Immunology and Cell Biology (2014) 92, 214–220 & 2014 Australasian Society for Immunology Inc. All rights reserved 0818-9641/14 www.nature.com/icb

REVIEW

The Ly49 natural killer cell receptors: a versatile tool for viral self-discrimination Richard Berry1, Jamie Rossjohn1,2 and Andrew G Brooks3 The activation of murine and human natural killer (NK) cells is regulated by families of receptors including the Ly49 and Killer immunoglobulin-like receptors, respectively, both of which contain activating and inhibitory members. The archetypal role of inhibitory Ly49 receptors is to attenuate NK cell responses to normal cells that express major histocompatibility complex (MHC) class-I molecules, in essence allowing for more robust responses to infected or cancerous cells that lack MHC-I on their cell surface. However, it is now evident that Ly49 receptors have an appreciably more sophisticated array of functions. In particular, some activating Ly49 receptors can bind directly to MHC-I-like viral gene products such as m157, whereas others recognize self-MHC-I but only in the presence of viral chaperones. Although Ly49 receptor recognition is centred on the MHC-I-like fold, these NK cell receptors can also engage related ligands in unexpected ways. Herein we review the varied strategies employed by Ly49 receptors to recognize both self and viral ligands, with particular emphasis on the recently determined mode of Ly49-m157 ligation, and highlight the versatile nature of this family in the control of viral infections. Immunology and Cell Biology (2014) 92, 214–220; doi:10.1038/icb.2013.100; published online 14 January 2014 Keywords: cytomegalovirus; MHC; NK cells

Natural killer (NK) cells are critical components of the innate immune response and accordingly, form part of the first line of defence to viral infections. NK cell activity and function are regulated via germline-encoded receptors that recognize major histocompatibility complex (MHC) class I (MHC-I) and MHC-I-like molecules. The NK cell receptors that mediate these functions belong to a variety of structurally distinct families (Figure 1). For example, the CD94NKG2 receptors are heterodimeric type II integral membrane proteins that comprise an invariant CD94 polypeptide covalently associated with a specific NKG2 subunit (NKG2A/B, C or E).1,2 These receptors are essentially monomorphic, highly conserved across species and recognize the MHC-Ib molecule HLA-E (Qa-1b in mice) and which like the receptors themsleves, displays little polymorphism.3–7 NKG2D is another highly conserved NK receptor with specificity for MHC-Ilike proteins. However, in contrast to the CD94-NKG2 family, NKG2D is a homodimer and is highly promiscuous, having multiple distinct ligands including MIC-A, MIC-B and the ULBP proteins in humans along with the RAE-1, MULT1 and H60 proteins in mice.8–17 In addition to the CD94-NKG2 and NKG2D receptors, both rodent and primate NK cells express additional receptor families that are primarily focused on recognition of MHC-I proteins. In humans, this function is largely mediated by the killer immunoglobulin-like receptor (KIR) family.18 In contrast to the lectin-like receptors described above, KIRs, which are encoded by a

large cluster of genes within a highly polymorphic region of chromosome 19, possess two or three immunoglobulin-like domains in their extracellular region.19,20 The first described KIR possessed intracellular tyrosine-based inhibitory motifs in their cytoplasmic regions that recruit and activate phosphatases to limit cellular activation.21–23 The structures of individual KIR in complex with their pMHC-I ligands have provided important insight into the molecular basis of their specificity.24–26 However, some KIR members have gained the capacity to activate NK cells,27 although the ligands for such activating KIRs are not well defined. The KIR family of receptors has evolved relatively recently and is absent in rodents where an equally large receptor family, termed Ly49, mediates apparently equivalent functions.28 There exists considerable variability in Ly49 gene and sequence composition between mouse strains that impacts on NK cell responses to both the loss of MHC-I on target cells and to virus-encoded ligands.29 This review will focus on the role of Ly49 receptors in combating viral infection with a particular emphasis on recent developments in our understanding of the recognition of virally encoded ligands by activating Ly49 receptors. THE LY49 RECEPTOR FAMILY Ly49 receptors are type II integral membrane proteins that form disulfide-linked homodimers on the cell surface.30,31 Like the CD94NKG2 and NKG2D receptors they are members of the C-type lectin

1Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, Victoria, Australia; 2Institute of Infection and Immunity, School of Medicine, Cardiff University, Cardiff, UK and 3Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria, Australia Correspondence: Professor J Rossjohn, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria. E-mail: [email protected] or Professor AG Brooks, Department of Microbiology and Immunology, University of Melbourne, Royal Parade, Parkville, Victoria 3010, Australia. E-mail: [email protected] Received 14 October 2013; accepted 10 November 2013; published online 14 January 2014

Ly49 NK cell receptors: tool for viral self-discrimination R Berry et al 215 70 Ly49

Cy

TM

CD94

Cy

TM

NKG2A-E

Cy

TM

KIR3D KIR2D

Ig

Ig

Ig

Ig

Ig

20 20

α1s α2s α3s 21 21

Mouse

lectin

lectin

Mouse/human

lectin

Mouse/human

TM

Cy

Human

TM

Cy

Human

Figure 1 Domain structure of NK cell receptors involved in the recognition of MHC-I and MHC-I-like molecules in mice and humans. The length of the stalk region linking the transmembrane (TM) segment to the first extracellular domain is indicated. For simplicity, the presence or absence of intracellular tyrosine-based inhibitory motif motifs in the cytoplasmic (Cy) regions are omitted. Ig, immunoglobulin.

