure 2.
Figure 2.
Testing of anti–MULT-1 mAb. (A) CV-1 cells were infected with three MOI of WT VV or MULT-1-VV per cell and stained with anti–MULT-1 mAb 14 h after infection. (B) BALB/3T3 cells were stained with PE-NKG2D tetramer, anti–RAE-1α,β,γ or anti–MULT-1 antibodies. (A and B) Cells incubated with second antibody were used as a negative control (dotted line). Each histogram represents 104 gated propidium iodide–negative cells. (C) Upon metabolic labeling with [35S]methionine, lysates of MULT-1-3T3 cells were immunoprecipitated using anti–MULT-1 mAb coupled to protein G–Sepharose. Before separation by 11.5% SDS-PAGE, the samples were mock treated (−) or digested with EndoH (+).
Figure 3.
Figure 3.
The m145 protein down-regulates MULT-1. (A and B) SVEC4-10 cells were infected with 1 PFU of GFP-positive viruses per cell or left uninfected. 12 h after infection, cells were collected and stained with anti–MULT-1 mAb. Cells incubated with the second antibody upon binding to irrelevant primary antibody were used as a negative control (dotted line). Each histogram represents 104 gated propidium iodide-negative, GFP-positive cells (infected) or GFP-negative (uninfected) cells. n.i., noninfected cells. (C) CV-1 cells were infected with three MOI of WT VV, m145-VV, or MULT-1-VV or coinfected with MULT-1-VV/WT VV or MULT-1-VV/m145-VV per cell and, 14 h after infection, were analyzed for the expression of membrane-associated MULT-1 by staining with anti– MULT-1 mAb. Cells incubated with the second antibody in the absence of the primary antibody were used as a negative control (dotted line). Each histogram represents 104 gated propidium iodide-negative cells.
Figure 4.
Figure 4.
MCMV infection stimulates MULT-1 expression. (A) SVEC4-10 cells were infected for 12 h with 1 PFU of the indicated GFP-expressing viruses per cell or were mock infected. Total RNA was isolated from the cells, and the expression of MULT-1 mRNA was quantified by real-time RT-PCR. MULT-1 cDNA copies in each sample were normalized by measuring the β-actin cDNA copy numbers. Bars represent the ratio of MULT-1 and β-actin cDNA copy numbers in each sample. (B) SVEC4-10 cells were infected with 1 PFU of the indicated GFP-expressing viruses per cell, or left untreated and, upon 12 h, harvested and stained with anti–MULT-1 mAb. Histogram represents 104 gated propidium iodide–negative, GFP-positive (infected) or GFP-negative (uninfected) cells. (C) SVEC4-10 cells were infected with 2 PFU/cell of the indicated GFP-expressing viruses and analyzed for the intracellular (bottom) or surface (top) expression of MULT-1. Infection was visualized by GFP expression.
Figure 5.
Figure 5.
The m145 does not affect ER export of the MULT-1 protein. CV-1 cells were infected with control-VV or coinfected with MULT-1-VV and control VV or with MULT-1-VV and m145-VV and analyzed 12 h after infection. The cells were metabolically labeled for 30 min and chased for the indicated times, and the lysates were immunoprecipitated using anti-HA antibodies coupled to protein A–Sepharose. Before separation by 10% SDS-PAGE, the samples were mock treated (−) or digested by EndoH (+). R, EndoH resistant; S, EndoH sensitive; D, EndoH digested.
Figure 6.
Figure 6.
Δm145 virus is attenuated in vivo in an NK-dependent fashion. (A and B) NK cell–depleted or undepleted BALB/c mice were injected i.v. with 2 × 105 PFU of indicated viruses. The mice were killed 4 d after infection, and viral titers in organs were determined. Titers in the spleen of individual mice (circles) and median values (horizontal bars) are shown. (C) BALB/c mice were injected i.p. with blocking anti-NKG2D mAb or left untreated. 4 d after i.v. infection with 2 × 105 PFU of indicated viruses, the mice were killed and viral titers in organs were determined. Titers in the spleen and lungs of individual mice (circles) and median values (horizontal bars) are shown.
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