The immunoevasive function encoded by the mouse cytomegalovirus gene m152 protects the virus against T cell control in vivo

Abstract

Cytomegaloviruses encode numerous functions that inhibit antigen presentation in the major histocompatibility complex (MHC) class I pathway in vitro. One example is the mouse cytomegalovirus (MCMV) glycoprotein gp40, encoded by the m152 gene, which selectively retains murine but not human MHC class I complexes in the endoplasmic reticulum-Golgi intermediate compartment/cis-Golgi compartment (Ziegler, H., R. Thäle, P. Lucin, W. Muranyi, T. Flohr, H. Hengel, H. Farrell, W. Rawlinson, and U.H. Koszinowski. 1997. Immunity. 6:57-66). To investigate the in vivo significance of this gene function during MCMV infection of the natural host, we constructed recombinants of MCMV in which the m152 gene was deleted, as were the corresponding virus revertants. We report on the following findings: Deletion of the m152 gene has no effect on virus replication in cell culture, whereas after infection of mice, the m152-deficient virus replicates to significantly lower virus titers. This attenuating effect is lifted by reinsertion of the gene into the mutant. Mutants and revertants grow to the same titer in animals deprived of the function targeted by the viral gene function, namely in mice deficient in beta2-microglobulin, mice deficient in the CD8 molecule, and mice depleted of T cells. Upon adoptive transfer of naive lymphocytes into infected mice, the absence of the m152 gene function sensitizes the virus to primary lymphocyte control. These results prove that MHC-reactive functions protect CMVs against attack by CD8(+) T lymphocytes in vivo.

Figure 1

Characterization of m152 recombinant viruses. (A) Genome structure of recombinant viruses. The HindIII cleavage map of the MCMV strain Smith genome is shown at the top, and the expanded HindIII E region of wild-type and recombinant viruses is shown below, with the HindIII (H) and EcoRI (E) cleavage sites indicated. The open box with the arrow depicts the position and orientation of the m152 gene, and the shaded boxes represent viral sequences that were used for homologous recombination. The positions of the loxP sites are indicated by asterisks (*). The marker genes used for selection, loxZ and gpt, are indicated. The probe used for Southern blot analysis is represented by a bar. The expected sizes of the HindIII and EcoRI fragments are indicated by arrows. (B) Southern blot analysis of the recombinant virus genomes. DNA was isolated from infected NIH 3T3 cells and digested with restriction enzymes HindIII and EcoRI, respectively. Sizes of the DNA fragments are indicated in kb.

Figure 1

Characterization of m152 recombinant viruses. (A) Genome structure of recombinant viruses. The HindIII cleavage map of the MCMV strain Smith genome is shown at the top, and the expanded HindIII E region of wild-type and recombinant viruses is shown below, with the HindIII (H) and EcoRI (E) cleavage sites indicated. The open box with the arrow depicts the position and orientation of the m152 gene, and the shaded boxes represent viral sequences that were used for homologous recombination. The positions of the loxP sites are indicated by asterisks (*). The marker genes used for selection, loxZ and gpt, are indicated. The probe used for Southern blot analysis is represented by a bar. The expected sizes of the HindIII and EcoRI fragments are indicated by arrows. (B) Southern blot analysis of the recombinant virus genomes. DNA was isolated from infected NIH 3T3 cells and digested with restriction enzymes HindIII and EcoRI, respectively. Sizes of the DNA fragments are indicated in kb.

Figure 2

Functional characterization of the m152 deletion mutants. (A) Normal maturation of newly synthesized MHC class I molecules in cells infected with the m152 deletion mutant. B12 cells were either mock infected or infected with wild-type MCMV or ΔMC95.21 and rMC96.27 recombinants. 6 h after infection, cells were pulse labeled for 1 h with [³⁵S]methionine, and newly synthesized molecules were chased for 2 h. K^d MHC class I complexes were precipitated from cell lysates with anti-K^d mAb MA-215. Half of the precipitates were digested with Endo H or mock treated before separation by 12.5% SDS-PAGE. The different glycosylation forms of the MHC class I heavy chains with regard to Endo H sensitivity are denoted as r, Endo H resistant; s, Endo H sensitive; or d, Endo H digested. MWM, molecular weight marker. (B) Restoration of MHC class I antigen presentation in cells infected with the m152 deletion mutant. BALB/c MEFs were infected with wild-type (w.t.) MCMV, the m152 deletion mutant ΔMC95.21, and the revertant virus rMC96.27 under conditions that allowed expression of only IE (▵) or IE and E viral proteins (•). Antigen presentation was tested with CTLs specific for the MCMV antigen pp89 at the indicated E/T ratios in a 4-h ⁵¹Cr-release assay.