superfamily and are comprised of a carboxy-terminal lectin domain (also referred to as a NK domain or NKD) that confers specificity for distinct allotypic groups of MHC-I and MHC-I-like molecules.32 However, in contrast to the other lectin-like receptors encoded within the NK cell gene complex on chromosome 6, the NKD of the Ly49 receptors is tethered to the cell membrane by an extended stalk region.33–36 This stalk is approximately 70 amino acids in length and comprised of three a-helical segments denoted as a1s (membrane proximal), a2s and a3s (membrane distal), each of which are separated by short loop regions (Figure 1a). To date, 23 Ly49 genes (Ly49A–W) have been identified, the majority of which encode inhibitory receptors (Table 1). Like the KIR family, there is considerable genetic variation both in which Ly49 genes are present and in the sequence of individual genes across different mouse strains.28 The inhibitory Ly49 receptors include the prototypical family members Ly49A and Ly49C and possess intracellular tyrosine-based inhibitory motifs within their cytoplasmic regions that, in response to ligand binding, recruit and activate phosphatases such as SHP-1 that serve to limit NK cell activation.21,37 In addition to the inhibitory members of the Ly49 family, there are more recently evolved Ly49 members such as Ly49D and Ly49H that have lost the motifs associated with the transduction of inhibitory signals and acquired the capacity to interact with the small disulfide bonded homodimers such as DAP12.38 This interaction facilitates the transduction of activation signals via ITAMs encoded within the cytoplasmic region of DAP12.39 More recently activating Ly49 receptors have been shown to associate with another homdimeric adaptor protein, DAP10. Critically, Ly49H þ NK cells from DAP10-deficient mice have an impaired proliferative response following infection with murine cytomegalovirus (MCMV) demonstrating the importance of the association with this adaptor protein.40 Although some activating Ly49 receptors have been reported to recognize virally encoded proteins, a large number of activating and even some inhibitory Ly49 receptors, nevertheless, remain orphans with no identified ligand. LY49-MEDIATED ‘MISSING SELF’ RECOGNITION The classical role of inhibitory Ly49 receptors is to ligate MHC-I molecules and transduce inhibitory signals that limit NK cell activation.32,41 Accordingly, in this ‘missing self’ model, NKmediated lysis is restricted to cells that lack surface expression of MHC-I such as occurs during malignant transformation or viral infection,32 although the exact response is determined by the balance of signals derived from both activating and inhibitory receptors expressed on the NK cell surface (Figure 2). The MHC-I ligand

Table 1 Summary of murine Ly49 receptors and their ligands Receptor

Activity

Self ligand (s)

Ly49A



Db,d,p,k, H2-M3

Ly49B Ly49C

 

Kb,d,k, Dd,b,k

Ly49D Ly49E

þ 

Dd

Ly49F Ly49G

 

Dd Dd, Ld

Ly49H Ly49I

þ 

K/Db,d,s,q,v

Ly49J Ly49K

 þ

Kb

Ly49L Ly49M

þ þ

Kk

Ly49N Ly49O

þ 

Ly49P Ly49Q

þ 

Dd

Ly49R Ly49S

þ 

Dd,k, Ld

Ly49T Ly49U

 þ

Db

Viral ligand

Self/viral ligand complex

m157

m157 m157

H2d, H2k, H2a, H2f/m04

Db,d,k, Ld

Ly49V



Db,d, Kk

Ly49W

þ

Dd, Kk

Dk, H2a, H2d/m04

H2d, H2k, H2f/m04

specificities of the inhibitory receptors are for the most part well documented (Table 1). However, it has recently been reported that Ly49A can also engage the non-classical MHC-I molecule H2-M3 and that this interaction can impact on the licensing of NK cells.42 Accordingly, it is possible that additional Ly49 receptor ligands outside the normal sphere of classical MHC-I molecules are yet to be identified. The general mechanism of Ly49-mediated MHC-I recognition has been extensively studied.34,36,43–46 Ly49 receptors engage MHC-I via their NKD, which interacts with a concave groove underneath the peptide-binding platform, making extensive contacts with the a1, a2, a3 domains and the b2-microglobulin subunit (Figure 3). This binding mode is distinct from that employed by abT-cell receptors47 and results in no direct contacts to the bound peptide, explaining why, for the most part, Ly49 recognition of MHC-I is peptide independent,48,49 although some element of peptide specificity has been reported for Ly49C50 and Ly49I.51 Instead, subtle differences in Immunology and Cell Biology

Ly49 NK cell receptors: tool for viral self-discrimination R Berry et al 216

NK cell

Target cell

Inhibition

Activation

Activation

Inhibition

?

Figure 2 Schematic representation of potential encounters between NK cells and their targets. In the case of a normal healthy cell, NK cell responses are attenuated due to ligation of inhibitory Ly49 receptors by self MHC-I (a). Target cells can trigger NK cell-mediated lysis if the MHC-I ligands for inhibitory Ly49 receptors are downregulated (b) or if the NK cell possesses an activating Ly49 receptor capable of recognizing the m157 gene product (c). If the NK cell expresses inhibitory Ly49 receptors that recognize m157, however, the infected cell escapes NK-mediated lysis (d). It is unclear whether Ly49 receptors could simultaneously engage MHC-I in cis and m157 in trans (e) and what the outcome of such a ligation event would be.

ligand specificity are conferred by minor alterations within the NKD, allowing distinct Ly49 receptors to recognize different MHC-I allotypes.43 Although the determination of multiple Ly49-MHC-I complexes has revealed the molecular details at the receptor–ligand interface, intriguingly they have also shown that the membrane proximal termini of both Ly49 and MHC-I seemingly extend in the same direction. How then do Ly49 receptors recognize MHC-I molecules expressed on the surface of target cells? Investigations from the Mariuzza and Held laboratories have shown that the membrane proximal stalks of Ly49L can ‘backfold’, passing between the two Ly49 NKDs, thereby allowing a single Ly49 homodimer to engage two MHC-I molecules on the surface of an opposing cell (in trans).33 The backfolded Ly49 conformation, also referred to as ‘open’ in reference to the spacing between the a2 helices (red in Figure 3b), is facilitated by the flexible Ls loop that connects the NKD to the helical stalk. However, some Ly49 receptors, including Ly49A in complex with H2Dd have also been observed to adopt an alternate, closed conformation whereby the NKDs are closely juxtaposed in a manner that would prohibit backfolding of the stalks.33,36,52 To date, all structures of Ly49 Immunology and Cell Biology

