The putative natural killer decoy early gene m04 (gp34) of murine cytomegalovirus encodes an antigenic peptide recognized by protective antiviral CD8 T cells

Several early genes of murine cytomegalovirus (MCMV) encode proteins that mediate immune evasion by interference with the major histocompatibility complex class I (MHC-I) pathway of antigen presentation to cytolytic T lymphocytes (CTL). Specifically, the m152 gene product gp37/40 causes retention of MHC-I molecules in the endoplasmic reticulum (ER)-Golgi intermediate compartment. Lack of MHC-I on the cell surface should activate natural killer (NK) cells recognizing the "missing self." The retention, however, is counteracted by the m04 early gene product gp34, which binds to folded MHC-I molecules in the ER and directs the complex to the cell surface. It was thus speculated that gp34 might serve to silence NK cells and thereby complete the immune evasion of MCMV. In light of these current views, we provide here results demonstrating an in vivo role for gp34 in protective antiviral immunity. We have identified an antigenic nonapeptide derived from gp34 and presented by the MHC-I molecule D(d). Besides the immunodominant immediate-early nonapeptide consisting of IE1 amino acids 168-176 (IE1(168-176)), the early nonapeptide m04(243-251) is the second antigenic peptide described for MCMV. The primary immune response to MCMV generates significant m04-specific CD8 T-cell memory. Upon adoptive transfer into immunodeficient recipients, an m04-specific CTL line controls MCMV infection with an efficacy comparable to that of an IE1-specific CTL line. Thus, gp34 is the first noted early protein of MCMV that escapes viral immune evasion mechanisms. These data document that MCMV is held in check by a redundance of protective CD8 T cells recognizing antigenic peptides in different phases of viral gene expression.     

A Novel MHC-I Surface Targeted for Binding by the MCMV m06 Immunoevasin Revealed by Solution NMR

As part of its strategy to evade detection by the host immune system, murine cytomegalovirus (MCMV) encodes three proteins that modulate cell surface expression of major histocompatibility complex class I (MHC-I) molecules: the MHC-I homolog m152/gp40 as well as the m02-m16 family members m04/gp34 and m06/gp48. Previous studies of the m04 protein revealed a divergent Ig-like fold that is unique to immunoevasins of the m02-m16 family. Here, we engineer and characterize recombinant m06 and investigate its interactions with full-length and truncated forms of the MHC-I molecule H2-L^d by several techniques. Furthermore, we employ solution NMR to map the interaction footprint of the m06 protein on MHC-I, taking advantage of a truncated H2-L^d, “mini-H2-L^d,” consisting of only the α1α2 platform domain. Mini-H2-L^d refolded in vitro with a high affinity peptide yields a molecule that shows outstanding NMR spectral features, permitting complete backbone assignments. These NMR-based studies reveal that m06 binds tightly to a discrete site located under the peptide-binding platform that partially overlaps with the β₂-microglobulin interface on the MHC-I heavy chain, consistent with in vitro binding experiments showing significantly reduced complex formation between m06 and β₂-microglobulin-associated MHC-I. Moreover, we carry out NMR relaxation experiments to characterize the picosecond-nanosecond dynamics of the free mini-H2-L^d MHC-I molecule, revealing that the site of interaction is highly ordered. This study provides insight into the mechanism of the interaction of m06 with MHC-I, suggesting a structural manipulation of the target MHC-I molecule at an early stage of the peptide-loading pathway.     

Expression of m157, a murine cytomegalovirus-encoded putative major histocompatibility class I (MHC-I)-like protein, is independent of viral regulation of host MHC-I

A murine cytomegalovirus (MCMV)-encoded protein, m157, has a putative major histocompatibility complex class I (MHC-I) structure and is recognized by the Ly49H NK cell activation receptor. Using a monoclonal antibody against m157, in this study we directly demonstrated that m157 is a cell surface-expressed glycophosphatidylinositol-anchored protein with early viral gene kinetics. Beta-2 microglobulin and TAP1 (transporter associated with antigen processing 1) were not required for its expression. MCMV-encoded proteins that down-regulate MHC-I did not affect the expression of m157. Thus, m157 is expressed on infected cells in a manner independent of viral regulation of host MHC-I.     

