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The Journal of Immunology, 2004, 172: 5504-5511.
Copyright © 2004 by The American Association of Immunologists

Determination of Structural Principles Underlying Three Different Modes of Lymphocytic Choriomeningitis Virus Escape from CTL Recognition1

Lucas Malard Velloso*, Jakob Michaëlsson{dagger}, Hans-Gustaf Ljunggren{ddagger}, Gunter Schneider* and Adnane Achour2,{ddagger}

* Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden; {dagger} Gladstone Institute of Virology and Immunology, University of California, San Francisco, CA 94141; and {ddagger} Center for Infectious Medicine, Department of Medicine, Karolinska Institute, Huddinge University Hospital, Stockholm, Sweden


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Lymphocytic choriomeningitis virus infection of H-2b mice generates a strong CD8+ CTL response mainly directed toward three immunodominant epitopes, one of which, gp33, is presented by both H-2Db and H-2Kb MHC class I molecules. This CTL response acts as a selective agent for the emergence of viral escape variants. These variants generate altered peptide ligands (APLs) that, when presented by class I MHC molecules, antagonize CTL recognition and ultimately allow the virus to evade the cellular immune response. The emergence of APLs of the gp33 epitope is particularly advantageous for LCMV, as it allows viral escape in the context of both H-2Db and H-2Kb MHC class I molecules. We have determined crystal structures of three different APLs of gp33 in complex with both H-2Db and H-2Kb. Comparison between these APL/MHC structures and those of the index gp33 peptide/MHC reveals the structural basis for three different strategies used by LCMV viral escape mutations: 1) conformational changes in peptide and MHC residues that are potential TCR contacts, 2) impairment of APL binding to the MHC peptide binding cleft, and 3) introduction of subtle changes at the TCR/pMHC interface, such as the removal of a single hydroxyl group.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
CD8+ T lymphocytes exert important immune control over different viral infections (1, 2). However, viruses can counteract CTL responses, for example by production of proteins that perturb the mechanisms of Ag processing and peptide transport or by selection of variants with mutated amino acids in the presented peptides (3). Mutation of viral epitopes that alters or abolishes CTL recognition altogether appears to be the most important immune escape mechanism, as the variation generated by viruses defies the limits of the T cell repertoire. Such evasion strategies present a considerable challenge to the antiviral T cell immune response, enabling viruses such as lymphocytic choriomeningitis virus (LCMV),3 hepatitis B virus, hepatitis C virus, EBV, influenza, HIV, and SIV to survive and persist in the host (4, 5). However, the rules governing immune escape and the ultimate limits of CTL capacity to deal with variant epitopes that currently circulate are not fully understood.

Infection of H-2b (C57BL/6) mice with LCMV induces a strong CTL response, mainly directed against three immunodominant epitopes, two of which (gp33 and gp276) are derived from the viral glycoprotein, and the third (np396) from the viral nucleoprotein (6, 7, 8, 9). More than 50% of the total LCMV-specific CTL activity is directed toward gp33 (KAVYNFATC), the only immunodominant epitope that is restricted by both H-2Db and H-2Kb (10). The importance of both MHC class I molecules in determining the course of LCMV infection has been demonstrated in studies of knockout mice (11). Upon CTL selection pressure, altered peptide ligand (APL) variants emerge, allowing the virus to escape CD8 T cell recognition. Some of these are based on a single amino acid exchange in the immunodominant epitope, yet they result in no recognition in the context of both H-2Db and H-2Kb (9, 12, 13). Three of the most common mutations in gp33 occur at positions 3 (V to L), 4 (Y to F), and 6 (F to L) of the peptide, resulting in escape from gp33-specific CTL response in the context of both H-2b class I molecules. Functional studies of the interaction between these APLs and H-2Kb/H-2Db have revealed that some of these viral escape mutations act by impairing the binding of the APL to H-2Kb and/or H-2Db, whereas others affect TCR recognition, as demonstrated by CTL assays or surface plasmon resonance (12, 14, 15, 16). Overall, results from peptide binding assays, TCR-MHC binding kinetics, functional T cell assays, and generation of viral escape variants in vivo suggest that mutations in LCMV genes play an important role in deciding the outcome of the viral infection (17).

To provide a structural basis for how single amino acid mutants in one epitope can allow LCMV to escape CTL-mediated recognition in the context of two different MHC class I molecules, we have solved the crystal structures of the three main gp33 APLs, pV3L, pY4F, and pF6L, in complex with H-2Db and H-2Kb. We have also improved the resolution of the previously published structures of H-2Db/gp33 and H-2Kb/gp33 to 2.2 and 2.0 Å, respectively. Comparison between the present APL/MHC structures and those of the gp33 index peptide/MHC (14, 18) reveals that the virus makes use of diverse strategies to escape direct CTL recognition. The introduced single mutations 1) affect the conformation of the peptide, altering the conformation of potential main TCR contacts and/or main anchor positions as well as leading to movements in the {alpha}1 domain, the {alpha}2 domain, or both; 2) impair the binding of the mutated epitope to the MHC peptide binding cleft; or 3) introduce subtle changes, such as the removal of a single hydroxyl group at the TCR/pMHC interface, reducing the affinity of the TCR for the pMHC. Thus, the crystal structures described within this study allow us to establish the molecular basis for how any given mutation in an immunodominant peptide, in the context of both H-2Kb and H-2Db, permits the evasion of this epitope and, ultimately, of the LCMV virus.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Preparation of H-2Db and H-2Kb MHC class I molecules in complex with variant peptides

The index peptide gp33 (KAVYNFATM) and the APLs gp33 (V3L), gp33 (Y4F), and gp33 (F6L) were purchased from Research Genetics (Huntsville, AL). The refolding and purification of H-2Db and H-2Kb in complex with the mutated peptides and mouse {beta}2-microglobulin ({beta}2m) were conducted as previously described (19).