receptors in the closed conformation have been derived from truncated expression constructs in which the stalks are absent, and thus it remains a possibility that this form represents a conformation that would not be encountered in vivo. However, such an arrangement, where the stalks presumably extend away from the NKDs, is consistent with a convincing body of data indicating that Ly49 receptors are capable of binding MHC-I within the plane of the same membrane (in cis), which would require a reorientation of the Ly49 NKD relative to the cell membrane.53 Indeed it has been reported that approximately 70% of Ly49A on the NK cell surface is associated with its H-2Dd ligand in cis, and that this population is incapable of binding H-2Dd in trans.54 This does not necessarily imply that the closed/extended Ly49 conformation is stable and unable to switch to the trans-binding backfolded conformation. Instead this observation may reflect that when released from cis interactions, a Ly49 receptor is more likely to re-engage the same ligand (in cis) rather than switch conformation and bind a different ligand in trans (the path of least resistance). Given that cis interactions do not result in productive signalling, the function of cis binding may be to reduce the number of receptors that are available for trans binding, thereby altering the threshold at which NK cells become activated.55 MCMV: A STEALTHY INVADER The crucial role of NK cells in the control of infection is perhaps best understood in the context of cytomegalovirus (CMV) infection. Whereas individual CMVs are specific for their particular host, as a genus they infect a wide range of animal species. In humans, CMV infection is highly prevalent, and although asymptomatic in healthy individuals, can persist throughout the host’s lifetime and cause considerable morbidity and even mortality if the immune system is compromised.56,57 The overarching evolutionary success of CMV is in part due to the large size of its genome, which affords the virus the opportunity to dedicate multiple genes to non-essential functions.58 A number of these viral genes encode proteins whose function is dedicated to the subversion of the host’s immune system. Such immunoevasion genes are perhaps best characterized in the MCMV system, where they cluster to defined regions of the genome, with the m02 family (genes m02 to m16) at the extreme left of the genome and the m145 family (m17, m145 to m158) at the extreme right.59 Intriguingly, despite often sharing little amino-acid sequence homology, a number of these CMV immunoevasins adopt MHC-I like folds, leading to the assumption that they function as molecular mimics. This is particularly true of members of the m145 family including m144,60 m152,61 m153,62 and m157,63 but also of the human CMV-encoded molecule UL18.64 Each of these immunoevasins possesses classical MHC-I features including a set of a-helices lying above a b-sheet platform and supported by an a3-like domain. However, they differ significantly in their ability to bind peptides and b2m. The mode of action of these CMV-encoded MHC-I-like proteins is also varied. For example, UL18 binds to the host inhibitory receptor LILRB1 (LIR-1/ILT2) with high affinity.65 The LILR bind to a broad range of classical and non-classical MHC-I molecules via the conserved a3 domain and the b2m subunit66–68 and these interactions are mimicked to a considerable degree by UL18.64 However, not all MHC-I like immunoevasins act via molecular mimicry. For example, m152 acts to downregulate the expression of both MHC-I69 and the NKG2D ligand RAE-1.70 Among the best characterized immunoevasins in MCMV is m157, a glycosylphosphatidylinositol-anchored MHC-I-like protein that does

Ly49 NK cell receptors: tool for viral self-discrimination R Berry et al 217

Ly49-MHC-I

Ly49-m157

Target cell

NK cell Open Ly49 conformation Backfolded stalks

Closed Ly49 conformation Extended stalks

Figure 3 Comparison of the mode of recognition of MHC-I and m157 by Ly49 receptors. (a) The points of contact between Ly49C and H2-Kb (PDB:1P1Z) and Ly49H and m157 (PDB:4JO8) are represented as spheres, with the exception of the Ly49 aromatic peg motif (Tyr115 and Trp123), which are represented as sticks. For simplicity, only the interactions between a single Ly49 monomer and H2-Kb (left) and a single m157 molecule with the Ly49 homodimer (right) are displayed. (b) Representation of Ly49-MHC-I and Ly49-m157 complexes at the NK-target cell interface. Ly49–MHC-I interactions are mediated by the Ly49 NKD (blue) and require an open Ly49 conformation whereby the stalks (light blue) backfold between the NKDs. In contrast, Ly49– m157 interactions occur via the Ly49 stalk region (light blue), which extend away from the NKDs and sits on top of the m157 (green) platform. The switch between backfolded and extended stalk arrangements is facilitated by the flexible Ls loop (dashed line). To emphasize the different domain juxtapositioning between the open and closed conformation, the Ly49 a2 helices are highlighted in red.

not bind peptides and does not associate with b2-microglobulin.63 Somewhat counterintuitively, m157 binds directly to the Ly49Hactivating receptor, leading to MCMV ‘resistance’ in mouse strains such as C57BL/6 (B6).71–76 This resistance is evident in reduced viral titres in the spleen and liver of mice during the acute phase of the infection but does not strictly correlate with lower levels of virus in the salivary gland, a key reservoir of virus with respect to transmission.72,77–79 However, the m157 protein can also be recognized by inhibitory Ly49 receptors such as Ly49I from the 129/J strain, raising the possibility that this interaction attenuates NK cell responses in mouse strains that lack Ly49H.71 Accordingly, it has been proposed that m157 originally evolved as a decoy receptor to ligate inhibitory Ly49 receptors in the absence of endogenous MHC-I, and that the activating Ly49H receptor evolved later to combat viral infection.71 A NOVEL STRATEGY FOR THE DETECTION OF VIRAL MHC MIMICS The molecular mechanisms underpinning Ly49H-mediated targeting of m157 have recently been elucidated.80 Remarkably, although m157 adopts an MHC-I fold, Ly49H does not utilize its NKD in a

manner analogous to that observed in Ly49–MHC-I interactions (Figure 3). Instead, m157 binding is mediated solely by the helical stalks, which allow a single Ly49 homodimer to simultaneously bind two m157 molecules.80 Here, the Ly49 stalks sit over the m157 platform, engaging the a1 and a2 helices of m157 in a manner more akin to T cell receptor (TCR)-mediated recognition of MHC-I.47 The Ly49–m157 interactions were mediated primarily by an ‘aromatic peg motif’ (a tyrosine and tryptophan residue spaced eight amino acids apart) in the membrane distal a3s segment of the Ly49 stalks. This motif is present in all Ly49 variants that recognize m157, and its disruption by mutagenesis abrogated m157 binding, suggesting a common strategy underlies recognition by both activating and inhibitory Ly49 receptors.80,81 Moreover, with two exceptions (Ly49IB6 and Ly49U129/J, discussed below), at least one of the aromatic residues within the motif was found to be absent in all Ly49 variants that have been reported to lack m157 binding. Precisely how and why m157 evolved an MHC-I-like fold but adopted a completely unique strategy to mediate its function is not entirely clear. One possible explanation is that by ‘tackling the legs’ of the receptor, m157 does not have to compete with MHC-I ligands for the Ly49-binding site. Immunology and Cell Biology