Effective inhibition of K(b)- and D(b)-restricted antigen presentation in primary macrophages by murine cytomegalovirus

Macrophages play an important role in murine cytomegalovirus (MCMV) infection in vivo, both in disseminating infection and in harboring latent virus. MCMV encodes three immune evasion genes (m4, m6, and m152) that interfere with the ability of cytotoxic T cells (CTL) to detect virus-infected fibroblasts, but the efficacy of immune evasion in macrophages has been controversial. Here we show that MCMV immune evasion genes function in H-2(b) primary bone marrow macrophages (BMMphi) in the same way that they do in fibroblasts. Metabolic labeling experiments showed that class I is retained in the endoplasmic reticulum by MCMV infection and associates with m4/gp34 to a similar extent in fibroblasts and BMMphi. We tested a series of K(b)- and D(b)-restricted CTL clones specific for MCMV early genes against a panel of MCMV wild-type virus and mutants lacking m152, m4, or m6. MCMV immune evasion genes effectively inhibited antigen presentation. m152 appeared sufficient to abolish D(b)-restricted presentation in infected macrophages, as has been previously observed in infected fibroblasts. However, for inhibition of recognition of infected macrophages by K(b)-restricted CTL, m4, m6, and m152 were all required. The contribution of m4 to inhibition of recognition appeared much more important in macrophages than in fibroblasts. Thus, MCMV immune evasion genes function effectively in primary macrophages to prevent CTL recognition of early antigens and show the same pattern of major histocompatibility complex class I allele discrimination as is seen in fibroblasts. Furthermore, for inhibition of K(b)-restricted presentation, a strong synergistic effect was noted among m152, m4, and m6.     

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.     

The Structure of the Cytomegalovirus-Encoded m04 Glycoprotein,a Prototypical Member of the m02 Family of Immunoevasins

The ability of CMVs to evade the immune system of the host is dependent on the expression of a wide array of glycoproteins, many of which interfere with natural killer cell function. In murine CMV, two large protein families mediate this immune-evasive function. Although it is established that the m145 family members mimic the structure of MHC-I molecules, the structure of the m02 family remains unknown. The most extensively studied m02 family member is m04, a glycoprotein that escorts newly assembled MHC-I molecules to the cell surface, presumably to avoid “missing self” recognition. Here we report the crystal structure of the m04 ectodomain, thereby providing insight into this large immunoevasin family. m04 adopted a β-sandwich immunoglobulin variable (Ig-V)-like fold, despite sharing very little sequence identity with the Ig-V superfamily. In addition to the Ig-V core, m04 possesses several unique structural features that included an unusual β-strand topology, a number of extended loops and a prominent α-helix. The m04 interior was packed by a myriad of hydrophobic residues that form distinct clusters around two conserved tryptophan residues. This hydrophobic core was well conserved throughout the m02 family, thereby indicating that murine CMV encodes a number of Ig-V-like molecules. We show that m04 binds a range of MHC-I molecules with low affinity in a peptide-independent manner. Accordingly, the structure of m04, which represents the first example of an murine CMV encoded Ig-V fold, provides a basis for understanding the structure and function of this enigmatic and large family of immunoevasins.     

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.     

The murine cytomegalovirus immunomodulatory gene m152 prevents recognition of infected cells by M45-specific CTL but does not alter the immunodominance of the M45-specific CD8 T cell response in vivo

Although in vitro studies have shown that herpesviruses, including murine CMV (MCMV), encode genes that interfere with the MHC class I pathway, their effects on the CTL response in vivo is unclear. We identified a D(b)-restricted CTL epitope from MCMV M45 by screening an MCMV genomic library using CTL clones isolated from mice infected with MCMV lacking m152. Because m152 severely inhibits CTL recognition of M45 in vitro, we questioned whether an M45-specific response would be generated in mice infected with wild-type MCMV expressing m152. Mice infected with wild-type MCMV or MCMVDelta(m)152 made similar responses to the M45 Ag. Moreover, we saw no skewing of the proportion of M45-specific CD8 T cells within the total MCMV-specific response after infection with MCMV with m152. Despite the profound effect m152 has on presentation of M45 in vitro, it does not affect the immunodominance of M45 in the CTL response in vivo.     