Crystallization of H-2Db and H-2Kb MHC class I complexes

Crystals for H-2Db in complex with the mutated peptides were obtained in hanging drops by vapor diffusion in 1.6–2.0 M ammonium sulfate and 0.1 M Tris-HCl (pH 8.5) at room temperature. Typically, 4 µl of a 6 mg/ml protein solution in 20 mM Tris-HCl (pH 8.0) was mixed in a 2:1 ratio with the crystallization reservoir solution. Crystals for the H-2Kb complexes were obtained in 1.7–1.8 M NaH2PO4/K2PO4 (pH 6.8–7.0) and 2% 2-methyl-2,4-pentanediol at room temperature. Drops consisted of 2 µl of a 5 mg/ml protein solution in 20 mM Tris-HCl (pH 7.0) mixed with equal volumes of the crystallization reservoir.

Data collection and processing

Data collection was performed under cryogenic conditions (temperature, 100°K). All crystals were soaked during a short time in a cryoprotectant solution containing 25% glycerol before data collection. X-ray data were collected at different synchrotron beam lines (Table I). The diffraction data from all crystals except H-2Kb (V3L) were processed with MOSFLM (20) and SCALA (21). Diffraction data from H-2Kb (V3L) were processed using the HKL2000 program package (22). Data collection statistics for all datasets are presented in Table I.


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  		Table I. Data collection and refinement statistics

 
Structure determination and refinement

The structures of the H-2Db and H-2Kb complexes were solved by molecular replacement using the AmoRe (23) or MolRep (24) program. The crystal structures of H-2Db/gp33 (PDB code 1N5A) and H-2Kb/gp33 (PDB code 1N59) were used as search models. Four percent of the reflections were set aside in all datasets for monitoring the refinement by Rfree. Refinement was performed with REFMAC5 (25). The structures were initially subjected to rigid body refinement, treating the {alpha}1{alpha}2 (residues 1–182), {alpha}3 (residues 183–274), and {beta}2m domains and the peptide as four separate units. Cycles of restrained refinement were alternated with model rebuilding using the program O (26). A clear electron density in the (Fo-Fc) map was observed inside the peptide binding cleft, and the peptides could be placed unambiguously in all MHC complexes, except for the first residue (p1K) in the H-2Kb/gp33, H-2Kb/gp33 (V3L), and H-2Kb/gp33 (Y4F) complexes. Refinement cycles of TLS parameters were also performed using REFMAC5 (25). Water molecules were added using the program arp/wARP (27). The stereochemistry of the models was analyzed with PROCHECK (28). Final refinement statistics are presented in Table I. All figures were prepared using the programs Bobscript (29) and Raster 3D (30).

Structural comparisons

In all structural comparisons, alignments are based on the superposition of the C{alpha} atoms of the {alpha}1{alpha}2 domains using the program LSQMAN with default parameters (31). The resolutions of the H-2Kb/gp33 and H-2Db/gp33 complexes were extended to 2.2 and 2.0 Å (Table I) from the 2.9 Å previously reported (19), allowing a more reliable assessment of the observed structural differences between the structures of index peptide and mutant escape variants.

Molecular model of the TCR

The molecular model of the p14 TCR specific for the H-2Db/gp33 MHC complex was created using the SWISS-MODEL Protein Modeling Server. The crystal structure of the TCR-2C/H-2Kb/DEV-8 peptide complex (PDB code 2CKB) (32) was used as a template for the creation of a preliminary model. This was then superimposed on the structures of H-2Db/gp33 variants/mouse {beta}2m solved in this study, and the entire complexes were subjected to several energy minimization rounds using the CNS suite of programs (33). The quality of the models was continuously assessed using the PROCHECK program (28). The coordinates of the TCR model will be provided upon request.

Peptide stabilization assays

Peptide binding was assessed by cell surface stabilization of H-2Kb and H-2Db on TAP-deficient RMA-S cells (34). RMA-S cells were incubated in serum-free medium (AIM-V; Life Technologies, Paisley, U.K.) supplemented with the indicated concentrations of synthetic peptides at 26°C for 15–20 h. Cells were subsequently washed and incubated in AIM-V medium at 37°C for 60 min in the absence of peptides. Cell surface expression was measured by FACS using FITC-conjugated mAbs specific for H-2Db (KH95) and H-2Kb (AF6-88.5; BD PharMingen/BD Biosciences, Mountain View, CA). Briefly, the cells were incubated in PBS supplemented with 1% FCS and the respective Abs (1 µg/ml) for 30 min on ice, washed three times with PBS/1% FCS, and analyzed directly using a FACScan (BD Biosciences).


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
The LCMV-derived immunodominant gp33 epitope is presented in diametrically opposite conformations by H-2Db and H-2Kb (Fig. 1) due to the requirements imposed by the peptide binding clefts of the respective MHC class I molecules (19). The gp33 index peptide has the characteristic conformation for H-2Db, in which it bulges from residues 6–8, with p1K, p7A, and particularly p4Y and p6F projecting up into the solvent (Fig. 1B). By contrast, the index peptide binds deeply in the cleft of H-2Kb and has a flatter conformation, with three residues available for interaction with TCR (p1K, p5N, and p8T; Fig. 1A). Comparison of the two different conformations of gp33 when bound to either H-2Db or H-2Kb revealed a relative shift in the positions of the peptide amino acids in each structure, leading, in turn, to the use of different amino acid side chains as anchors in different pockets of the two MHC complexes. Thus, gp33 binds H-2Db with p5N and p9M as main and p3V as secondary anchors (Fig. 1B), whereas H-2Kb uses two main (p6F and p9M) and two secondary anchors (p3V and p4Y; Fig. 1A). It should be noted that residues p4Y and p6F, which act as potential TCR contact residues when gp33 is presented by H-2Db, are used as anchor residues when the peptide is presented by H-2Kb (Fig. 1).



