Characterization of a murine cytomegalovirus class I major histocompatibility complex (MHC) homolog: comparison to MHC molecules and to the human cytomegalovirus MHC homolog

Abstract

Both human and murine cytomegaloviruses (HCMV and MCMV) down-regulate expression of conventional class I major histocompatibility complex (MHC) molecules at the surfaces of infected cells. This allows the infected cells to evade recognition by cytotoxic T cells but leaves them susceptible to natural killer cells, which lyse cells that lack class I molecules. Both HCMV and MCMV encode class I MHC heavy-chain homologs that may function in immune response evasion. We previously showed that a soluble form of the HCMV class I homolog (U(L)18) expressed in Chinese hamster ovary cells binds the class I MHC light-chain beta2-microglobulin and a mixture of endogenous peptides (M. L. Fahnestock, J. L. Johnson, R. M. R. Feldman, J. M. Neveu, W. S. Lane, and P. J. Bjorkman, Immunity 3:583-590, 1995). Consistent with this observation, sequence comparisons suggest that U(L)18 contains the well-characterized groove that serves as the binding site in MHC molecules for peptides derived from endogenous and foreign proteins. By contrast, the MCMV homolog (m144) contains a substantial deletion within the counterpart of its alpha2 domain and might not be expected to contain a groove capable of binding peptides. We have now expressed a soluble version of m144 and verified that it forms a heavy chain-beta2-microglobulin complex. By contrast to U(L)18 and classical class I MHC molecules, m144 does not associate with endogenous peptides yet is thermally stable. These results suggest that U(L)18 and m144 differ structurally and might therefore serve different functions for their respective viruses.

FIG. 1

Comparison of MCMV and HCMV class I homologs with class I MHC molecules. (A) Sequence alignment of the mature extracellular regions of m144 with a murine class I molecule (muMHC) and of U_L18 with a human class I MHC molecule (huMHC) (based on data from Fig. 1 in reference 14). Numbering is with reference to class I MHC molecules. Crystallographically determined secondary-structural elements in class I MHC molecules (3) are shown above the sequences as arrows for β strands (strands 1 through 8 within the α1 and α2 domains are labeled β1 to β8, and strands 1 through 7 within the α3 domain are labeled A to G) and spirals for α-helical regions. Positions of conserved tyrosines in the pocket that accommodates peptide N termini (pocket A in class I MHC molecules [45, 47]) are marked with an asterisk, and potential N-linked glycosylation sites are underlined. (B) Locations of U_L18 and m144 sequence insertions and deletions on the class I MHC structure. Ribbon diagrams of the carbon-α backbone of the α1 and α2 domains of HLA-A2 (4, 45) are shown with the locations of U_L18 or m144 insertions indicated by asterisks; class I regions that are deleted in U_L18 or m144 are indicated by dashed lines. Conserved tyrosines shared between U_L18 and class I molecules are highlighted in the left panel. This figure was generated by using Molscript (28) and Raster-3D (34).

FIG. 1

Comparison of MCMV and HCMV class I homologs with class I MHC molecules. (A) Sequence alignment of the mature extracellular regions of m144 with a murine class I molecule (muMHC) and of U_L18 with a human class I MHC molecule (huMHC) (based on data from Fig. 1 in reference 14). Numbering is with reference to class I MHC molecules. Crystallographically determined secondary-structural elements in class I MHC molecules (3) are shown above the sequences as arrows for β strands (strands 1 through 8 within the α1 and α2 domains are labeled β1 to β8, and strands 1 through 7 within the α3 domain are labeled A to G) and spirals for α-helical regions. Positions of conserved tyrosines in the pocket that accommodates peptide N termini (pocket A in class I MHC molecules [45, 47]) are marked with an asterisk, and potential N-linked glycosylation sites are underlined. (B) Locations of U_L18 and m144 sequence insertions and deletions on the class I MHC structure. Ribbon diagrams of the carbon-α backbone of the α1 and α2 domains of HLA-A2 (4, 45) are shown with the locations of U_L18 or m144 insertions indicated by asterisks; class I regions that are deleted in U_L18 or m144 are indicated by dashed lines. Conserved tyrosines shared between U_L18 and class I molecules are highlighted in the left panel. This figure was generated by using Molscript (28) and Raster-3D (34).

FIG. 2

SDS-PAGE (15% polyacrylamide) analysis of U_L18 and the two forms of m144. Proteins were purified from the supernatants of transfected CHO cells by passage over an immunoaffinity column followed by size exclusion chromatography. The heavy chains of both heterodimers migrate with a higher apparent molecular mass than is suggested by the mass of their protein backbones due to the addition of N-linked glycosides (U_L18 contains 13, and m144 contains 4, potential N-linked glycosylation sites).

FIG. 3

Thermal denaturation profiles. The CD signal at 223 nm is plotted as molar ellipticity per mean residue after smoothing as a function of increasing temperature. T_ms (indicated by arrows) were determined by taking the maximum of a plot of dθ/dT versus T (where θ is ellipticity) after averaging the data with a moving window of 5 points. (A) m144-hβ₂m and m144-mβ₂m melting curves. (B) K^d-hβ2m melting curves in the presence and absence of added peptide (see also references and 13). (C) U_L18 melting curves in the presence and absence of added peptide.

FIG. 3

Thermal denaturation profiles. The CD signal at 223 nm is plotted as molar ellipticity per mean residue after smoothing as a function of increasing temperature. T_ms (indicated by arrows) were determined by taking the maximum of a plot of dθ/dT versus T (where θ is ellipticity) after averaging the data with a moving window of 5 points. (A) m144-hβ₂m and m144-mβ₂m melting curves. (B) K^d-hβ2m melting curves in the presence and absence of added peptide (see also references and 13). (C) U_L18 melting curves in the presence and absence of added peptide.

FIG. 3

Thermal denaturation profiles. The CD signal at 223 nm is plotted as molar ellipticity per mean residue after smoothing as a function of increasing temperature. T_ms (indicated by arrows) were determined by taking the maximum of a plot of dθ/dT versus T (where θ is ellipticity) after averaging the data with a moving window of 5 points. (A) m144-hβ₂m and m144-mβ₂m melting curves. (B) K^d-hβ2m melting curves in the presence and absence of added peptide (see also references and 13). (C) U_L18 melting curves in the presence and absence of added peptide.

FIG. 4

Far-UV CD spectra of peptide-filled U_L18, peptide-filled K^d, FcRn, and m144 expressed as ellipticity per mean residue. CD spectra of the partially empty versions of U_L18 and K^d superimpose almost perfectly upon spectra of their peptide-filled counterparts (data not shown).