Murine Cytomegalovirus Perturbs Endosomal Trafficking of Major Histocompatibility Complex Class I Molecules in the Early Phase of Infection

Murine cytomegalovirus (MCMV) functions interfere with protein trafficking in the secretory pathway. In this report we used Δm138-MCMV, a recombinant virus with a deleted viral Fc receptor, to demonstrate that MCMV also perturbs endosomal trafficking in the early phase of infection. This perturbation had a striking impact on cell surface-resident major histocompatibility complex class I (MHC-I) molecules due to the complementary effect of MCMV immunoevasins, which block their egress from the secretory pathway. In infected cells, constitutively endocytosed cell surface-resident MHC-I molecules were arrested and retained in early endosomal antigen 1 (EEA1)-positive and lysobisphosphatidic acid (LBPA)-negative perinuclear endosomes together with clathrin-dependent cargo (transferrin receptor, Lamp1, and epidermal growth factor receptor). Their progression from these endosomes into recycling and degradative routes was inhibited. This arrest was associated with a reduction of the intracellular content of Rab7 and Rab11, small GTPases that are essential for the maturation of recycling and endolysosomal domains of early endosomes. The reduced recycling of MHC-I in Δm138-MCMV-infected cells was accompanied by their accelerated loss from the cell surface. The MCMV function that affects cell surface-resident MHC-I was activated in later stages of the early phase of viral replication, after the expression of known immunoevasins. MCMV without the three immunoevasins (the m04, m06, and m152 proteins) encoded a function that affects endosomal trafficking. This function, however, was not sufficient to reduce the cell surface expression of MHC-I in the absence of the transport block in the secretory pathway.Herpesviruses are well known to interfere with major histocompatibility complex class I (MHC-I) molecules in order to ensure evasion from immune recognition. A majority of evidence so far indicates that they target MHC-I maturation events and MHC-I trafficking in the secretory pathway (33), although evidence exists suggesting that herpesviruses could also interfere with MHC-I functions in endosomal pathways (8). Murine cytomegalovirus (MCMV), a member of the herpesvirus family, dedicates a substantial part of its genome to encoding nonessential genes for the modulation of cellular functions (40), including MHC-I trafficking in the secretory pathway (24, 27, 45, 48, 49, 52). All known immune evasion functions encoded by MCMV are based on a direct interaction of viral gene products with MHC-I complexes in the secretory pathway. The egress of nascent MHC-I complexes to the cell surface of MCMV-infected cells is abolished as a consequence of their retention in the endoplasmic reticulum (ER)-cis-Golgi intermediate compartment (ERGIC) by the m152 gene product (10, 19, 24, 52, 56) as well as redirection of those that escape into the Golgi compartment toward late endosomes (LEs) for degradation by the m06 MCMV gene product (45). These effects are opposed by gp34, a product of the MCMV m04 gene, which associates with MHC-I complexes and reaches the cell surface (24, 27).The loss of MHC-I from the cell surface is an expected consequence of the activity of m152 and m06, which act in the secretory pathway. The level of cell surface MHC-I is substantially reduced at later times of infection (10, 19, 24, 48, 52), and cells stably transfected with either the m152 or m06 gene do not display MHC-I at the cell surface (20, 24). If the loss of MHC-I from the cell surface is a consequence of the prevented egress from the secretory pathway, then the cell surface loss should follow the kinetics of the constitutive internalization of MHC-I complexes in the endosomal pathway. Given that the constitutive internalization is the net result of cell surface supply from the secretory pathway, endocytic uptake, and endocytic recycling, it is a slow process that occurs in normal fibroblasts at a rate of ∼6 to 8% per hour (36). Therefore, the effect of MCMV immunoevasins on cell surface MHC-I should be expected at later times of infection. However, several reports demonstrated that the level of MHC-I surface expression was already reduced in the early phase of infection (10, 45, 48, 52). Thus, it would be reasonable to expect that MCMV contributes with a function that causes the accelerated retrieval of cell surface-resident MHC-I complexes.In this report we demonstrate that MCMV perturbs endosomal trafficking very early in infection by acting on distal parts of early endosome (EE) route and affecting the trafficking of both clathrin-dependent and clathrin-independent cargoes. Clathrin-dependent cargo does not share primary endocytic carriers with MHC-I proteins (12, 14), which enter the cell via the nonclathrin Arf6-associated endocytic carriers (12, 14, 41, 42, 53), but they meet in the proximal part of the common early endocytic route and redirect to distal endocytic carriers around the cell center (12, 14). The perturbation of the distal part of the EE route has dramatic consequences on MHC-I, since it supplements the viral mechanisms that act in the secretory pathway. The net result of this perturbation is a complete loss of MHC-I molecules from the cell surface.     