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  		FIGURE 1. Electron density maps for the gp33 wild-type and variants in complex with either H-2Kb or H-2Db. The 2Fo-Fc map of gp33 wild-type and variants when bound to H-2Kb (left panel) and H-2Db (right panel) contoured at 1.0 {sigma}. The final models are displayed for comparison. A, H-2Kb/index gp33; B, H-2Db/index gp33; C, H-2Kb/gp33 (F6L); D, H-2Db/gp33 (F6L); E, H-2Kb/gp33 (Y4F); F, H-2Db/gp33 (Y4F); G, H-2Kb/gp33 (V3L); H, H-2Db/gp33 (V3L). The peptides are depicted with their N termini to the left and their C termini to the right, and the sequences of the index peptide and the APLs are displayed, with the point mutations highlighted in red. Each of the residues of the index peptide is labeled, with the residues acting as anchor positions highlighted in blue and underlined.

 
There are at least three mutations in gp33 that lead to viral escape in the context of both H-2Db and H-2Kb restriction elements: p3V to L, p4Y to F, and p6F to L. These mutations are conservative, but still permit the virus to efficiently evade recognition by specific CD8+ T cells. The index and variant epitopes bind with variable affinities to either H-2Kb or H-2Db in vitro (Fig. 2). With the exception of the pF6L mutant, the APLs stabilized the expression of H-2Kb molecules on the surface of RMA-S cells as well as the index gp33 peptide, indicating that the mutations pV3L and pY4F do not affect binding of substituted peptides to H-2Kb (Fig. 2A). In contrast, the only mutation that led to a slight decrease in the stabilization of H-2Db by the gp33 peptide was the pV3L substitution. The comparison of the structures of the variant gp33 epitopes bound to H-2Db and H-2Kb (Fig. 1, C–H) with the structures of the index gp33 peptide bound to both class I molecules provides a structural basis for these escape mechanisms. We have improved the resolution of the structures of H-2Db and H-2Kb in complex with the index peptide gp33 to 2.2 and 2.0 Å, respectively. The structural differences introduced by the APLs are described in detail below, first with respect to H-2Kb, then to H-2Db.



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  		FIGURE 2. Peptide stabilization assays. Cell surface expression of H-2Db and H-2Kb on TAP-deficient RMA-S cells after stabilization with wild-type and mutant gp33 peptides. RMA-S cells were incubated at 26°C overnight in increasing concentrations of the peptides gp33, gp33 (V3L), gp33 (Y4F), and gp33 (F6L), respectively. Cells were subsequently washed twice to remove unbound peptides and incubated for 1 h at 37°C in serum-free cell culture medium. Cell surface expression was measured by flow cytometry using H-2Db- and H-2Kb-specific antibodies. Data points are means of triplicate determinations ± 1 SD.

 
The pF6L substitution impairs binding of the variant peptide to H-2Kb

In H-2Kb/index gp33, the phenylalanine residue at position 6 of gp33 (p6F) projects down into the C pocket, composed of residues V9, F74, V97, S99, Q114, and Y116 (Fig. 3A). A change from F to L at this position results in a shorter and less bulky residue that does not fit the hydrophobic C pocket as well as phenylalanine, impairing the binding of the mutated peptide (Fig. 3A). The H-2Kb/gp33 (F6L) is the only structure within this study that displays a significant difference in average B factors for the peptide (40 Å2) vs MHC (23 Å2; for residues 1–182), consistent with the poor binding of pF6L to H-2Kb observed in the peptide stabilization assays (Fig. 2A). A comparison of the interface between the peptide and the H-2Kb binding cleft in both the index and the pF6L structures also reveals the presence of a large cavity within the C pocket in the latter structure that is not present in H-2Kb/index gp33 (Fig. 3A). Thus, although the pF6L mutation does not introduce major structural changes at the surface of the MHC/peptide interface, it alters the optimal dwell of interaction between the TCR and the MHC/peptide complex (35). Only two other examples of MHC class I molecules in complex with weak binding peptides have been previously described. The structures of HLA-A2 and H-2Kb in complex with the tumor-associated HER2/neu GP2 and MUC1 epitopes, respectively, revealed that unlike other class I structures, the center of the peptides (residue p5 or p6) did not make stabilizing contacts with the peptide binding cleft (36, 37, 38). Both low affinity tumor-associated epitopes bound with the same overall features as the high affinity peptides, with a conserved number of hydrogen bonds between the main chain of the peptide and residues of the H chain, but with deviations occurring within the central region of the peptide. The presence of a cavity at the side of the C pocket contributed most to the low affinity and stability of the two peptides to either H-2Kb or HLA-A2. Thus, it appears that there is a direct correlation between noncanonical peptide binding with respect to the presence of large cavities within or around the C pocket and high B values for peptide anchors at p5/p6. Optimal binding to H-2Kb is critically dependent upon filling the C pocket, which is not the case with low affinity peptides such as MUC1 (37) or gp33 (F6L) presented in this study.



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  		FIGURE 3. Detailed description of the structural consequences for each single mutation in the H-2Kb/gp33 complex. The peptide binding grooves of both index and variant H-2Kb structures are superposed and viewed in different orientations. The carbon backbones for the index and substituted peptide are colored in yellow and rose, respectively. Oxygen, nitrogen, and sulfur atoms are colored in red, blue, and green, respectively. All residues are labeled. Hydrogen bonds appear as dotted lines. Cavities are displayed as red grids (A) and were calculated using the program SURFNET (52 ). A, H-2Kb/gp33 (F6L); B, H-2Kb/gp33 (Y4F); C, H-2Kb/gp33 (V3L).

