Involvement of an NKG2D ligand H60c in epidermal dendritic T cell-mediated wound repair

Dendritic epidermal T cells (DETCs) found in mouse skin are NKG2D-positive γδ T cells involved in immune surveillance and wound repair. It is assumed that the interaction of an NKG2D receptor on DETCs and an MHC class I-like NKG2D ligand on keratinocytes activates DETCs, which then secrete cytokines promoting wound repair. However, direct evidence that DETC activation through NKG2D signaling promotes wound repair is not available. In the present study, we generated mAbs for an NKG2D ligand H60c previously suggested to be expressed specifically on skin keratinocytes. Local administration of H60c-specific mAb inhibited activation of DETCs and significantly delayed wound repair. Likewise, administration of NKG2D-specific mAb impaired wound repair to a similar extent. The delay in wound closure resulting from the blockade of the NKG2D pathway was comparable to that observed in γδ T cell-deficient mice. These results indicate that H60c/NKG2D interactions play a critical role in wound repair. Reassessment of binding affinities showed that H60c monomers bind to NKG2D with affinity (K(d) = 26 ± 3.2 nM) comparable to those of other high-affinity NKG2D ligands. H60c is transcribed not only in skin but also in tissues such as tongue and female reproductive tract known to contain epithelium-resident γδ T cells expressing invariant TCRs, suggesting a more general role for H60c in the maintenance of epithelial integrity.     

Systemic NKG2D down-regulation impairs NK and CD8 T cell responses in vivo

The immunoreceptor NKG2D stimulates activation of cytotoxic lymphocytes upon engagement with MHC class I-related NKG2D ligands of which at least some are expressed inducibly upon exposure to carcinogens, cell stress, or viruses. In this study, we investigated consequences of a persistent NKG2D ligand expression in vivo by using transgenic mice expressing MHC class I chain-related protein A (MICA) under control of the H2-K(b) promoter. Although MICA functions as a potent activating ligand of mouse NKG2D, H2-K(b)-MICA mice appear healthy without aberrations in lymphocyte subsets. However, NKG2D-mediated cytotoxicity of H2-K(b)-MICA NK cells is severely impaired in vitro and in vivo. This deficiency concurs with a pronounced down-regulation of surface NKG2D that is also seen on activated CD8 T cells. As a consequence, H2-K(b)-MICA mice fail to reject MICA-expressing tumors and to mount normal CD8 T cell responses upon Listeria infection emphasizing the importance of NKG2D in immunity against tumors and intracellular infectious agents.     

Structure of the HCMV UL16-MICB Complex Elucidates Select Binding of a Viral Immunoevasin to Diverse NKG2D Ligands

The activating immunoreceptor NKG2D promotes elimination of infected or malignant cells by cytotoxic lymphocytes through engagement of stress-induced MHC class I-related ligands. The human cytomegalovirus (HCMV)-encoded immunoevasin UL16 subverts NKG2D-mediated immune responses by retaining a select group of diverse NKG2D ligands inside the cell. We report here the crystal structure of UL16 in complex with the NKG2D ligand MICB at 1.8 Å resolution, revealing the molecular basis for the promiscuous, but highly selective, binding of UL16 to unrelated NKG2D ligands. The immunoglobulin-like UL16 protein utilizes a three-stranded β-sheet to engage the α-helical surface of the MHC class I-like MICB platform domain. Intriguingly, residues at the center of this β-sheet mimic a central binding motif employed by the structurally unrelated C-type lectin-like NKG2D to facilitate engagement of diverse NKG2D ligands. Using surface plasmon resonance, we find that UL16 binds MICB, ULBP1, and ULBP2 with similar affinities that lie in the nanomolar range (12–66 nM). The ability of UL16 to bind its ligands depends critically on the presence of a glutamine (MICB) or closely related glutamate (ULBP1 and ULBP2) at position 169. An arginine residue at this position however, as found for example in MICA or ULBP3, would cause steric clashes with UL16 residues. The inability of UL16 to bind MICA and ULBP3 can therefore be attributed to single substitutions at key NKG2D ligand locations. This indicates that selective pressure exerted by viral immunoevasins such as UL16 contributed to the diversification of NKG2D ligands.     