Figure 2

Functional characterization of the m152 deletion mutants. (A) Normal maturation of newly synthesized MHC class I molecules in cells infected with the m152 deletion mutant. B12 cells were either mock infected or infected with wild-type MCMV or ΔMC95.21 and rMC96.27 recombinants. 6 h after infection, cells were pulse labeled for 1 h with [³⁵S]methionine, and newly synthesized molecules were chased for 2 h. K^d MHC class I complexes were precipitated from cell lysates with anti-K^d mAb MA-215. Half of the precipitates were digested with Endo H or mock treated before separation by 12.5% SDS-PAGE. The different glycosylation forms of the MHC class I heavy chains with regard to Endo H sensitivity are denoted as r, Endo H resistant; s, Endo H sensitive; or d, Endo H digested. MWM, molecular weight marker. (B) Restoration of MHC class I antigen presentation in cells infected with the m152 deletion mutant. BALB/c MEFs were infected with wild-type (w.t.) MCMV, the m152 deletion mutant ΔMC95.21, and the revertant virus rMC96.27 under conditions that allowed expression of only IE (▵) or IE and E viral proteins (•). Antigen presentation was tested with CTLs specific for the MCMV antigen pp89 at the indicated E/T ratios in a 4-h ⁵¹Cr-release assay.

Figure 3

In vitro growth of recombinant viruses. NIH 3T3 cells were infected with wild-type MCMV (○), ΔMC95.24 (▴), or ΔMC95.21 (•) recombinants at a multiplicity of infection of 0.1 PFU per cell. Supernatants (A) and cells (B) were harvested at the indicated time points after infection (p.i.), and virus titers were determined.

Figure 5

Attenuation of the m152 deletion mutant is T cell dependent. (A) 4-d-old BALB/c and C57BL/6 mice were depleted of CD4⁺ and CD8⁺ T lymphocytes or were left untreated, and they were then infected with 1,000 PFU i.p. of the ΔMC95.24 (○) or rMC96.27 viruses (•). 10 d after infection, virus titers were determined. Titers of individual mice (circles) and median values (horizontal bars) are shown. There was a significant difference in virus titers between ΔMC95.24 and rMC96.27 in both mouse strains (P < 0.005; left panels). Depletion of T cells abrogated the difference (right panels). (B) 8-wk-old B cell–deficient mice (μMT^−/−, BALB/c background) were depleted of CD8⁺ T cells or both CD4⁺ and CD8⁺ T lymphocytes, or they were left untreated. Mice were infected with 2 × 10⁵ PFU i.p. of the ΔMC95.24 and rMC96.27 viruses, and virus titers were determined 10 d after infection. Titers in individual animals and median values (horizontal bars) are shown. The differences in virus titers between the groups of nondepleted mice infected with ΔMC95.24 and rMC96.27 were significant (P < 0.005) for titers in lungs and spleens (left panels). Depletion of both T cell subsets abrogated the differences between the two recombinants in both organs tested (right panels). Depletion of only CD8⁺ T cells reduced but did not abolish the differences between the two viruses (P < 0.05; center panels). DL, detection limit.

Figure 4

Reduced virulence and replication capacity of the m152 deletion mutant in vivo. (A) Newborn BALB/c mice were inoculated with 100 PFU i.p. of wild-type (w.t.) MCMV (○), ΔMC95.24 (filled gray triangles), or rMC96.27 (•) virus 12 h post partum, and their survival was monitored daily. (B) Newborn BALB/c mice were infected as shown in A, and virus titers were determined 8 d after infection. Data represent the mean value of at least five mice.

Figure 4

Reduced virulence and replication capacity of the m152 deletion mutant in vivo. (A) Newborn BALB/c mice were inoculated with 100 PFU i.p. of wild-type (w.t.) MCMV (○), ΔMC95.24 (filled gray triangles), or rMC96.27 (•) virus 12 h post partum, and their survival was monitored daily. (B) Newborn BALB/c mice were infected as shown in A, and virus titers were determined 8 d after infection. Data represent the mean value of at least five mice.

Figure 6

No growth difference of m152 deletion and revertant viruses in β₂m^−/− and CD8^−/− mice. Normal C57BL/6, β₂m^−/−, and CD8^−/− mice (all 4 d old) were inoculated with 1,000 PFU i.p. of the ΔMC95.24 (open bars) or rMC96.27 (shaded bars) recombinant viruses. Shown are virus titers in lungs 10 d after infection. Data represent the mean value of five mice. There was a significant difference in virus titers between ΔMC95.24 and rMC96.27 viruses (P < 0.005). The titer difference between the two viruses in β₂m^−/− and CD8^−/− mice is not significant.

Figure 7

Susceptibility of the m152 deletion mutant to MCMV-primed and naive T lymphocytes. 8-wk-old BALB/c gamma-irradiated mice were injected with 10⁵ PFU of ΔMC95.24 or rMC96.27 virus. 2 × 10⁵ T lymphocytes were obtained from latently infected or uninfected BALB/c mice, and cells were transferred intravenously into recipients immediately after infection. Mice that did not receive cell transfer were used as negative controls. Shown are titers in individual recipients, measured 13 d after transfer and infection. Horizontal bars indicate the median values. DL, detection limit.