 
The pY4F mutation induces changes in potential TCR contact residues in the {alpha}2 domain of H-2Kb

Tyrosine at position 4 (p4Y) in gp33 projects down into the D pocket of H-2Kb formed by residues Q114, E152, L156, and Y159, where it is involved in the formation of three hydrogen bonds with the side chains of E152 and R155 (Fig. 3B). The side chain of p4Y is further stabilized by hydrophobic interactions with the side chains of Y159 and p6F, whereas the main chain forms a hydrogen bond with N70. The structures of H-2Kb in complex with either the gp33 index peptide or the gp33 (Y4F) variant escape are very similar, with a root mean square deviation (r.m.s.d.) of 0.25 Å for the C{alpha} atoms of H chain residues 1–182. Overall, the peptides are identical in both complexes, with the largest shift at the C{alpha} level for residue p3V of 0.35 Å. Removal of the tyrosine hydroxyl group due to the pY4F mutation results in changes in the neighboring residues, E152, E154, and R155, on the {alpha}2 domain. The hydroxyl group of p4Y forms a hydrogen bond with the NH1 atom of R155. The pY4F mutation breaks this hydrogen bond, allowing the R155 side chain to take a slightly different conformation, with the N{epsilon} atom moving 1.4 Å toward the C terminus of the peptide. This movement, in turn, also affects the position of the NH1 and NH2 atoms. These changes induce a C{alpha} shift of 0.4 Å for residue E154. The side chain of E154 is also affected by this C{alpha} shift, with atoms CD, OE1, and OE2 moving by >1 Å. Residues R155 and R154 have been previously identified as important TCR contacts in the H-2Kb/TCR crystal structures (32, 39). The loss of the hydroxyl group also affects the side chain of residue E152, which forms a hydrogen bond with the tyrosine hydroxyl in the H-2Kb/gp33 index peptide structure (19). Atoms OE1 and OE2 of residue E152 move by 0.5 Å away from the p3F side chain. Thus, the pY4F substitution does not result in any major structural changes at the MHC/peptide interface besides subtle movements of {alpha}2 TCR contact residues.

The pV3L mutation introduces subtle changes in the variant peptide, which are amplified to H-2Kb residues in the {alpha}1 and {alpha}2 domains

The side chain of residue p3V in the gp33 index peptide points toward the negatively charged B pocket of H-2Kb composed of residues Y7, V9, E24, Y45, and N70. Residues E63 and K66 are situated above this pocket; K66 is involved in hydrogen bond interactions with the backbone carbonyl of p3V (Fig. 3C). Here again, both H-2Kb structures are very similar, with an r.m.s.d. of 0.14 Å for the C{alpha} atoms of residues 1–182 that correspond to the {alpha}1 and {alpha}2 regions of the H chain. However subtle local structural modifications appear in {alpha}1 residues in the mutated structure. To accommodate the larger leucine side chain, the main chain of the N terminus of the mutated peptide moves slightly toward the bottom of the peptide binding cleft, with the largest C{alpha} shift (0.4 Å) observed at pV3L. The increase in the side chain size introduced by the pV3L mutation leads to a series of changes in the neighboring residues of the {alpha}2 domain. The side chain of residue E63 is forced upward by the leucine side chain, resulting in a movement of the OE1 and OE2 atoms of this residue by >1.5 Å. This, in turn, results in a movement of the side chain of residue K66 away from the side chain of residue E63. These two residues are involved in hydrogen bond interactions, and this movement leads to a change in hydrogen bond acceptor atom from the OE2 to the OE1 atom of residue E63. The side chain of residue Y45 also undergoes a rearrangement by rotating 62° around its {chi} torsion angle to move away from the p3L side chain. The C{alpha} shift observed for residue p3L leads to a subtle movement in the side chain of residue p4Y toward the {alpha}2 domain. All the hydrogen bond interactions described in the structure of H-2Kb/index gp33 are retained in the structure of H-2Kb/gp33 (V3L). As a consequence, H chain residue R155 undergoes a small movement to maintain its hydrogen bond interactions with the hydroxyl group of p4Y (Fig. 3C). The changes observed for the side chain of residue R155 in the pV3L mutation are similar to those described for pY4F, but with a smaller magnitude, where the N{epsilon} atom moves 1.0 Å toward the C terminus of the peptide. Residues E63, K66, and R155, which are affected by the pV3L mutation, have been shown to act as TCR contacts in crystal structures of H-2Kb/TCR complexes (32, 39).

In conclusion, all the three conserved mutations introduced by LCMV to escape recognition by CD8+ T cells do not result in any major structural change in the context of H-2Kb. The changes observed are subtle and result either in an impairment of the binding affinity of the modified gp33 epitope to the peptide binding cleft of H-2Kb or small conformational modifications of {alpha}1 or {alpha}2 H-2Kb residues, previously described as TCR contacts.

Differential structural effects of pY4F and pF6L substitutions on both gp33 and H-2Db H chains

In the H-2Db/gp33 complex, both residues p4Y and p6F project out of the peptide binding cleft, acting as potential main TCR contacts (14, 18, 19). The escape mutants pY4F and pF6L could thus be explained by impaired TCR recognition rather than by impaired MHC binding. However, the structural impacts of these two conservative substitutions are different. The pY4F mutation does not result in any modifications other than the removal of the tyrosine hydroxyl group, whereas the conserved pF6L substitution results in changes in the potential TCR contact residues that probably affect the sensitive regions of the TCR through direct sterical clashes and/or the introduction of cavities at the TCR/pMHC interface.

The pF6L mutation results in shifts in the central portion variant peptide (residues p4 to p7). The introduction of a less bulky leucine side chain causes a subtle movement of this portion of the peptide toward the {alpha}1 domain. The largest movements take place in residues p4Y and p6L (Fig. 4A). Residue p6L is shifted by over 1.2 Å at the C{alpha} level. The r.m.s.d. between the main chains of the index peptide and the gp33 (F6L) variant is 0.9 Å (including peptide residues p1 to p9), compared with an r.m.s.d. of 0.13–0.25 Å when the gp33 peptides from three independent structure determinations of the H-2Db/gp33 index peptide are superposed (14, 18, 19). The side chain of p6L is forced upward partly toward the {alpha}1 domain of H-2Db, whereas the side chain of p4Y moves slightly toward the {alpha}1 domain (Fig. 4A). The movements of residues p4 and p6 would most likely result in the creation of cavities at the pMHC/p14TCR interface and/or clashes with the CDR3{alpha} or CDR3{beta} of the TCR. Finally, the shift introduced in the main chain of residue p6 induces small movements in residues S150, A152, and H155 of the {alpha}2 domain toward the mutated peptide. Most of these residues have been identified as TCR contacts (32).