Making sense of the diverse ligand recognition by NKG2D

NKG2D recognizes multiple diverse ligands. Despite recent efforts in determining the crystal structures of NKG2D-ligand complexes, the principle governing this receptor-ligand recognition and hence the criteria for identifying unknown ligands of NKG2D remain central issues to be resolved. Here we compared the molecular recognition between NKG2D and three of the known ligands, UL16 binding protein (ULBP), MHC class I-like molecule, and retinoic acid early inducible gene as observed in the ligand-complexed crystal structures. The comparison shows that while the receptor uses a common interface region to bind the three diverse ligands, each ligand forms a distinct, but overlapping, set of hydrogen bonds, hydrophobic interactions, and salt bridges, illustrating the underlying principle of NKG2D-ligand recognition being the conservation in overall shape complementarity and binding energy while permitting variation in ligand sequence through induced fit recognition. To further test this hypothesis and to distinguish between diverse recognition and promiscuous ligand binding, four ULBP3 interface mutations, H21A, E76A, R82M, and D169A, were generated to each disrupt a single hydrogen bond or salt bridge. All mutant ULBP3 displayed reduced receptor binding, suggesting a specific, rather than promiscuous, receptor-ligand recognition. Mutants with severe loss of binding affect the receptor interactions that are mostly buried. Finally, a receptor-ligand recognition algorithm was developed to assist the identification of diverse NKG2D ligands based on evaluating the potential hydrogen bonds, hydrophobic interactions, and salt bridges at the receptor-ligand interface.     

Immunogenetics of the NKG2D ligand gene family

NKG2D ligands (NKG2DLs) are a group of major histocompatibility complex (MHC) class I-like molecules, the expression of which is induced by cellular stresses such as infection, tumorigenesis, heat shock, tissue damage, and DNA damage. They act as a molecular danger signal alerting the immune system for infected or neoplastic cells. Mammals have two families of NKG2DL genes: the MHC-encoded MIC gene family and the ULBP gene family encoded outside the MHC region in most mammals. Rodents such as mice and rats lack the MIC family of ligands. Interestingly, some mammals have NKG2DL-like molecules named MILL that are phylogenetically related to MIC, but do not function as NKG2DLs. In this paper, we review our current knowledge of the MIC, ULBP, and MILL gene families in representative mammalian species and discuss the origin and evolution of the NKG2DL gene family.     

Comparative genomic analysis of mammalian NKG2D ligand family genes provides insights into their origin and evolution

NKG2D is a major activating receptor of natural killer cells. Its ligands are major histocompatibility complex (MHC) class I-like molecules whose expression is induced by cellular stresses such as infections and tumorigenesis. Humans have two families of NKG2D ligands (NKG2DL): MHC class I-related chains (MIC) encoded in the MHC and UL16-binding proteins (ULBP) encoded outside the MHC. By contrast, mice have only the latter family of ligands; instead, they have non-MHC-encoded MILL molecules that are closely related to MIC, but do not function as NKG2DL. To gain insights into the origin and evolution of MIC, ULBP, and MILL gene families, we conducted comparative genomic analysis of NKG2DL family genes in five mammalian species. In the opossum MHC, we identified a ULBP-like gene adjacent to a previously described MIC-like gene, suggesting that ULBP genes were originally encoded in the MHC. The opossum genome also contained a transcribed MILL-like gene in a region syntenic to the rodent regions encoding MILL molecules. These observations indicate that MIC-, ULBP-, and MILL-like genes emerged before the divergence of placental and marsupial mammals. Comparison of the human, cattle, rat, mouse, and opossum genomes indicates that after emigration from the MHC, ULBP genes underwent extensive duplications in each species. In mice, some of the ULBP genes appear to have been translocated telomerically on the same chromosome, forming a major cluster of existent NKG2DL genes.     

Cutting edge: the minor histocompatibility antigen H60 peptide interacts with both H-2Kb and NKG2D

Minor histocompatibility Ags elicit cell-mediated immune responses and graft rejection in individuals receiving MHC-matched tissues. H60 represents a dominant Ag that elicits a strong CTL response in C57BL/6 mice immunized against BALB.B. An 8-aa peptide in the H60 protein is presented by H-2K(b) and this is recognized by the TCR as an alloantigen. The intact H60 glycoprotein is a ligand for the costimulatory NKG2D receptor that is expressed by activated CD8(+) T cells. Thus, H60 may provide both an allogeneic peptide and its own costimulation. We show that mutation of an H-2K(b)-binding anchor residue in the H60 peptide completely abrogates binding of H60 glycoprotein to NKG2D and a synthetic H60 peptide partially blocks the binding of NKG2D to its ligand. Ligands of the human NKG2D receptor are remarkably polymorphic, suggesting that these may also serve as minor histocompatibility Ags.     