Ly49 NK cell receptors: tool for viral self-discrimination R Berry et al 218

THE IMPACT OF LY49 CONFORMATION ON M157 BINDING The Ly49 aromatic peg motif resides in the same segment of the stalk that is buried between the NKDs in the backfolded Ly49 conformation.33 Accordingly, the backfolded Ly49 conformation responsible for engaging MHC-I in trans is incompatible with m157 ligation. Despite being present within the crystal, the Ly49H NKD was not visible in the Ly49H–m157 complex structure.80 This was presumably due to the flexibility in the precise location of this domain, which is connected to the stalk (and by extension m157) via the flexible Ls loop. Thus, although the Ly49H NKD cannot be directly observed, the location of m157 adjacent to the stalks and the presence of a large ‘empty’ cavity in the crystal packing emanating from the C-termini of the Ly49 stalks are consistent with the view that Ly49 receptors engage m157 using the closed conformation where the stalks extend away from the Ly49 NKD. Evidence of two Ly49 conformations, one that permits m157 binding and the other that prohibits it, is supported by the location of natural sequence variations and targeted mutations within the Ly49 NKD.82 Before a number of advances including the observation of the backfolded Ly49 conformation and the elucidation of the mode of Ly49-m157 recognition, Kielczewska et al. performed a search for amino-acid residues within the NKD that discriminated between m157-binding and non-binding receptors,82 leading to the identification of amino-acid residues 146 and 151, both of which are present at the Ly49 homodimer interface. Mutation of these residues in m157-binding receptors such as Ly49H abrogated m157 recognition, whereas replacement with the corresponding Ly49H residue (His146 or Gly151) in non-binding Ly49 variants such as Ly49IB6 or Ly49U129/J conferred m157-binding capacity.82 In light of recent structural advances, the molecular basis for these observations has now been clarified. In the structure of backfolded Ly49-L4, residues 146 and 151 either make or are ideally positioned to make (depending on the side chain character) interactions with the backfolded a3s stalk. Accordingly, variations in these residues could conceivably stabilize or destabilize the backfolded Ly49 conformation, and thus indirectly affect the capacity to recognize m157. Thus, it appears that the residues lining the Ly49 homodimer interface comprise a second ‘hidden’ m157-binding determinant that does not directly affect Ly49–m157 interactions, but impacts m157 targeting indirectly by impacting on the Ly49 conformation. OCCAM’S RAZOR Receptor stalk regions are often considered as innocuous linkers that possess no intrinsic biological function. Accordingly, the finding that m157 recognition is mediated by the Ly49 stalks was unexpected, and may prompt re-examination of studies in which chimeric receptors, where the NKD of Ly49s are fused to the intracellular, transmembrane and stalk regions of an activating Ly49 receptor.82,83 For example, it was previously reported that m157-immunoglobulin fusion proteins stain reporter cells expressing the Ly49H NKD fused to the Ly49U stalk, but not the Ly49U NKD fused to the Ly49H stalk.82 Quite reasonably, the authors concluded that m157-binding capacity resided within the Ly49H NKD. However, it appears that in this situation, Occam’s razor, which states that the simplest solution is often the correct one, may not always hold true. One possible alternate explanation may be that the lack of m157 binding to the Ly49H stalk could be attributed to masking of the a3s region by the backfolded conformation engendered by the residues within the Ly49U NKD. Moreover, binding of the Ly49H NKD/Ly49U stalk chimera could be due to a lower propensity of the Ly49H NKD to adopt a backfolded conformation, thereby Immunology and Cell Biology

making the Ly49U stalk (which contains the aromatic peg motif) accessible for interactions with m157. UTILIZING SELF-MHC-I TO DETECT MCMV INFECTION It is also evident that m157 is not the only MCMV-encoded protein to be targeted by activating NK cell receptors. The observation that some mouse strains such as Ma/My that lack the Ly49H receptor also display enhanced control of viral replication in an NK cell-dependent manner suggested the presence of other key NK cell complex-encoded receptors.84,85 Intriguingly, this resistance relies on the presence of the activating Ly49P receptor, which specifically recognizes MCMVinfected cells in the presence of the H2k haplotype.86 Such a recognition event requires the presence of the virus-encoded glycoprotein m04, which binds to MHC-I molecules in the endoplasmic reticulum (ER) and on the cell surface.87,88 Unlike the related m06 protein that binds MHC-I and directs them to the lysosome for degradation,89 m04 escorts newly assembled MHC-I molecules to the cell surface. It has since been demonstrated that numerous activating receptors including Ly49LBALB, Ly49P1NOD and Ly49W1NOD can recognize MCMV-infected cells of certain H2 haplotypes (including H2d, H2k, H2a and H2f ) in an m04-specific manner.83 Accordingly, unlike Ly49H-mediated recognition of m157, which is largely restricted to B6 mice, m04-specific H2-dependent recognition of MCMV-infected cells appears to represent a more common mechanism of host defence. So far, relatively little is known regarding the precise molecular mechanism underpinning this novel viral recognition strategy, although the interaction between m04 and H2-Kb appears to reside within the m04 transmembrane region.90 Indeed, it is likely that there are yet to be further currently unidentified factors involved, as m04 is reportedly necessary but not sufficient for recognition of infection by Ly49PMa/My-expressing reporter cells.83 PERSPECTIVES Over the last decade, it has become increasingly apparent that the function of Ly49 receptors is not limited to MHC-I recognition. Instead, during the course of evolution, this large receptor family has become the target of a number of viral immunoevasins. In particular, targeting of the Ly49 stalks by the MCMV-encoded molecule m157 has highlighted the versatility of viruses and the host response, in the battle for immune evasion. In the future, it will be interesting to see whether targeting of membrane-bound receptor stalks represents a common strategy utilized by a diverse range of pathogens. Although the genetic divergence between MCMV and HCMV suggests that there may be no strict homologue of m157 in HCMV, the identity of virus-encoded ligands that ligate inhibitory or activating human NK cell receptors remains the ultimate goal of the field. Despite growing evidence of expansion of NK cell populations bearing activating receptors such as CD94-NKG2C and KIR2DS1/2 and 4 in CMVseropositive individuals,91,92 the identity of bona fide virus-encoded ligands for these receptors remains elusive.