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  		FIGURE 4. Detailed description of the structural consequences for each single mutation in the H-2Db/gp33 complex. The peptide binding grooves of both index and variant H-2Db structures are superposed and viewed in different orientations. The carbon backbones for the index and substituted peptide are colored in yellow and rose, respectively. Oxygen, nitrogen, and sulfur atoms are colored in red, blue, and green, respectively. All residues are labeled. The TCR V{alpha} is colored in light blue in E and F, respectively, whereas V{beta} is colored in green. Hydrogen bonds appear as dotted lines. A, H-2Db/gp33 (F6L); B, H-2Db/gp33 (Y4F); C, H-2Db/gp33 (V3L).

 
It should be noted that the pF6L mutation does not affect any of the main or secondary peptide anchor positions, as the side chains of the different anchor positions take very similar conformations. Despite the observed differences, the binding affinity of the gp33 (F6L) variant for H-2Db is similar to that of the index peptide (Fig. 4A).

In contrast, the crystal structures of H-2Db/index gp33 and H-2Db/gp33 (Y4F) are remarkably similar (Fig. 4B). Thus, the only significant difference between the two structures is the removal of the tyrosine hydroxyl group at p4 in the mutant escape peptide (Fig. 4A). Nonetheless, this minor structural difference leads to a 100-fold decrease in binding affinity of the p14 TCR, specific for H-2Db/index gp33 (14), abolishing CTL activity toward cells infected with this variant of the virus (9). This is the first structural evidence of such an effective viral escape mechanism due to removal of a single hydroxyl group.

X-ray crystallographic structures of {alpha}{beta}TCR/peptide/MHC complexes have revealed that exceedingly small changes in the pMHC/TCR interface in response to APLs can transmit themselves as different signals (i.e., agonism vs antagonism) (40) through the TCR signaling complex. It has been proposed, based on several measurements of the half-life of pMHC/TCR complexes, that these differences act by extending (in the case of agonists) or reducing (in the case of antagonists) the half-life of the complex, which then leads to agonistic or antagonistic signaling, respectively (35, 40, 41). Models of the p14 TCR based on the crystal structure of the 2C TCR in complex with H-2Kb (32, 42) and on the H-2Db/gp33 (19) or H-2Db/APLs structures described in this study suggest that p4Y and p6F, the two potential TCR contacts of H-2Db/gp33, are located at the interface between the TCR and H-2Db, with p4Y protruding directly into the most sensitive part of the TCR (the so-called functional hot spot), which is located between the CDR3 domains of the V{alpha} and V{beta} chains (43). These models would explain why the single removal of a hydroxyl group can have such a dramatic effect on the affinity of the p14TCR for the pMHC (14) and consequently on the CTL response against this epitope (9).

Furthermore, the movements of the potential main TCR contacts following the substitutions of p6F to a leucine probably result in the creation of cavities at the pMHC/TCR interface. Similar cavities have been described in the crystal structures of four different complexes of A6 TCR with HLA-A2 presenting agonistic and antagonistic peptides (41, 44, 45). These structures were remarkably similar even though the MHC/TCR contact resulted in very different T cell signals. The antagonist peptide displayed no structural changes relative to the wild-type peptide, except for a mutation form P to A at position 6. This mutation resulted in the creation of a cavity at the interface between the antagonistic MHC/peptide complex and the TCR (41). In a follow-up study, the T cell antagonist peptide was converted into an agonist by stepwise repair of the defect in the TCR/MHC interface using N-alkylated amino acids that progressively filled up the cavity, restoring {alpha}{beta}TCR binding affinity and resulting in agonistic signaling (46).

Viruses such as LCMV can make use of subtle modifications, such as the removal of the hydroxyl group from the tyrosine side chain (pY4F mutation), to avoid recognition by CD8+ T cells (Fig. 4B). In contrast, using a conserved substitution such as pF6L, LCMV can also alter the conformation of the mutated peptide as well as of sections of the H-2Db {alpha}1 and {alpha}2 domains (Fig. 4B). Thus, the virus is able to make use of conservative mutations to induce subtle conformational changes with dramatic effects on CTL recognition.

The pV3L substitution affects the conformation of p4Y, a potential TCR contact

The side chain of the secondary anchor position p3V projects down into the C-pocket of H-2Db formed by residues Q97, S99, L114, H155, Y156, and Y159 (19). The structures of H-2Db/index gp33 and H-2Db/gp33 (V3L) H chains are fairly similar. However, the pV3L mutation results in a reduced binding of the peptide to H-2Db (Fig. 2B), in accordance with other mutational results from V to A at this position (12). Introduction of the larger leucine side chain through the pV3L mutation results in a small movement of the peptide toward the {alpha}1 domain. The C{alpha} shifts in the peptide occur in residues p2A, p3L, and p4Y (Fig. 4C). The V3L substitution also affects the positioning of the side chain of p4Y, a potential main TCR interacting peptide residue that moves slightly upward (Fig. 4C). Indeed, the pV3L mutation reduces the affinity of the H-2Db/gp33/p14 TCR interaction by >40-fold (14). The pV3L mutation also leads to the movement of the side chain of residue H155 within and over the peptide binding cleft, keeping its hydrogen bond interaction with the carbonyl of p4Y.