Shed NKG2D ligand boosts NK cell immunity

Ligands for natural killer (NK) cell activating receptors can be released from tumor cells and are believed to promote tumor growth by acting as decoys for effector lymphocytes. In a recent paper published in Science, Deng et al. report another scenario in which a shed form of the MULT1 mouse NKG2D ligand boosts NK cell functions.Natural killer (NK) cells are cytolytic and cytokine-producing lymphocytes of the innate immune system that participate in the control of tumor growth and microbial infections¹. NK cell effector activities are tightly controlled by a fine balance of inhibitory and activating signals delivered by surface receptors. Activating receptors can recognize two types of ligands, self-molecules encoded by the host''s own genome whose expression is upregulated upon cellular stress, or exogenous molecules produced by microbes during infection. NKG2D, one of the best characterized activating receptor expressed by NK and T cells, binds to several different ligands in human and mouse². NKG2D ligands are poorly expressed on the vast majority of normal cell surfaces, but are upregulated on tumor and virus-infected cells. In addition, NKG2D ligands can be released by both surface cleavage and exosome excretion. It has been reported that shed ligands can block tumor cell recognition by effector cells by preventing NKG2D interaction with its ligands³. However, several reports do not correlate the presence of soluble ligands with decreased NKG2D expression nor functional activities.Deng et al.⁴ focused their analysis on the NKG2D mouse ligand MULT1, which is commonly overexpressed on primary tumor cells. They first showed that MULT1-transduced fibroblast can cleave MULT1 from the plasma membrane, resulting in a released shed form in the supernatant. Shed MULT1 is of high affinity to NKG2D (∼13 nM) similar to recombinant MULT1. They further reveal the presence of shed MULT1 in the serum of mice developing spontaneous MULT1⁺ tumors. Interestingly, the authors detected a very high concentration of shed MULT1 in the sera of Apoe^−/− mice exhibiting severe atherosclerosis and liver inflammation. Given that these autoimmune injuries observed in this mouse model depend on NKG2D activity⁵, it was unlikely that shed MULT1 exert an inhibitory effect on immunity.Surprisingly, the authors further showed that mouse tumor cells engineered to release a secreted form of MULT1 (secMULT1) similar to the shed MULT1 were rejected when injected into syngenic mice. Tumor rejection is dependent on NK cells as cells grow in NK but not in CD8⁺ T cell-depleted host and requires NKG2D. Importantly, the controlled release of secMULT1 from tumors harboring inducible secMULT1 promotes tumor rejection. To rule out the possibility that tumor cell rejection was due to intrinsic modifications of tumor cells, the author monitored the rejection of a mixture of 9:1 secMULT1⁻: secMULT1⁺ tumor cells and showed an improved antitumoral effect on both secMULT1⁺ and, importantly, secMULT1⁻ tumors. In addition, direct intratumoral injection of recombinant MULT1 promotes tumor rejection. These results suggested that soluble MULT1 mobilizes or activates anti-tumor effector cells. Deng et al. further reported increased frequencies of cytotoxic and IFN-γ-secreting NK cells associated with secMULT1⁺ tumors as compared to control tumor cells. Altogether, these data suggest that a shed NKG2D ligand can promote tumor rejection by boosting NK cell effector functions.Shed MULT1 could crosslink NKG2D and thus activate NK cells. However, shed and secMULT1 are monomeric molecules similar to the recombinant MULT1 which fails to activate NK cells in vitro. Formation of multivalent structures in vivo was not detected. In addition, whereas the transmembrane form of MULT1 can activate NK cells by crosslinking NKG2D and induces NKG2D downregulation, soluble MULT1 upregulates NKG2D on the NK cell surface. This upregulation is probably due do a decreased downregulation of NKG2D surface expression because no increase in NKG2D mRNA or protein was observed. Based on these findings, the authors hypothesized that NKG2D ligands expressed on non-tumor host cell membrane continuously engage NKG2D on NK cells, leading to NKG2D downregulation and NK cell desensitization, whereas soluble MULT1 blocks these interactions to increase NK cell responsiveness (Figure 1). Along this line, NK cells from mutant mice genetically deficient for the NKG2D ligand expressed by tumor-associated myeloid cells are not desensitized.Open in a separate windowFigure 1Tumor-associated cells express NKG2DL which can desensitize NK cells. Tumor shedding of MULT1 delivers soluble MULT1 that outcompetes for NKG2D binding and prevents NK cell desensitization. Boosted NK cell functions lead to improved tumor cell rejection by other activating receptors.The induction of cell desensitization by a frequent or even constant stimulation is a very common mechanism across living objects. Regarding NK cells, another example of tuning via desensitization resides in the impact of the long lasting absence of MHC class I molecules in their environment. Indeed, NK cells are hyporesponsive in a MHC-I-deficient host⁶. There are accumulating data indicating that in the absence of engagement of inhibitory receptors for MHC class I molecules, NK cells get desensitized due to their chronic interaction with endogenous stimulating ligands⁷. Indeed, in the absence of engagement of this inhibitory pathway, NK cell activation would be unleashed⁸. This scenario is supported by a series of in vitro and in vivo experiments in which NK cells are desensitized following chronic exposure to stimulatory molecules expressed at the surface of interacting cells^9,10. Thus, the induction of MHC class I downregulation or NKG2D ligand upregulation boosts NK cell function, whereas the sustained lack of MHC class I or expression of NKG2D ligands impairs NK cell reactivity. This tuning of immune response as a function of the speed of change of the stimuli detected by lymphocytes is at the center of the recently proposed Discontinuity Theory¹¹.Finally, consistent with their findings with secMULT1 but somewhat counter-intuitively, Deng et al. also show that NKG2D receptor deficiency or blockade using anti-NKG2D monoclonal antibodies mimics the effect of soluble MULT1. Indeed, in both conditions, NK cell effector functions are boosted, resulting in improved tumor rejection. Similarly, blocking other NK activating receptors, such as NKp46, may also lead to NK cell desensitization¹². Checkpoint inhibitory receptors are revolutionizing the treatment of cancers by inhibiting the inhibitory receptors. The findings reported by Deng et al. together with earlier results propose alternative strategies of cancer treatment using antibodies that are directed against activating receptors. In the case of NKG2D, the chronic engagement of NK cells with membrane-bound NKG2D ligand affects not only NKG2D-dependent but also NKG2D-independent signaling pathways⁹. The blockade of NKG2D desensitization by antibodies directed against NKG2D should thus also boost NK cell activation via other pathways, such as antibody-dependent cell cytotoxicity. However, the precise identification of the ligand-receptor pair involved in the control of tumors by NK cells will be a limiting factor to these innovative therapeutic approaches. Indeed, antibodies against activating receptors should be designed to boost NK cell reactivity but should not block the recognition of the tumors by NK cells. Finally, as the tuning of NK cell reactivity by soluble NKG2D ligands depends on their affinity for NKG2D, the pre-clinical development of this new class of drug candidates might reveal novel pharmacokinetics and the pharmacodynamics guidelines.     