Role of murine cytomegalovirus US22 gene family members in replication in macrophages

The large cytomegalovirus (CMV) US22 gene family, found in all betaherpesviruses, comprises 12 members in both human cytomegalovirus (HCMV) and murine cytomegalovirus (MCMV). Conserved sequence motifs suggested a common ancestry and related functions for these gene products. Two members of this family, m140 and m141, were recently shown to affect MCMV replication on macrophages. To test the role of all US22 members in cell tropism, we analyzed the growth properties in different cell types of MCMV mutants carrying transposon insertions in all 12 US22 gene family members. When necessary, additional targeted mutants with gene deletions, ATG deletions, and ectopic gene revertants were constructed. Mutants with disruption of genes M23, M24, m25.1, m25.2, and m128 (ie2) showed no obvious growth phenotype, whereas growth of M43 mutants was reduced in a number of cell lines. Genes m142 and m143 were shown to be essential for virus replication. Growth of mutants with insertions into genes M36, m139, m140, and m141 in macrophages was severely affected. The common phenotype of the m139, m140, and m141 mutants was explained by an interaction at the protein level. The M36-dependent macrophage growth phenotype could be explained by the antiapoptotic function of the gene that was required for growth on macrophages but not for growth on other cell types. Together, the comprehensive set of mutants of the US22 gene family suggests that individual family members have diverged through evolution to serve a variety of functions for the virus.     

Coordinated function of murine cytomegalovirus genes completely inhibits CTL lysis

Murine CMV (MCMV) encodes three viral genes that interfere with Ag presentation (VIPRs) to CD8 T cells, m04, m06, and m152. Because the functional impact of these genes during normal infection of C57BL/6 mice is surprisingly modest, we wanted to determine whether the VIPRs are equally effective against the entire spectrum of H-2(b)-restricted CD8 T cell epitopes. We also wanted to understand how the VIPRs interact at a functional level. To address these questions, we used a panel of MCMV mutants lacking each VIPR in all possible combinations, and CTL specific for 15 H-2(b)-restricted MCMV epitopes. Only expression of all three MCMV VIPRs completely inhibited killing by CTL specific for all 15 epitopes, but removal of any one VIPR enabled lysis by at least some CTL. The dominant interaction between the VIPRs was cooperation: m06 increased the inhibition of lysis achieved by either m152 or m04. However, for 1 of 15 epitopes m04 functionally antagonized m152. There was little differential impact of any of the VIPRs on K(b) vs D(b), but a surprising degree of differential impact of the three VIPRs for different epitopes. These epitope-specific differences did not correlate with functional avidity, or with timing of VIPR expression in relation to Ag expression in the virus replication cycle. Although questions remain about the molecular mechanism and in vivo role of these genes, we conclude that the coordinated function of MCMV's three VIPRs results in a powerful inhibition of lysis of infected cells by CD8 T cells.     