1 Brooks AG, Posch PE, Scorzelli CJ, Borrego F, Coligan JE. NKG2A complexed with CD94 defines a novel inhibitory natural killer cell receptor. J Exp Med 1997; 185: 795–800. 2 Lazetic S, Chang C, Houchins JP, Lanier LL, Phillips JH. Human natural killer cell receptors involved in MHC class I recognition are disulfide-linked heterodimers of CD94 and NKG2 subunits. J Immunol 1996; 157: 4741–4745. 3 Borrego F, Ulbrecht M, Weiss EH, Coligan JE, Brooks AG. Recognition of human histocompatibility leukocyte antigen (HLA)-E complexed with HLA class I signal sequence-derived peptides by CD94/NKG2 confers protection from natural killer cell-mediated lysis. J Exp Med 1998; 187: 813–818.

Ly49 NK cell receptors: tool for viral self-discrimination R Berry et al 219 4 Braud VM, Allan DS, O’Callaghan CA, Soderstrom K, D’Andrea A, Ogg GS et al. HLA-E binds to natural killer cell receptors CD94/NKG2A, B and C. Nature 1998; 391: 795–799. 5 Petrie EJ, Clements CS, Lin J, Sullivan LC, Johnson D, Huyton T et al. CD94-NKG2A recognition of human leukocyte antigen (HLA)-E bound to an HLA class I leader sequence. J Exp Med 2008; 205: 725–735. 6 Sullivan LC, Clements CS, Beddoe T, Johnson D, Hoare HL, Lin J et al. The heterodimeric assembly of the CD94-NKG2 receptor family and implications for human leukocyte antigen-E recognition. Immunity 2007; 27: 900–911. 7 Zeng L, Sullivan LC, Vivian JP, Walpole NG, Harpur CM, Rossjohn J et al. A structural basis for antigen presentation by the MHC class Ib molecule, Qa-1b. J Immunol 2012; 188: 302–310. 8 Bacon L, Eagle RA, Meyer M, Easom N, Young NT, Trowsdale J. Two human ULBP/ RAET1 molecules with transmembrane regions are ligands for NKG2D. J Immunol 2004; 173: 1078–1084. 9 Bauer S, Groh V, Wu J, Steinle A, Phillips JH, Lanier LL et al. Activation of NK cells and T cells by NKG2D, a receptor for stress-inducible MICA. Science 1999; 285: 727–729. 10 Carayannopoulos LN, Naidenko OV, Fremont DH, Yokoyama WM. Cutting edge: murine UL16-binding protein-like transcript 1: a newly described transcript encoding a highaffinity ligand for murine NKG2D. J Immunol 2002; 169: 4079–4083. 11 Cerwenka A, Bakker AB, McClanahan T, Wagner J, Wu J, Phillips JH et al. Retinoic acid early inducible genes define a ligand family for the activating NKG2D receptor in mice. Immunity 2000; 12: 721–727. 12 Cosman D, Mullberg J, Sutherland CL, Chin W, Armitage R, Fanslow W et al. ULBPs, novel MHC class I-related molecules, bind to CMV glycoprotein UL16 and stimulate NK cytotoxicity through the NKG2D receptor. Immunity 2001; 14: 123–133. 13 Groh V, Rhinehart R, Randolph-Habecker J, Topp MS, Riddell SR, Spies T. Costimulation of CD8alphabeta T cells by NKG2D via engagement by MIC induced on virusinfected cells. Nat Immunol 2001; 2: 255–260. 14 Groh V, Steinle A, Bauer S, Spies T. Recognition of stress-induced MHC molecules by intestinal epithelial gammadelta T cells. Science 1998; 279: 1737–1740. 15 Li P, McDermott G, Strong RK. Crystal structures of RAE-1beta and its complex with the activating immunoreceptor NKG2D. Immunity 2002; 16: 77–86. 16 Li P, Morris DL, Willcox BE, Steinle A, Spies T, Strong RK. Complex structure of the activating immunoreceptor NKG2D and its MHC class I-like ligand MICA. Nat Immunol 2001; 2: 443–451. 17 Radaev S, Rostro B, Brooks AG, Colonna M, Sun PD. Conformational plasticity revealed by the cocrystal structure of NKG2D and its class I MHC-like ligand ULBP3. Immunity 2001; 15: 1039–1049. 18 Long EO, Burshtyn DN, Clark WP, Peruzzi M, Rajagopalan S, Rojo S et al. Killer cell inhibitory receptors: diversity, specificity, and function. Immunol Rev 1997; 155: 135–144. 19 Colonna M, Samaridis J. Cloning of immunoglobulin-superfamily members associated with HLA-C and HLA-B recognition by human natural killer cells. Science 1995; 268: 405–408. 20 Wagtmann N, Biassoni R, Cantoni C, Verdiani S, Malnati MS, Vitale M et al. Molecular clones of the p58 NK cell receptor reveal immunoglobulin-related molecules with diversity in both the extra- and intracellular domains. Immunity 1995; 2: 439–449. 21 Burshtyn DN, Scharenberg AM, Wagtmann N, Rajagopalan S, Berrada K, Yi T et al. Recruitment of tyrosine phosphatase HCP by the killer cell inhibitor receptor. Immunity 1996; 4: 77–85. 22 Campbell KS, Dessing M, Lopez-Botet M, Cella M, Colonna M. Tyrosine phosphorylation of a human killer inhibitory receptor recruits protein tyrosine phosphatase 1C. J Exp Med 1996; 184: 93–100. 23 Fry AM, Lanier LL, Weiss A. Phosphotyrosines in the killer cell inhibitory receptor motif of NKB1 are required for negative signaling and for association with protein tyrosine phosphatase 1C. J Exp Med 1996; 184: 295–300. 24 Boyington JC, Motyka SA, Schuck P, Brooks AG, Sun PD. Crystal structure of an NK cell immunoglobulin-like receptor in complex with its class I MHC ligand. Nature 2000; 405: 537–543. 25 Fan QR, Long EO, Wiley DC. Crystal structure of the human natural killer cell inhibitory receptor KIR2DL1-HLA-Cw4 complex. Nat Immunol 2001; 2: 452–460. 26 Vivian JP, Duncan RC, Berry R, O’Connor GM, Reid HH, Beddoe T et al. Killer cell immunoglobulin-like receptor 3DL1-mediated recognition of human leukocyte antigen B. Nature 2011; 479: 401–405. 27 Moretta A, Sivori S, Vitale M, Pende D, Morelli L, Augugliaro R et al. Existence of both inhibitory (p58) and activatory (p50) receptors for HLA-C molecules in human natural killer cells. J Exp Med 1995; 182: 875–884. 28 Carlyle JR, Mesci A, Fine JH, Chen P, Belanger S, Tai LH et al. Evolution of the Ly49 and Nkrp1 recognition systems. Semin Immunol 2008; 20: 321–330. 29 Makrigiannis AP, Anderson SK. Ly49 gene expression in different inbred mouse strains. Immunol Res 2000; 21: 39–47. 30 Wong S, Freeman JD, Kelleher C, Mager D, Takei F. Ly-49 multigene family. New members of a superfamily of type II membrane proteins with lectin-like domains. J Immunol 1991; 147: 1417–1423. 31 Yokoyama WM, Jacobs LB, Kanagawa O, Shevach EM, Cohen DI. A murine T lymphocyte antigen belongs to a supergene family of type II integral membrane proteins. J Immunol 1989; 143: 1379–1386. 32 Karlhofer FM, Ribaudo RK, Yokoyama WM. MHC class I alloantigen specificity of Ly-49 þ IL-2-activated natural killer cells. Nature 1992; 358: 66–70.