ULBP4 is a novel ligand for human NKG2D

The ULBPs are a family of MHC class I-related molecules. We have previously shown that ULBPs 1, 2, and 3 are functional ligands of the NKG2D/DAP10 receptor complex on human natural killer (NK) cells. Here, we describe a new member of the ULBP family, ULBP4, which contains predicted transmembrane and cytoplasmic domains, unlike the other ULBPs, which are GPI-linked proteins. Transduction of ULBP4 into EL4 cells confers the ability to bind recombinant NKG2D and mediates increased cytotoxic activity by human NK cells, consistent with the role of ULBPs as ligands for the NKG2D/DAP10 activating receptors. Tissue expression of ULBP4 differs from other members of the family, in that it is expressed predominantly in the skin.     

Importance of NKG2D-NKG2D ligands interaction for cytolytic activity of natural killer cell

In this study, we investigate the relationship between natural killer (NK) cell susceptibility and the surface markers of cancer cells. Through phenotypic analysis, we found evidence that more susceptible cancer cell lines (K562 and Jurkat) express more NKG2D ligands. Major histocompatibility complex (MHC) class I chain-related A/B (MIC-A/B) and UL16 binding protein (ULBP) 1-5 molecules are typical ligands of NKG2D. The high killing activity of NK cells against K562 was abolished through the addition of a NKG2D blocking antibody. Upon in vitro stimulation with quercetin, low susceptible cancer cells increased NKG2D ligand expression, leading to enhancement of NK cell cytolytic activity. These results suggested that the anti-cancer activity of NK cells is not dependent on the origin and growth style of the target cells, but is dependent on the surface markers of the target cells.     