NK cell receptor/H2-Dk-dependent host resistance to viral infection is quantitatively modulated by H2q inhibitory signals

The cytomegalovirus resistance locus Cmv3 has been linked to an epistatic interaction between two loci: a Natural Killer (NK) cell receptor gene and the major histocompatibility complex class I (MHC-I) locus. To demonstrate the interaction between Cmv3 and H2(k), we generated double congenic mice between MA/My and BALB.K mice and an F(2) cross between FVB/N (H-2(q)) and BALB.K (H2(k)) mice, two strains susceptible to mouse cytomegalovirus (MCMV). Only mice expressing H2(k) in conjunction with Cmv3(MA/My) or Cmv3(FVB) were resistant to MCMV infection. Subsequently, an F(3) cross was carried out between transgenic FVB/H2-D(k) and MHC-I deficient mice in which only the progeny expressing Cmv3(FVB) and a single H2-D(k) class-I molecule completely controlled MCMV viral loads. This phenotype was shown to be NK cell-dependent and associated with subsequent NK cell proliferation. Finally, we demonstrated that a number of H2(q) alleles influence the expression level of H2(q) molecules, but not intrinsic functional properties of NK cells; viral loads, however, were quantitatively proportional to the number of H2(q) alleles. Our results support a model in which H-2(q) molecules convey Ly49-dependent inhibitory signals that interfere with the action of H2-D(k) on NK cell activation against MCMV infection. Thus, the integration of activating and inhibitory signals emanating from various MHC-I/NK cell receptor interactions regulates NK cell-mediated control of viral load.     

Complete DNA sequence of the rat cytomegalovirus genome

We have determined the complete genome sequence of the Maastricht strain of rat cytomegalovirus (RCMV). The RCMV genome has a length of 229,896 bp and is arranged as a single unique sequence flanked by 504-bp terminal direct repeats. RCMV was found to have counterparts of all but one of the open reading frames (ORFs) that are conserved between murine CMV (MCMV) and human CMV (HCMV). Like HCMV, RCMV lacks homologs of the genes belonging to the MCMV m02 glycoprotein gene family. However, RCMV contains 15 ORFs with homology to members of the MCMV m145 glycoprotein gene family. Four ORFs are predicted to encode homologs of host proteins; R33 and R78 both putatively encode G protein-coupled receptors, whereas r144 and r131 encode homologs of major histocompatibility class I heavy chains and CC chemokines, respectively. An intriguing feature of the RCMV genome is the presence of an ORF, r127, with similarity to the rep gene of parvoviruses as well as ORF U94 of human herpesvirus 6A (HHV-6A) and HHV-6B. Counterparts of these ORFs have not been found in the other sequenced herpesviruses.     

Double-stranded RNA binding by a heterodimeric complex of murine cytomegalovirus m142 and m143 proteins

In response to viral infection, cells activate a variety of antiviral responses, including several that are triggered by double-stranded (ds) RNA. Among these are the protein kinase R and oligoadenylate synthetase/RNase L pathways, both of which result in the shutoff of protein synthesis. Many viruses, including human cytomegalovirus, encode dsRNA-binding proteins that prevent the activation of these pathways and thereby enable continued protein synthesis and viral replication. We have extended these analyses to another member of the beta subfamily of herpesviruses, murine cytomegalovirus (MCMV), and now report that products of the m142 and m143 genes together bind dsRNA. Coimmunoprecipitation experiments demonstrate that these two proteins interact in infected cells, consistent with their previously reported colocalization. Jointly, but not individually, the proteins rescue replication of a vaccinia virus mutant with a deletion of the dsRNA-binding protein gene E3L (VVDeltaE3L). Like the human cytomegalovirus dsRNA-binding protein genes TRS1 and IRS1, m142 and m143 are members of the US22 gene family. We also found that two other members of the MCMV US22 family, M23 and M24, encode dsRNA-binding proteins, but they do not rescue VVDeltaE3L replication. These results reveal that MCMV, like many other viruses, encodes dsRNA-binding proteins, at least two of which can inhibit dsRNA-activated antiviral pathways. However, unlike other well-studied examples, the MCMV proteins appear to act in a heterodimeric complex.     