Concluding remarks

There are a range of conceivable mechanisms through which minor changes in the sequence of a peptide could affect TCR recognition, from localized surface perturbations of amino acid side chains to concerted main chain shifts in the peptide and/or the MHC (47). Our analyses of the structural differences between MHC class I complexes for a series of mutants demonstrates that subtle substitutions are sufficient to affect TCR recognition in a number of ways. Most of these mutations result in shifts in the peptide, which, in turn, can induce small changes in residues of the {alpha}1 and {alpha}2 domains, which either interact with the peptide or act as TCR contacts. This is in agreement with previous structural studies using viral APL escape variants of HIV-1 and HLA-B8 (48). The binding affinity of some peptides is reduced through the introduction of cavities between the epitope and specific binding sites, such as the C pocket in H-2Kb. Additionally, potential sterical clashes between side chains of the mutated peptide and specific TCR regions as well as the introduction of cavities at the pMHC/TCR interface may impair the binding affinity between pMHC and TCR (41, 46). An efficient T cell activation requires an optimal dwell-time of interaction between the TCR and the pMHC complex (35). All the escape mutations introduced by LCMV decrease the stability of the interaction with the TCR, resulting in an antagonist message.

In both LCMV and other viruses, such as HIV, CTLs are main mediators of antiviral immunity, and so these lymphocytes have been of central interest to many scientists who study viral immunopathogenesis and design vaccines (1, 49, 50, 51). Combined study of the interaction of LCMV peptides and H-2b MHC class I molecules by in vitro functional CTL assays and measurement of peptide binding affinities has led to development of molecular theories to explain viral escape through selected amino acid substitution. The determination of crystal structures of these APL-MHC complexes enables a reassessment of these observations in a three-dimensional context. The structures presented in this study highlight three molecular mechanisms used by LCMV to escape CTL recognition: 1) impairment of binding of the APL to the MHC; 2) conformational changes in both peptide and H chain, affecting potential TCR contact residues; and 3) chemical changes, such as the removal of a single peptide hydroxyl group, that do not affect the peptide or MHC conformation, but clearly have an effect on TCR/pMHC interface, reducing the affinity of the TCR for the pMHC and abolishing CTL activation.

Several antigenic ligands exist for any given virus, each one recognized by a subpopulation of CTLs. The relative Ag specificities of these CTL subpopulations differ. Thus, the efficiency of antagonistic viral escape variants in subverting the host’s antiviral immune response and establishing a chronic and persistent infection depends on the relative immunodominance of the epitope being antagonized. By limiting the number of potentially immunodominant viral peptides (three in the case of LCMV) and by directing the host’s immune response toward these epitopes, LCMV can efficiently secure its escape from a CTL response through single mutations. The emergence of viral variants in vivo clearly has a replicative advantage in such a way that the balance between viral replication and host immune control may be shifted to allow host survival and viral persistence (52).


   Acknowledgments
 
We thank Dr. Tatyana Sandalova for help with data collection, and Dr. Robert A. Harris for assistance with manuscript preparation. We gratefully acknowledge access to synchrotron radiation at beamline 711 at MAX Laboratory, Lund University (Lund, Sweden), and at beamlines ID29 and BM14, European Synchrotron Radiation Facility (Grenoble, France).


   Footnotes
 
1 This work was supported by grants from the Swedish Foundation for Strategic Research, The Åke Wibergs Stiftelse, The Alex and Eva Wallströms Stiftelse, The Magnus Bergwalls Stiftelse, and the Swedish Research Council. Back

2 Address correspondence and reprint requests to Dr. Adnane Achour, Center for Infectious Medicine, F59, Department of Medicine, Karolinska Institute, Huddinge University Hospital, 141 86 Stockholm, Sweden. E-mail address: adnane.achour{at}medhs.ki.se Back

3 Abbreviations used in this paper: LCMV, lymphocytic choriomeningitis virus; APL, altered peptide ligand; {beta}2m, {beta}2-microglobulin; r.m.s.d., root mean square deviation. Back

Received for publication November 30, 2003. Accepted for publication February 18, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

