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

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.

Figure 1.

MCMV infection down-regulates H60. 3T3 cells were infected with wild-type MCMV (Smith), DMS94.5 (Δm150-165), or ΔMC96.24 (Δm152) viruses at an MOI of 2. 48 h after infection, cells were stained with control IgG2a (dotted histograms) or anti-H60 mAb (bold histograms).

Figure 2.

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.

Figure 3.

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.

Figure 4.

Depletion of NK cells or blockade of NKG2D restores virulence of Dm155. (A) BALB/c mice were injected i.p. with control rat IgG, anti-asialo GM1 antisera, or anti-NKG2D mAb (CX5). After antibody treatment, mice were infected with 10⁵ PFUs (A) or 2 × 10⁵ PFUs (B) of Smith, Rqm155 (revertant), or Rvm155 (Dm155). 3 d after infection, spleens and livers were harvested. Plaque assays were performed on organ homogenates to determine viral titers. In each experiment, three mice were used in each experimental group and error bars represent the standard deviation of the viral titers from these three mice. Three independent experiments were performed and data from two experiments using 2 × 10⁵ PFUs were combined for presentation in B. The limit of detection was 1 PFU/ml organ homogenate.