Murine cytomegalovirus interference with antigen presentation has little effect on the size or the effector memory phenotype of the CD8 T cell response

As with most herpesviruses, CMVs encode viral genes that inhibit Ag presentation to CD8 T cells (VIPRs). VIPR function has been assumed to be essential for CMV to establish its characteristic lifetime infection of its host. We compared infection of C57BL/6 mice with wild-type murine CMV (MCMV) and a virus lacking each of MCMV's three known VIPRs: m4, m6, and m152. During acute infection, there was very little difference between the two viruses with respect to the kinetics of viral replication and clearance, or in the size and kinetics of the virus-specific CD8 T cell response. During chronic infection, a large, effector memory, virus-specific CD8 T cell population (CD8(low)CD62L(-)CD11c(+)NKG2A(+)) was maintained in both infections; the size and phenotype of the CD8 T cell response to both viruses was remarkably similar. The characteristic effector memory phenotype of the CD8 T cells suggested that both wild-type and Deltam4+m6+m152 virus continued to present Ag to CD8 T cells during the chronic phase of infection. During the chronic phase of infection, MCMV cannot be isolated from immunocompetent mice. However, upon immunosuppression, both Deltam4+m6+m152 and wild-type virus could be reactivated from mice infected for 6 wk. Thus, restoring the ability of CD8 T cells to detect MCMV had little apparent effect on the course of MCMV infection and on the CD8 T cell response to it. These results challenge the notion that VIPR function is necessary for CMV persistence in the host.     

Macrophages escape inhibition of major histocompatibility complex class I-dependent antigen presentation by cytomegalovirus

The mouse cytomegalovirus (MCMV) m152- and m06-encoded glycoproteins gp40 and gp48, respectively, independently downregulate major histocompatibility complex (MHC) class I surface expression during the course of productive MCMV infection in fibroblasts. As a result, presentation of an immediate-early protein pp89-derived nonapeptide to H-2L(d)-restricted CD8(+) cytotoxic T cells is completely prevented in fibroblasts. Here we demonstrate that MCMV-infected primary bone marrow macrophages and the macrophage cell line J774 constitutively present pp89 peptides during permissive MCMV infection to cytotoxic T lymphocytes (CTL). In contrast to fibroblasts, expression of the m152 and m06 genes in macrophages does not affect surface expression of MHC class I. Assessment of pp89 synthesis and quantification of extracted peptide revealed a significantly higher efficiency of macrophages than of fibroblasts to process pp89 into finally trimmed peptide. The yield of pp89 peptide determined in MCMV-infected tissues of bone marrow chimeras confirmed that bone marrow-derived cells represent a prime source of pp89 processing in parenchymal organs. The finding that macrophages resist the viral control of MHC I-dependent antigen presentation reconciles the paradox of efficient induction of CMV-specific CD8(+) CTL in vivo despite extensive potential of CMVs to subvert MHC class I.     

Crystal structure of the murine cytomegalovirus MHC-I homolog m144

Large DNA viruses of the herpesvirus family produce proteins that mimic host MHC-I molecules as part of their immunoevasive strategy. The m144 glycoprotein, expressed by murine cytomegalovirus, is thought to be an MHC-I homolog whose expression prolongs viral survival in vivo by preventing natural killer cell activation. To explore the structural basis of this m144 function, we have determined the three-dimensional structure of an m144/beta2-microglobulin (beta2m) complex at 1.9A resolution. This structure reveals the canonical features of MHC-I molecules including readily identifiable alpha1, alpha2, and alpha3 domains. A unique disulfide bond links the alpha1 helix to the beta-sheet floor, explaining the known thermal stability of m144. Close juxtaposition of the alpha1 and alpha2 helices and the lack of critical residues that normally contribute to anchoring the peptide N and C termini eliminates peptide binding. A region of 13 amino acid residues, corresponding to the amino-terminal portion of the alpha2 helix, is missing in the electron density map, suggesting an area of structural flexibility that may be involved in ligand binding.     

