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The cytomegalovirus m155 gene product subverts natural killer cell antiviral protection by disruption of H60-NKG2D interactions.

Lodoen MB, Abenes G, Umamoto S, Houchins JP, Liu F, Lanier LL - J. Exp. Med. (2004)

Bottom Line: Treatment with the proteasome inhibitors lactacystin or epoxomicin reversed m155 down-regulation of H60.An MCMV mutant virus lacking m155 was severely attenuated in BALB/c mice; however, treatment with neutralizing anti-NKG2D monoclonal antibody or with NK-depleting anti-asialo GM1 antisera restored virulence of the mutant virus.Thus, down-regulation of H60 by m155 is a powerful mechanism of inhibiting NKG2D-mediated antiviral function.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Cancer Research Institute, University of California, San Francisco, San Francisco, CA 94143, USA.

ABSTRACT
Natural killer (NK) cells are an important early mediator of host immunity to murine cytomegalovirus (MCMV) infection. However, MCMV has evolved mechanisms to elude recognition and clearance by NK cells. We have identified an MCMV immune evasion protein that impairs NKG2D-mediated NK cell antiviral activity. Infection of BALB/c 3T3 cells with the Smith strain of MCMV resulted in strong down-regulation of H60, a high affinity ligand for NKG2D, from the surface of virus-infected cells. The MCMV m155 protein specifically down-regulated H60 without affecting expression of the other known NKG2D ligands, RAE-1 and MULT-1. Treatment with the proteasome inhibitors lactacystin or epoxomicin reversed m155 down-regulation of H60. An MCMV mutant virus lacking m155 was severely attenuated in BALB/c mice; however, treatment with neutralizing anti-NKG2D monoclonal antibody or with NK-depleting anti-asialo GM1 antisera restored virulence of the mutant virus. Thus, down-regulation of H60 by m155 is a powerful mechanism of inhibiting NKG2D-mediated antiviral function.

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m155 down-regulates H60, but not RAE-1 or MULT-1. (A) 293T cells were transfected with a vector encoding H60, MULT-1, or RAE-1, and either a control vector (dotted histograms) or a vector encoding m155 (bold histograms). 48 h after transfection, cells were stained with anti-H60, anti–MULT-1, or anti–RAE-1 mAbs. H60, MULT-1, and RAE-1 were encoded on vectors carrying an IRES-GFP, whereas a non-GFP vector was used for m155 cDNA and for the control vector. Histograms show GFP+ populations. Results are representative of three independent experiments. (B) 3T3 cells were infected with Rqm155-Rq155 (the m155 revertant virus) or Dm155 (Δm155) virus at an MOI of 2. 48 h after infection, cells were stained with control IgG2a (dotted histograms), anti-H60, anti–RAE-1, anti–MULT-1, or anti–β1 integrin (bold histograms). This experiment was performed several times with comparable results.
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fig2: m155 down-regulates H60, but not RAE-1 or MULT-1. (A) 293T cells were transfected with a vector encoding H60, MULT-1, or RAE-1, and either a control vector (dotted histograms) or a vector encoding m155 (bold histograms). 48 h after transfection, cells were stained with anti-H60, anti–MULT-1, or anti–RAE-1 mAbs. H60, MULT-1, and RAE-1 were encoded on vectors carrying an IRES-GFP, whereas a non-GFP vector was used for m155 cDNA and for the control vector. Histograms show GFP+ populations. Results are representative of three independent experiments. (B) 3T3 cells were infected with Rqm155-Rq155 (the m155 revertant virus) or Dm155 (Δm155) virus at an MOI of 2. 48 h after infection, cells were stained with control IgG2a (dotted histograms), anti-H60, anti–RAE-1, anti–MULT-1, or anti–β1 integrin (bold histograms). This experiment was performed several times with comparable results.

Mentions: By transiently transfecting human 293T cells with vectors encoding H60 and vectors encoding each of the MCMV ORFs, we were able to examine the effect of individual MCMV gene products on H60 expression. H60 was expressed in a vector containing an IRES–enhanced GFP element, permitting visualization of GFP+ cells that express H60 upstream of the IRES. Cotransfection of H60 with m155 resulted in a substantial decrease in H60 expressed on the surface (Fig. 2 A). We also considered the possibility that m155 may affect other ligands for NKG2D, such as RAE-1 or MULT-1. m155 did not affect of any of the other known NKG2D ligands because expression of these molecules was not changed by cotransfection with m155 (Fig. 2 A). In addition, m155 did not cause global down-regulation of cell surface receptors because the level of MHC class I was unaltered on the surface of 293T cells transfected with m155 (not depicted).