NKG2D is a costimulatory receptor for human naive CD8+ T cells

In humans, all alpha beta CD8+ T cells express NKG2D, but in mouse, it is only expressed by activated and memory CD8+ T cells. We purified human naive CD8+ T cells to show that NKG2D serves as a costimulatory receptor for TCR induced Ca2+ mobilization and proliferation. The resulting effector cells are skewed toward a type 1 phenotype and produce high levels of IFN-gamma and TNF-alpha. NKG2D ligands, MHC class I chain-related (MIC)A, MICB, and UL16-binding proteins are expressed on the proliferating cells and NKG2D is down-regulated. The addition of the homeostatic cytokines IL-7 and IL-15 to the culture medium not only enhances proliferation but also counteracts the down-regulation of NKG2D, more so than the addition of IL-2. These results indicate that NKG2D can regulate the priming of human naive CD8+ T cells, which may provide an alternative mechanism for potentiating and channeling the immune response.     

Structural studies of allelic diversity of the MHC class I homolog MIC-B,a stress-inducible ligand for the activating immunoreceptor NKG2D

MIC-A and MIC-B are distant MHC class I homologs that serve as stress-inducible Ags on epithelial and epithelially derived cells. They are ligands for the widely expressed activating immunoreceptor NKG2D. To define the structural and functional consequences of sequence differences between MIC-A and MIC-B and between alleles of MIC-A and alleles of MIC-B, we determined the crystal structure of one allele of human MIC-B. Comparisons between the two previously reported MIC-A crystal structures and the MIC-B crystal structure show that, as expected, MIC-B is very similar in structure to MIC-A and likely interacts with NKG2D in an analogous manner. The interdomain flexibility observed in the MIC-A structures, a feature unique to MIC proteins among MHC class I proteins and homologs, is also displayed by MIC-B, with an interdomain relationship intermediate between the two examples of MIC-A structures. Mapping sequence variations onto the structures of MIC-A and MIC-B reveals patterns completely distinct from those displayed by classical MHC class I proteins, with a number of substitutions falling on positions likely to affect interactions with NKG2D, but with other positions lying distant from the NKG2D binding sites or buried within the core of the proteins.     

MHC class I-like genes in cattle,<Emphasis Type="Italic"> MHCLA,</Emphasis> with similarity to genes encoding NK cell stimulatory ligands

A comparative genomics approach for mining databases of expressed sequence tags (ESTs) was used to identify two members of a novel MHC class I gene family in cattle. These paralogous genes, named MHC class I-like gene family A1 ( MHCLA1) and MHCLA2, were shown by phylogenetic analysis to be related to human and mouse genes encoding NK cell stimulatory ligands, ULBP, RAET, H60 and Raet-1. Radiation hybrid mapping placed cattle MHCLA1 on BTA9, which, on the basis of existing comparative mapping data, identified the ULBP, RAET1, H60 and Raet1 genes as homologues of the cattle MHCLA genes. However, the human and mouse orthologues of MHCLA1 and MHCLA2 could not be defined due to extensive sequence divergence from all known members of the ULBP1/ RAET1/H60/Raet1 gene family. The cattle MHCLA1 molecule is predicted to be missing an alpha(3) domain, similar to the human and mouse homologues. Like the human ULBP genes, MHCLA1 was found to be transcribed constitutively in a variety of fetal and adult tissues by RT-PCR. The patterns of hybridization obtained by Southern blotting using MHCLA1 as a probe and DNA from 14 species representing five mammalian orders suggests that the MHCLA genes evolved rapidly in the Cetartiodactyla. Previous findings demonstrating that ULBPs serve as ligands for the NK cell NKG2D stimulatory receptor, and that this interaction can be blocked by a human cytomegalovirus glycoprotein that binds to ULBPs, suggests that the extensive divergence found among the cattle, human and mouse MHCLA homologues is due to selection exerted by viral pathogens.     