33 Back J, Malchiodi EL, Cho S, Scarpellino L, Schneider P, Kerzic MC et al. Distinct conformations of Ly49 natural killer cell receptors mediate MHC class I recognition in trans and cis. Immunity 2009; 31: 598–608. 34 Dam J, Guan R, Natarajan K, Dimasi N, Chlewicki LK, Kranz DM et al. Variable MHC class I engagement by Ly49 natural killer cell receptors demonstrated by the crystal structure of Ly49C bound to H-2K(b). Nat Immunol 2003; 4: 1213–1222. 35 Dimasi N, Sawicki MW, Reineck LA, Li Y, Natarajan K, Margulies DH et al. Crystal structure of the Ly49I natural killer cell receptor reveals variability in dimerization mode within the Ly49 family. J Mol Biol 2002; 320: 573–585. 36 Tormo J, Natarajan K, Margulies DH, Mariuzza RA. Crystal structure of a lectin-like natural killer cell receptor bound to its MHC class I ligand. Nature 1999; 402: 623–631. 37 Nakamura MC, Niemi EC, Fisher MJ, Shultz LD, Seaman WE, Ryan JC. Mouse Ly-49A interrupts early signaling events in natural killer cell cytotoxicity and functionally associates with the SHP-1 tyrosine phosphatase. J Exp Med 1997; 185: 673–684. 38 Smith KM, Wu J, Bakker AB, Phillips JH, Lanier LL. Ly-49D and Ly-49H associate with mouse DAP12 and form activating receptors. J Immunol 1998; 161: 7–10. 39 Lanier LL, Corliss BC, Wu J, Leong C, Phillips JH. Immunoreceptor DAP12 bearing a tyrosine-based activation motif is involved in activating NK cells. Nature 1998; 391: 703–707. 40 Orr MT, Sun JC, Hesslein DG, Arase H, Phillips JH, Takai T et al. Ly49H signaling through DAP10 is essential for optimal natural killer cell responses to mouse cytomegalovirus infection. J Exp Med 2009; 206: 807–817. 41 Natarajan K, Boyd LF, Schuck P, Yokoyama WM, Eliat D, Margulies DH. Interaction of the NK cell inhibitory receptor Ly49A with H-2Dd: identification of a site distinct from the TCR site. Immunity 1999; 11: 591–601. 42 Andrews DM, Sullivan LC, Baschuk N, Chan CJ, Berry R, Cotterell CL et al. Recognition of the nonclassical MHC class I molecule H2-M3 by the receptor Ly49A regulates the licensing and activation of NK cells. Nat Immunol 2012; 13: 1171–1177. 43 Deng L, Cho S, Malchiodi EL, Kerzic MC, Dam J, Mariuzza RA. Molecular architecture of the major histocompatibility complex class I-binding site of Ly49 natural killer cell receptors. J Biol Chem 2008; 283: 16840–16849. 44 Choi T, Ferris ST, Matsumoto N, Poursine-Laurent J, Yokoyama WM. Ly49-dependent NK cell licensing and effector inhibition involve the same interaction site on MHC ligands. J Immunol 2011; 186: 3911–3917. 45 Matsumoto N, Mitsuki M, Tajima K, Yokoyama WM, Yamamoto K. The functional binding site for the C-type lectin-like natural killer cell receptor Ly49A spans three domains of its major histocompatibility complex class I ligand. J Exp Med 2001; 193: 147–158. 46 Wang J, Whitman MC, Natarajan K, Tormo J, Mariuzza RA, Margulies DH. Binding of the natural killer cell inhibitory receptor Ly49A to its major histocompatibility complex class I ligand. Crucial contacts include both H-2Dd AND beta 2-microglobulin. J Biol Chem 2002; 277: 1433–1442. 47 Gras S, Burrows SR, Turner SJ, Sewell AK, McCluskey J, Rossjohn J. A structural voyage toward an understanding of the MHC-I-restricted immune response: lessons learned and much to be learned. Immunol Rev 2012; 250: 61–81. 48 Correa I, Raulet DH. Binding of diverse peptides to MHC class I molecules inhibits target cell lysis by activated natural killer cells. Immunity 1995; 2: 61–71. 49 Roth C, Kourilsky P, Ojcius DM. Ly-49-independent inhibition of natural killer cellmediated cytotoxicity by a soluble major histocompatibility complex class I molecule. Eur J Immunol 1994; 24: 2110–2114. 50 Franksson L, Sundback J, Achour A, Bernlind J, Glas R, Karre K. Peptide dependency and selectivity of the NK cell inhibitory receptor Ly-49C. Eur J Immunol 1999; 29: 2748–2758. 51 Hanke T, Takizawa H, McMahon CW, Busch DH, Pamer EG, Miller JD et al. Direct assessment of MHC class I binding by seven Ly49 inhibitory NK cell receptors. Immunity 1999; 11: 67–77. 52 Dam J, Baber J, Grishaev A, Malchiodi EL, Schuck P, Bax A et al. Variable dimerization of the Ly49A natural killer cell receptor results in differential engagement of its MHC class I ligand. J Mol Biol 2006; 362: 102–113. 53 Back J, Angelov GS, Mariuzza RA, Held W. The interaction with H-2D(d) in cis is associated with a conformational change in the Ly49A NK cell receptor. Front Immunol2: 55. 54 Back J, Chalifour A, Scarpellino L, Held W. Stable masking by H-2Dd cis ligand limits Ly49A relocalization to the site of NK cell/target cell contact. Proc Natl Acad Sci USA 2007; 104: 3978–3983. 55 Doucey MA, Scarpellino L, Zimmer J, Guillaume P, Luescher IF, Bron C et al. Cis association of Ly49A with MHC class I restricts natural killer cell inhibition. Nat Immunol 2004; 5: 328–336. 56 Reddehase MJ. Antigens and immunoevasins: opponents in cytomegalovirus immune surveillance. Nat Rev Immunol 2002; 2: 831–844. 57 Sissons JG, Carmichael AJ, McKinney N, Sinclair JH, Wills MR. Human cytomegalovirus and immunopathology. Springer Semin Immunopathol 2002; 24: 169–185. 58 Dunn W, Chou C, Li H, Hai R, Patterson D, Stolc V et al. Functional profiling of a human cytomegalovirus genome. Proc Natl Acad Sci USA 2003; 100: 14223–14228. 59 Rawlinson WD, Farrell HE, Barrell BG. Analysis of the complete DNA sequence of murine cytomegalovirus. J Virol 1996; 70: 8833–8849. 60 Natarajan K, Hicks A, Mans J, Robinson H, Guan R, Mariuzza RA et al. Crystal structure of the murine cytomegalovirus MHC-I homolog m144. J Mol Biol 2006; 358: 157–171. 61 Wang R, Natarajan K, Revilleza MJ, Boyd LF, Zhi L, Zhao H et al. Structural basis of mouse cytomegalovirus m152/gp40 interaction with RAE1gamma reveals a paradigm