  1. O’Connor, D., T. Friedrich, A. Hughes, T. M. Allen, D. Watkins. 2001. Understanding cytotoxic T-lymphocyte escape during simian immunodeficiency virus infection. Immunol. Rev. 183:115.[Medline]
  2. Klenerman, P., Y. Wu, R. Phillips. 2002. HIV: current opinion in escapology. Curr. Opin. Microbiol. 5:408.[Medline]
  3. Tortorella, D., B. E. Gewurz, M. H. Furman, D. J. Schust, H. L. Ploegh. 2000. Viral subversion of the immune system. Annu. Rev. Immunol. 18:861.[Medline]
  4. Lucas, M., U. Karrer, A. Lucas, P. Klenerman. 2001. Viral escape mechanisms: escapology taught by viruses. Int. J. Exp. Pathol. 82:269.[Medline]
  5. Borrow, P., G. M. Shaw. 1998. Cytotoxic T-lymphocyte escape viral variants: how important are they in viral evasion of immune clearance in vivo?. Immunol. Rev. 164:37.[Medline]
  6. Gairin, J. E., M. B. Oldstone. 1992. Design of high-affinity major histocompatibility complex-specific antagonist peptides that inhibit cytotoxic T-lymphocyte activity: implications for control of viral disease. J. Virol. 66:6755.[Abstract/Free Full Text]
  7. Klavinskis, L. S., J. L. Whitton, E. Joly, M. B. Oldstone. 1990. Vaccination and protection from a lethal viral infection: identification, incorporation, and use of a cytotoxic T lymphocyte glycoprotein epitope. Virology 178:393.[Medline]
  8. Oldstone, M. B., J. L. Whitton, H. Lewicki, A. Tishon. 1988. Fine dissection of a nine amino acid glycoprotein epitope, a major determinant recognized by lymphocytic choriomeningitis virus-specific class I-restricted H-2Db cytotoxic T lymphocytes. J. Exp. Med. 168:559.[Abstract/Free Full Text]
  9. Pircher, H., D. Moskophidis, U. Rohrer, K. Burki, H. Hengartner, R. M. Zinkernagel. 1990. Viral escape by selection of cytotoxic T cell-resistant virus variants in vivo. Nature 346:629.[Medline]
  10. Hudrisier, D., M. B. Oldstone, J. E. Gairin. 1997. The signal sequence of lymphocytic choriomeningitis virus contains an immunodominant cytotoxic T cell epitope that is restricted by both H-2Db and H-2Kb molecules. Virology 234:62.[Medline]
  11. Perarnau, B., M. F. Saron, B. R. San Martin, N. Bervas, H. Ong, M. J. Soloski, A. G. Smith, J. M. Ure, J. E. Gairin, F. A. Lemonnier. 1999. Single H2Kb, H2Db and double H2KbDb knockout mice: peripheral CD8+ T cell repertoire and anti-lymphocytic choriomeningitis virus cytolytic responses. Eur. J. Immunol. 29:1243.[Medline]
  12. Puglielli, M. T., A. J. Zajac, R. G. van der Most, J. L. Dzuris, A. Sette, J. D. Altman, R. Ahmed. 2001. In vivo selection of a lymphocytic choriomeningitis virus variant that affects recognition of the GP33–43 epitope by H-2Db but not H-2Kb. J. Virol. 75:5099.[Abstract/Free Full Text]
  13. Aebischer, T., D. Moskophidis, U. H. Rohrer, R. M. Zinkernagel, H. Hengartner. 1991. In vitro selection of lymphocytic choriomeningitis virus escape mutants by cytotoxic T lymphocytes. Proc. Natl. Acad. Sci. USA 88:11047.[Abstract/Free Full Text]
  14. Tissot, A. C., C. Ciatto, P. R. Mittl, M. G. Grutter, A. Pluckthun. 2000. Viral escape at the molecular level explained by quantitative T-cell receptor/peptide/MHC interactions and the crystal structure of a peptide/MHC complex. J. Mol. Biol. 302:873.[Medline]
  15. Lewicki, H., A. Tishon, P. Borrow, C. F. Evans, J. E. Gairin, K. M. Hahn, D. A. Jewell, I. A. Wilson, M. B. Oldstone. 1995. CTL escape viral variants. I. Generation and molecular characterization. Virology 210:29.[Medline]
  16. Lewicki, H. A., M. G. Von Herrath, C. F. Evans, J. L. Whitton, M. B. Oldstone. 1995. CTL escape viral variants. II. Biologic activity in vivo. Virology 211:443.[Medline]
  17. Oldstone, M. B.. 2002. Biology and pathogenesis of lymphocytic choriomeningitis virus infection. Curr. Top. Microbiol. Immunol. 263:83.[Medline]
  18. Wang, B., A. Sharma, R. Maile, M. Saad, E. J. Collins, J. A. Frelinger. 2002. Peptidic termini play a significant role in TCR recognition. J. Immunol. 169:3137.[Abstract/Free Full Text]
  19. Achour, A., J. Michaelsson, R. A. Harris, J. Odeberg, P. Grufman, J. K. Sandberg, V. Levitsky, K. Karre, T. Sandalova, G. Schneider. 2002. A structural basis for LCMV immune evasion: subversion of H-2Db and H-2Kb presentation of gp33 revealed by comparative crystal structure: analyses. Immunity 17:757.[Medline]
  20. Leslie, A. G. W. 1992. Recent changes to the MOSFLM package for processing film and image plate data. Joint CCP4 ESF-EACBM Newslett. 26.
  21. Evans, P. R.. 1997. Scala. Joint CCP4 ESF-EACBM Newslett. 33:22.
  22. Otwinowski, Z., W. Minor. 1997. Processing of x-ray diffraction data collected in oscillation mode. Methods Enzymol. 276:307.
  23. Navaza, J.. 2001. Implementation of molecular replacement in AMoRe. Acta Crystallogr. D Biol. Crystallogr. 57:1367.[Medline]
  24. Vagin, A., A. Teplyakov. 1997. MOLREP: an automated program for molecular replacement. J. Appl. Crystallogr. 30:1022.
  25. Winn, M. D., M. N. Isupov, G. N. Murshudov. 2001. Use of TLS parameters to model anisotropic displacements in macromolecular refinement. Acta Crystallogr. D Biol. Crystallogr. 57:122.[Medline]
  26. Jones, T. A., J. Y. Zou, S. W. Cowan, S. W. Kjeldgaard. 1991. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47:110.
  27. Perrakis, A., M. Harkiolaki, K. S. Wilson, V. S. Lamzin. 2001. ARP/wARP and molecular replacement. Acta Crystallogr. D Biol. Crystallogr. 57:1445.[Medline]
  28. Laskowski, R. A., D. S. Moss, J. M. Thornton. 1993. Main-chain bond lengths and bond angles in protein structures. J. Mol. Biol. 231:1049.[Medline]