Activation of V gamma 9V delta 2 T cells by NKG2D

Human Vgamma9 Vdelta2 T cells recognize phosphorylated nonpeptide Ags (so called phosphoantigens), certain tumor cells, and cells treated with aminobisphosphonates. NKG2D, an activating receptor for NK cells, has been described as a potent costimulatory receptor in the Ag-specific activation of gammadelta and CD8 T cells. This study provides evidence that Vgamma9 Vdelta2 T cells may also be directly activated by NKG2D. Culture of PBMC with immobilized NKG2D-specific mAb or NKG2D ligand MHC class I related protein A (MICA) induces the up-regulation of CD69 and CD25 in NK and Vgamma9 Vdelta2 but not in CD8 T cells. Furthermore, NKG2D triggers the production of TNF-alpha but not of IFN-gamma, as well as the release of cytolytic granules by Vgamma9 Vdelta2 T cells. Purified Vgamma9 Vdelta2 T cells kill MICA-transfected RMA mouse cells but not control cells. Finally, DAP10, which mediates NKG2D signaling in human NK cells, was detected in resting and activated Vgamma9 Vdelta2 T cells. These remarkable similarities in NKG2D function in NK and Vgamma9 Vdelta2 T cells may open new perspectives for Vgamma9 Vdelta2 T cell-based immunotherapy, e.g., by Ag-independent killing of NKG2D ligand-expressing tumors.     

The role of NKG2D signaling in inhibition of cytotoxic T-lymphocyte lysis by the Murine cytomegalovirus immunoevasin m152/gp40

Three proteins encoded by murine cytomegalovirus (MCMV) -- gp34, encoded by m04 (m04/gp34), gp48, encoded by m06 (m06/gp48), and gp40, encoded by m152 (m152/gp40) -- act together to powerfully impact the ability of primed cytotoxic CD8 T lymphocytes (CTL) to kill virus-infected cells. Of these three, the impact of m152/gp40 on CTL lysis appears greater than would be expected based on its impact on cell surface major histocompatibility complex (MHC) class I. In addition to MHC class I, m152/gp40 also downregulates the RAE-1 family of NKG2D ligands, which can provide costimulation for CD8 T cells. We hypothesized that m152/gp40 may impact CTL lysis so profoundly because it inhibits both antigen presentation and NKG2D-mediated costimulation. We therefore tested the extent to which m152/gp40's ability to inhibit CTL lysis of MCMV-infected cells could be accounted for by its inhibition of NKG2D signaling. As was predictable from the results reported in the literature, NKG2D ligands were not detected by NKG2D tetramer staining of cells infected with wild-type MCMV, whereas those infected with MCMV lacking m152/gp40 displayed measurable levels of the NKG2D ligand. To determine whether NKG2D signaling contributed to the ability of CTL to lyse these cells, we used a blocking anti-NKG2D antibody. Blocking NKG2D signaling did affect the killing of MCMV-infected cells for some epitopes. However, for all epitopes, the impact of m152/gp40 on CTL lysis was much greater than the impact of inhibition of NKG2D signaling. We conclude that the downregulation of NKG2D ligands by MCMV makes only a small contribution to the impact of m152/gp40 on CTL lysis and only for a small subset of CTL.     

Selective down-regulation of the NKG2D ligand H60 by mouse cytomegalovirus m155 glycoprotein

Both human and mouse cytomegaloviruses (CMVs) encode proteins that inhibit the activation of NK cells by down-regulating cellular ligands for the activating NK cell receptor NKG2D. Up to now, three ligands for the NKG2D receptor, named RAE-1, H60, and MULT-1, have been identified in mice. The resistance of mouse strains to murine CMV (MCMV) infection is determined by their ability to generate an effective NK cell response. The MCMV gene m152, a member of the m145 gene family, down-regulates the expression of RAE-1 in order to avoid NK cell control in vivo. Here we report that the m155 gene, another member of the m145 gene family, encodes a protein that interferes with the expression of H60 on the surfaces of infected cells. Deletion of the m155 gene leads to an only partial restoration of H60 expression on the cell surface, suggesting the involvement of another, so far unknown, viral inhibitor. In spite of this, an m155 deletion mutant virus shows NK cell-dependent attenuation in vivo. The acquisition of endo-beta-N-acetylglucosaminidase H resistance and the preserved half-life of H60 in MCMV-infected cells indicate that the m155-mediated effect must take place in a compartment after H60 exits from the ERGIC-cis-Golgi compartment.