Immunology and Cell Biology

Ly49 NK cell receptors: tool for viral self-discrimination R Berry et al 220

62

63

64

65

66

67

68

69

70

71

72

73

74

75

76

for MHC/MHC interaction in immune evasion. Proc Natl Acad Sci USA 2012; 109: E3578–E3587. Mans J, Natarajan K, Balbo A, Schuck P, Eikel D, Hess S et al. Cellular expression and crystal structure of the murine cytomegalovirus major histocompatibility complex class I-like glycoprotein, m153. J Biol Chem 2007; 282: 35247–35258. Adams EJ, Juo ZS, Venook RT, Boulanger MJ, Arase H, Lanier LL et al. Structural elucidation of the m157 mouse cytomegalovirus ligand for Ly49 natural killer cell receptors. Proc Natl Acad Sci USA 2007; 104: 10128–10133. Yang Z, Bjorkman PJ. Structure of UL18, a peptide-binding viral MHC mimic, bound to a host inhibitory receptor. Proc Natl Acad Sci USA 2008; 105: 10095–10100. Cosman D, Fanger N, Borges L, Kubin M, Chin W, Peterson L et al. A novel immunoglobulin superfamily receptor for cellular and viral MHC class I molecules. Immunity 1997; 7: 273–282. Chapman TL, Heikeman AP, Bjorkman PJ. The inhibitory receptor LIR-1 uses a common binding interaction to recognize class I MHC molecules and the viral homolog UL18. Immunity 1999; 11: 603–613. Shiroishi M, Kuroki K, Rasubala L, Tsumoto K, Kumagai I, Kurimoto E et al. Structural basis for recognition of the nonclassical MHC molecule HLA-G by the leukocyte Ig-like receptor B2 (LILRB2/LIR2/ILT4/CD85d). Proc Natl Acad Sci USA 2006; 103: 16412–16417. Willcox BE, Thomas LM, Bjorkman PJ. Crystal structure of HLA-A2 bound to LIR-1, a host and viral major histocompatibility complex receptor. Nat Immunol 2003; 4: 913–919. Ziegler H, Thale R, Lucin P, Muranyi W, Flohr T, Hengel H et al. A mouse cytomegalovirus glycoprotein retains MHC class I complexes in the ERGIC/cis-Golgi compartments. Immunity 1997; 6: 57–66. Lodoen M, Ogasawara K, Hamerman JA, Arase H, Houchins JP, Mocarski ES et al. NKG2D-mediated natural killer cell protection against cytomegalovirus is impaired by viral gp40 modulation of retinoic acid early inducible 1 gene molecules. J Exp Med 2003; 197: 1245–1253. Arase H, Mocarski ES, Campbell AE, Hill AB, Lanier LL. Direct recognition of cytomegalovirus by activating and inhibitory NK cell receptors. Science 2002; 296: 1323–1326. Brown MG, Dokun AO, Heusel JW, Smith HR, Beckman DL, Blattenberger EA et al. Vital involvement of a natural killer cell activation receptor in resistance to viral infection. Science 2001; 292: 934–937. Daniels KA, Devora G, Lai WC, O’Donnell CL, Bennett M, Welsh RM. Murine cytomegalovirus is regulated by a discrete subset of natural killer cells reactive with monoclonal antibody to Ly49H. J Exp Med 2001; 194: 29–44. Lee SH, Girard S, Macina D, Busa M, Zafer A, Belouchi A et al. Susceptibility to mouse cytomegalovirus is associated with deletion of an activating natural killer cell receptor of the C-type lectin superfamily. Nat Genet 2001; 28: 42–45. McWhorter AR, Smith LM, Masters LL, Chan B, Shellam GR, Redwood AJ. Natural killer cell dependent within-host competition arises during multiple MCMV infection: consequences for viral transmission and evolution. PLoS Pathog 2013; 9: e1003111. Scalzo AA, Fitzgerald NA, Simmons A, La Vista AB, Shellam GR. Cmv-1, a genetic locus that controls murine cytomegalovirus replication in the spleen. J Exp Med 1990; 171: 1469–1483.

