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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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Proteasome inhibition reverses m155 down-regulation of H60. (A) Lysates were generated from untreated cells or cells treated with 10 μM lactacystin for 14 h and immunoprecipitated with control IgG2a or anti-H60 mAb. Western blotting was performed for H60 or β1 integrin protein. (B) Untreated 3T3 or CT498 cells (3T3 cells stably transfected with m155) and 3T3 or CT498 cells treated with 10 μM lactacystin or 10 μM epoxomicin for 14 h were stained with a control IgG2a (dotted histograms), anti-H60, or anti–RAE-1 mAbs (bold histograms) and propidium iodide. Histograms show propidium iodide–negative cells (>95% of total cell population). (C) Lysates were generated from uninfected 3T3 cells, 3T3 cells infected with Rvm155, or 3T3 cells infected with Rqm155, and immunoprecipitated and Western blotted as described in B.
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fig3: Proteasome inhibition reverses m155 down-regulation of H60. (A) Lysates were generated from untreated cells or cells treated with 10 μM lactacystin for 14 h and immunoprecipitated with control IgG2a or anti-H60 mAb. Western blotting was performed for H60 or β1 integrin protein. (B) Untreated 3T3 or CT498 cells (3T3 cells stably transfected with m155) and 3T3 or CT498 cells treated with 10 μM lactacystin or 10 μM epoxomicin for 14 h were stained with a control IgG2a (dotted histograms), anti-H60, or anti–RAE-1 mAbs (bold histograms) and propidium iodide. Histograms show propidium iodide–negative cells (>95% of total cell population). (C) Lysates were generated from uninfected 3T3 cells, 3T3 cells infected with Rvm155, or 3T3 cells infected with Rqm155, and immunoprecipitated and Western blotted as described in B.

Mentions: In addressing the mechanism by which m155 down-regulates H60, we considered the possibility that m155 may affect H60 transcription. However, when we analyzed H60 RNA levels in 3T3 cells compared with 3T3 cells stably transfected with m155 (designated CT498 cells), we found a less than twofold difference in H60 transcription (Fig. S2, which is available at http://www.jem.org/cgi/content/full/jem.20040583/DC1). This is not surprising, considering that m155 encodes a potential membrane glycoprotein. Although H60 transcription was not substantially affected by expression of m155, H60 protein was strongly down-regulated from the surface of cells expressing m155 (Figs. 2 A and 3 B). We then hypothesized that m155 may cause degradation of H60. To examine this possibility, we treated 3T3 and CT498 cells with lactacystin, a cell-permeable, irreversible proteasome inhibitor, and immunoprecipitated H60 from lysates of treated or untreated cells. As revealed by Western blotting, an ∼80-kD band, representing H60, was immunoprecipitated from lysates of 3T3 cells (Fig. 3 B). No H60 protein was detected when lysates were immunoprecipitated with a control rat IgG2a, indicating that the 80-kD band is specific for H60. In lysates from CT498 cells, the 80-kD band was no longer present, consistent with the absence of H60 from the surface of these cells. However, treatment of CT498 cells with lactacystin restored H60 protein. We also observed restoration of H60 protein in lysates from CT498 cells treated with epoxomicin, another specific inhibitor of the proteasome (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)

Proteasome inhibition reverses m155 down-regulation of H60. (A) Lysates were generated from untreated cells or cells treated with 10 μM lactacystin for 14 h and immunoprecipitated with control IgG2a or anti-H60 mAb. Western blotting was performed for H60 or β1 integrin protein. (B) Untreated 3T3 or CT498 cells (3T3 cells stably transfected with m155) and 3T3 or CT498 cells treated with 10 μM lactacystin or 10 μM epoxomicin for 14 h were stained with a control IgG2a (dotted histograms), anti-H60, or anti–RAE-1 mAbs (bold histograms) and propidium iodide. Histograms show propidium iodide–negative cells (>95% of total cell population). (C) Lysates were generated from uninfected 3T3 cells, 3T3 cells infected with Rvm155, or 3T3 cells infected with Rqm155, and immunoprecipitated and Western blotted as described in B.
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Related In: Results  -  Collection

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fig3: Proteasome inhibition reverses m155 down-regulation of H60. (A) Lysates were generated from untreated cells or cells treated with 10 μM lactacystin for 14 h and immunoprecipitated with control IgG2a or anti-H60 mAb. Western blotting was performed for H60 or β1 integrin protein. (B) Untreated 3T3 or CT498 cells (3T3 cells stably transfected with m155) and 3T3 or CT498 cells treated with 10 μM lactacystin or 10 μM epoxomicin for 14 h were stained with a control IgG2a (dotted histograms), anti-H60, or anti–RAE-1 mAbs (bold histograms) and propidium iodide. Histograms show propidium iodide–negative cells (>95% of total cell population). (C) Lysates were generated from uninfected 3T3 cells, 3T3 cells infected with Rvm155, or 3T3 cells infected with Rqm155, and immunoprecipitated and Western blotted as described in B.
Mentions: In addressing the mechanism by which m155 down-regulates H60, we considered the possibility that m155 may affect H60 transcription. However, when we analyzed H60 RNA levels in 3T3 cells compared with 3T3 cells stably transfected with m155 (designated CT498 cells), we found a less than twofold difference in H60 transcription (Fig. S2, which is available at http://www.jem.org/cgi/content/full/jem.20040583/DC1). This is not surprising, considering that m155 encodes a potential membrane glycoprotein. Although H60 transcription was not substantially affected by expression of m155, H60 protein was strongly down-regulated from the surface of cells expressing m155 (Figs. 2 A and 3 B). We then hypothesized that m155 may cause degradation of H60. To examine this possibility, we treated 3T3 and CT498 cells with lactacystin, a cell-permeable, irreversible proteasome inhibitor, and immunoprecipitated H60 from lysates of treated or untreated cells. As revealed by Western blotting, an ∼80-kD band, representing H60, was immunoprecipitated from lysates of 3T3 cells (Fig. 3 B). No H60 protein was detected when lysates were immunoprecipitated with a control rat IgG2a, indicating that the 80-kD band is specific for H60. In lysates from CT498 cells, the 80-kD band was no longer present, consistent with the absence of H60 from the surface of these cells. However, treatment of CT498 cells with lactacystin restored H60 protein. We also observed restoration of H60 protein in lysates from CT498 cells treated with epoxomicin, another specific inhibitor of the proteasome (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