The cytomegalovirus m155 gene product subverts natural killer cell antiviral protection by disruption of H60-NKG2D interactions.

Lodoen MB, Abenes G, Umamoto S, Houchins JP, Liu F, Lanier LL - J. Exp. Med. (2004)

m155 down-regulates H60, but not RAE-1 or MULT-1. (A) 293T cells were transfected with a vector encoding H60, MULT-1, or RAE-1, and either a control vector (dotted histograms) or a vector encoding m155 (bold histograms). 48 h after transfection, cells were stained with anti-H60, anti–MULT-1, or anti–RAE-1 mAbs. H60, MULT-1, and RAE-1 were encoded on vectors carrying an IRES-GFP, whereas a non-GFP vector was used for m155 cDNA and for the control vector. Histograms show GFP+ populations. Results are representative of three independent experiments. (B) 3T3 cells were infected with Rqm155-Rq155 (the m155 revertant virus) or Dm155 (Δm155) virus at an MOI of 2. 48 h after infection, cells were stained with control IgG2a (dotted histograms), anti-H60, anti–RAE-1, anti–MULT-1, or anti–β1 integrin (bold histograms). This experiment was performed several times with comparable results.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2211837&req=5

fig2: m155 down-regulates H60, but not RAE-1 or MULT-1. (A) 293T cells were transfected with a vector encoding H60, MULT-1, or RAE-1, and either a control vector (dotted histograms) or a vector encoding m155 (bold histograms). 48 h after transfection, cells were stained with anti-H60, anti–MULT-1, or anti–RAE-1 mAbs. H60, MULT-1, and RAE-1 were encoded on vectors carrying an IRES-GFP, whereas a non-GFP vector was used for m155 cDNA and for the control vector. Histograms show GFP+ populations. Results are representative of three independent experiments. (B) 3T3 cells were infected with Rqm155-Rq155 (the m155 revertant virus) or Dm155 (Δm155) virus at an MOI of 2. 48 h after infection, cells were stained with control IgG2a (dotted histograms), anti-H60, anti–RAE-1, anti–MULT-1, or anti–β1 integrin (bold histograms). This experiment was performed several times with comparable results.
Mentions: By transiently transfecting human 293T cells with vectors encoding H60 and vectors encoding each of the MCMV ORFs, we were able to examine the effect of individual MCMV gene products on H60 expression. H60 was expressed in a vector containing an IRES–enhanced GFP element, permitting visualization of GFP+ cells that express H60 upstream of the IRES. Cotransfection of H60 with m155 resulted in a substantial decrease in H60 expressed on the surface (Fig. 2 A). We also considered the possibility that m155 may affect other ligands for NKG2D, such as RAE-1 or MULT-1. m155 did not affect of any of the other known NKG2D ligands because expression of these molecules was not changed by cotransfection with m155 (Fig. 2 A). In addition, m155 did not cause global down-regulation of cell surface receptors because the level of MHC class I was unaltered on the surface of 293T cells transfected with m155 (not depicted).

Bottom Line: Treatment with the proteasome inhibitors lactacystin or epoxomicin reversed m155 down-regulation of H60.An MCMV mutant virus lacking m155 was severely attenuated in BALB/c mice; however, treatment with neutralizing anti-NKG2D monoclonal antibody or with NK-depleting anti-asialo GM1 antisera restored virulence of the mutant virus.Thus, down-regulation of H60 by m155 is a powerful mechanism of inhibiting NKG2D-mediated antiviral function.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Cancer Research Institute, University of California, San Francisco, San Francisco, CA 94143, USA.

ABSTRACT
Natural killer (NK) cells are an important early mediator of host immunity to murine cytomegalovirus (MCMV) infection. However, MCMV has evolved mechanisms to elude recognition and clearance by NK cells. We have identified an MCMV immune evasion protein that impairs NKG2D-mediated NK cell antiviral activity. Infection of BALB/c 3T3 cells with the Smith strain of MCMV resulted in strong down-regulation of H60, a high affinity ligand for NKG2D, from the surface of virus-infected cells. The MCMV m155 protein specifically down-regulated H60 without affecting expression of the other known NKG2D ligands, RAE-1 and MULT-1. Treatment with the proteasome inhibitors lactacystin or epoxomicin reversed m155 down-regulation of H60. An MCMV mutant virus lacking m155 was severely attenuated in BALB/c mice; however, treatment with neutralizing anti-NKG2D monoclonal antibody or with NK-depleting anti-asialo GM1 antisera restored virulence of the mutant virus. Thus, down-regulation of H60 by m155 is a powerful mechanism of inhibiting NKG2D-mediated antiviral function.

Show MeSH
Related in: MedlinePlus