Immunology and Cell Biology

77 Andrews DM, Estcourt MJ, Andoniou CE, Wikstrom ME, Khong A, Voigt V et al. Innate immunity defines the capacity of antiviral T cells to limit persistent infection. J Exp Med 2010; 207: 1333–1343. 78 Smith HR, Heusel JW, Mehta IK, Kim S, Dorner BG, Naidenko OV et al. Recognition of a virus-encoded ligand by a natural killer cell activation receptor. Proc Natl Acad Sci USA 2002; 99: 8826–8831. 79 Voigt V, Forbes CA, Tonkin JN, Degli-Esposti MA, Smith HR, Yokoyama WM et al. Murine cytomegalovirus m157 mutation and variation leads to immune evasion of natural killer cells. Proc Natl Acad Sci USA 2003; 100: 13483–13488. 80 Berry R, Ng N, Saunders PM, Vivian JP, Lin J, Deuss FA et al. Targeting of a natural killer cell receptor family by a viral immunoevasin. Nat Immunol 2013; 14: 699–705. 81 Corbett AJ, Coudert JD, Forbes CA, Scalzo AA. Functional consequences of natural sequence variation of murine cytomegalovirus m157 for Ly49 receptor specificity and NK cell activation. J Immunol 2011; 186: 1713–1722. 82 Kielczewska A, Kim HS, Lanier LL, Dimasi N, Vidal SM. Critical residues at the Ly49 natural killer receptor’s homodimer interface determine functional recognition of m157, a mouse cytomegalovirus MHC class I-like protein. J Immunol 2007; 178: 369–377. 83 Pyzik M, Charbonneau B, Gendron-Pontbriand EM, Babic M, Krmpotic A, Jonjic S et al. Distinct MHC class I-dependent NK cell-activating receptors control cytomegalovirus infection in different mouse strains. J Exp Med 2011; 208: 1105–1117. 84 Desrosiers MP, Kielczewska A, Loredo-Osti JC, Adam SG, Makrigiannis AP, Lemieux S et al. Epistasis between mouse Klra and major histocompatibility complex class I loci is associated with a new mechanism of natural killer cell-mediated innate resistance to cytomegalovirus infection. Nat Genet 2005; 37: 593–599. 85 Scalzo AA, Lyons PA, Fitzgerald NA, Forbes CA, Yokoyama WM, Shellam GR. Genetic mapping of Cmv1 in the region of mouse chromosome 6 encoding the NK gene complex-associated loci Ly49 and musNKR-P1. Genomics 1995; 27: 435–441. 86 Kielczewska A, Pyzik M, Sun T, Krmpotic A, Lodoen MB, Munks MW et al. Ly49P recognition of cytomegalovirus-infected cells expressing H2-Dk and CMV-encoded m04 correlates with the NK cell antiviral response. J Exp Med 2009; 206: 515–523. 87 Kavanagh DG, Koszinowski UH, Hill AB. The murine cytomegalovirus immune evasion protein m4/gp34 forms biochemically distinct complexes with class I MHC at the cell surface and in a pre-Golgi compartment. J Immunol 2001; 167: 3894–3902. 88 Kleijnen MF, Huppa JB, Lucin P, Mukherjee S, Farrell H, Campbell AE et al. A mouse cytomegalovirus glycoprotein, gp34, forms a complex with folded class I MHC molecules in the ER which is not retained but is transported to the cell surface. EMBO J 1997; 16: 685–694. 89 Reusch U, Muranyi W, Lucin P, Burgert HG, Hengel H, Koszinowski UH. A cytomegalovirus glycoprotein re-routes MHC class I complexes to lysosomes for degradation. EMBO J 1999; 18: 1081–1091. 90 Lu X, Kavanagh DG, Hill AB. Cellular and molecular requirements for association of the murine cytomegalovirus protein m4/gp34 with major histocompatibility complex class I molecules. J Virol 2006; 80: 6048–6055. 91 Beziat V, Liu LL, Malmberg JA, Ivarsson MA, Sohlberg E, Bjorklund AT et al. NK cell responses to cytomegalovirus infection lead to stable imprints in the human KIR repertoire and involve activating KIRs. Blood 2013; 121: 2678–2688. 92 Guma M, Angulo A, Vilches C, Gomez-Lozano N, Malats N, Lopez-Botet M. Imprint of human cytomegalovirus infection on the NK cell receptor repertoire. Blood 2004; 104: 3664–3671.