  29. Esnouf, R. M.. 1999. Further additions to MolScript version 1.4, including reading and contouring of electron-density maps. Acta Crystallogr. D Biol. Crystallogr. 55:938.[Medline]
  30. Merritt, E. A., D. J. Bacon. 1997. Raster3D: photorealistic molecular graphics. Methods Enzymol. 277:505.[Medline]
  31. Kleywegt, G. J.. 1999. Experimental assessment of differences between related protein crystal structures. Acta Crystallogr. D Biol. Crystallogr. 55:1878.[Medline]
  32. Garcia, K. C., M. Degano, L. R. Pease, M. Huang, P. A. Peterson, L. Teyton, I. A. Wilson. 1998. Structural basis of plasticity in T cell receptor recognition of a self peptide-MHC antigen. Science 279:1166.[Abstract/Free Full Text]
  33. Brunger, A. T., P. D. Adams, G. M. Clore, W. L. DeLano, P. Gros, R. W. Grosse-Kunstleve, J. S. Jiang, J. Kuszewski, M. Nilges, N. S. Pannu, et al 1998. Crystallography and NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D Biol. Crystallogr. 54:905.[Medline]
  34. Ljunggren, H. G., N. J. Stam, C. Ohlen, J. J. Neefjes, P. Hoglund, M. T. Heemels, J. Bastin, T. N. Schumacher, A. Townsend, K. Karre, et al 1990. Empty MHC class I molecules come out in the cold. Nature 346:476.[Medline]
  35. Kalergis, A. M., N. Boucheron, M. A. Doucey, E. Palmieri, E. C. Goyarts, Z. Vegh, I. F. Luescher, S. G. Nathenson. 2001. Efficient T cell activation requires an optimal dwell-time of interaction between the TCR and the pMHC complex. Nat. Immunol. 2:229.[Medline]
  36. Kuhns, J. J., M. A. Batalia, S. Yan, E. J. Collins. 1999. Poor binding of a HER-2/neu epitope (GP2) to HLA-A2.1 is due to a lack of interactions with the center of the peptide. J. Biol. Chem. 274:36422.[Abstract/Free Full Text]
  37. Apostolopoulos, V., M. Yu, A. L. Corper, L. Teyton, G. A. Pietersz, I. F. McKenzie, I. A. Wilson, M. Plebanski. 2002. Crystal structure of a non-canonical low-affinity peptide complexed with MHC class I: a new approach for vaccine design. J. Mol. Biol. 318:1293.[Medline]
  38. Sharma, A. K., J. J. Kuhns, S. Yan, R. H. Friedline, B. Long, R. Tisch, E. J. Collins. 2001. Class I major histocompatibility complex anchor substitutions alter the conformation of T cell receptor contacts. J. Biol. Chem. 276:21443.[Abstract/Free Full Text]
  39. Reiser, J. B., C. Darnault, A. Guimezanes, C. Gregoire, T. Mosser, A. M. Schmitt-Verhulst, J. C. Fontecilla-Camps, B. Malissen, D. Housset, G. Mazza. 2000. Crystal structure of a T cell receptor bound to an allogeneic MHC molecule. Nat. Immunol. 1:291.[Medline]
  40. Rudolph, M. G., I. A. Wilson. 2002. The specificity of TCR/pMHC interaction. Curr. Opin. Immunol. 14:52.[Medline]
  41. Ding, Y. H., B. M. Baker, D. N. Garboczi, W. E. Biddison, D. C. Wiley. 1999. Four A6-TCR/peptide/HLA-A2 structures that generate very different T cell signals are nearly identical. Immunity 11:45.[Medline]
  42. Garcia, K. C., M. Degano, R. L. Stanfield, A. Brunmark, M. R. Jackson, P. A. Peterson, L. Teyton, I. A. Wilson. 1996. An {alpha}{beta} T cell receptor structure at 2.5 A and its orientation in the TCR-MHC complex. Science 274:209.[Abstract/Free Full Text]
  43. Degano, M., K. C. Garcia, V. Apostolopoulos, M. G. Rudolph, L. Teyton, I. A. Wilson. 2000. A functional hot spot for antigen recognition in a superagonist TCR/MHC complex. Immunity 12:251.[Medline]
  44. Garboczi, D. N., P. Ghosh, U. Utz, Q. R. Fan, W. E. Biddison, D. C. Wiley. 1996. Structure of the complex between human T-cell receptor, viral peptide and HLA-A2. Nature 384:134.[Medline]
  45. Ding, Y. H., K. J. Smith, D. N. Garboczi, U. Utz, W. E. Biddison, D. C. Wiley. 1998. Two human T cell receptors bind in a similar diagonal mode to the HLA-A2/Tax peptide complex using different TCR amino acids. Immunity 8:403.[Medline]
  46. Baker, B. M., S. J. Gagnon, W. E. Biddison, D. C. Wiley. 2000. Conversion of a T cell antagonist into an agonist by repairing a defect in the TCR/peptide/MHC interface: implications for TCR signaling. Immunity 13:475.[Medline]
  47. Batalia, M. A., E. J. Collins. 1997. Peptide binding by class I and class II MHC molecules. Biopolymers 43:281.[Medline]
  48. Reid, S. W., S. McAdam, K. J. Smith, P. Klenerman, C. A. O’Callaghan, K. Harlos, B. K. Jakobsen, A. J. McMichael, J. I. Bell, D. I. Stuart, E. Y. Jones. 1996. Antagonist HIV-1 Gag peptides induce structural changes in HLA B8. J. Exp. Med. 184:2279.[Abstract/Free Full Text]
  49. Moudgil, K. D., E. E. Sercarz, I. S. Grewal. 1998. Modulation of the immunogenicity of antigenic determinants by their flanking residues. Immunol. Today 19:217.[Medline]
  50. Arnold, P. Y., N. L. La Gruta, T. Miller, K. M. Vignali, P. S. Adams, D. L. Woodland, D. A. Vignali. 2002. The majority of immunogenic epitopes generate CD4+ T cells that are dependent on MHC class II-bound peptide-flanking residues. J. Immunol. 169:739.[Abstract/Free Full Text]
  51. O’Connor, D., T. Allen, D. I. Watkins. 2001. Vaccination with CTL epitopes that escape: an alternative approach to HIV vaccine development?. Immunol. Lett. 79:77.[Medline]
  52. Hunziker, L., M. Recher, A. Ciurea, M. M. Martinic, B. Odermatt, H. Hengartner, R. M. Zinkernagel. 2002. Antagonistic variant virus prevents wild-type virus-induced lethal immunopathology. J. Exp. Med. 196:1039.[Abstract/Free Full Text]
  53. Laskowski, R. A.. 1995. SURFNET: a program for visualizing molecular surfaces, cavities and intermolecular interactions. J. Mol. Graph. 13:323.[Medline]



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