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

We have recently demonstrated that the murine CMV (MCMV) gene m4 is an immune evasion gene that protects MCMV-infected targets from some virus-specific CTL clones. m4 encodes m4/gp34, a 34-kDa glycoprotein that binds to major histocompatibility complex class I in the endoplasmic reticulum and forms a detergent-stable complex that is exported to the surface of the cell. To investigate how m4/gp34 promotes CTL evasion, we analyzed the assembly and export of m4/gp34-K(b) complexes. We found that 50-70% of K(b) exported over the course of MCMV infection was m4/gp34 associated. Because these complexes are present at the cell surface, it is possible that m4 mediates CTL evasion by interfering with contact between class I and receptors on the T cell. In addition, we found that K(b) retained by the MCMV immune evasion gene m152 formed a novel type of complex with Endo H-sensitive m4/gp34; these complexes are distinguished from the exported complexes by being stable in 1% digitonin and unstable in 1% Nonidet P-40. Because this association occurs in a pre-Golgi compartment, m4/gp34 might also interfere with Ag presentation by affecting some aspect of class I assembly, such as peptide loading. Although m4/gp34 requires beta(2)-microglobulin to bind class I, there was no significant binding of m4/gp34 to beta(2)-microglobulin in the absence of class I H chain, demonstrating that m4/gp34 forms Nonidet P-40-stable complexes specifically with folded conformations of class I. We conclude that m4/gp34 promotes immune evasion by a novel mechanism involving altered assembly and/or T cell recognition of class I molecules.     

Molecular architecture of the major histocompatibility complex class I-binding site of Ly49 natural killer cell receptors

Natural killer (NK) cells play a vital role in the detection and destruction of virally infected and tumor cells during innate immune responses. The highly polymorphic Ly49 family of NK receptors regulates NK cell function by sensing major histocompatibility complex class I (MHC-I) molecules on target cells. Despite the determination of two Ly49-MHC-I complex structures, the molecular features of Ly49 receptors that confer specificity for particular MHC-I alleles have not been identified. To understand the functional architecture of Ly49-binding sites, we determined the crystal structures of Ly49C and Ly49G and completed refinement of the Ly49C-H-2K(b) complex. This information, combined with mutational analysis of Ly49A, permitted a structure-based classification of Ly49s that we used to dissect the binding site into three distinct regions, each having different roles in MHC recognition. One region, located at the center of the binding site, has a similar structure across the Ly49 family and mediates conserved interactions with MHC-I that contribute most to binding. However, the preference of individual Ly49s for particular MHC-I molecules is governed by two regions that flank the central region and are structurally more variable. One of the flanking regions divides Ly49s into those that recognize both H-2D and H-2K versus only H-2D ligands, whereas the other discriminates among H-2D or H-2K alleles. The modular design of Ly49-binding sites provides a framework for predicting the MHC-binding specificity of Ly49s that have not been characterized experimentally.     

Membrane-anchored beta 2-microglobulin stabilizes a highly receptive state of MHC class I molecules

The magnitude of response elicited by CTL-inducing vaccines correlates with the density of MHC class I (MHC-I)-peptide complexes formed on the APC membrane. The MHC-I L chain, beta2-microglobulin (beta2m), governs complex stability. We reasoned that genetically converting beta2m into an integral membrane protein should exert a marked stabilizing effect on the resulting MHC-I molecules and enhance vaccine efficacy. In the present study, we show that expression of membranal human beta2m (hbeta2m) in mouse RMA-S cells elevates MHC-I thermal stability. RMA-S transfectants bind an exogenous peptide at concentrations 10(4)- to 10(6)-fold lower than parental RMA-S, as detected by complex-specific Abs and by T cell activation. Moreover, saturation of the transfectants' MHC-I by exogenous peptide occurs within 1 min, as compared with approximately 1 h required for parental cells. At saturation, however, level of peptide bound by modified cells is only 3- to 5-fold higher. Expression of native hbeta2m only results in marginal effect on the binding profile. Soluble beta2m has no effect on the accelerated kinetics, but the kinetics of transfectants parallel that of parental cells in the presence of Abs to hbeta2m. Ab inhibition and coimmunoprecipitation analyses suggest that both prolonged persistence of peptide-receptive H chain/beta2m heterodimers and fast heterodimer formation via lateral diffusion may contribute to stabilization. In vivo, peptide-loaded transfectants are considerably superior to parental cells in suppressing tumor growth. Our findings support the role of an allosteric mechanism in determining ternary MHC-I complex stability and propose membranal beta2m as a novel scaffold for CTL induction.