Figure 7.
Figure 7. MATp1 is necessary for infected cell recognition by activating Ly49 receptors, which led to its high variability among field MCMV isolates and loss of recognition. (A–C) Reporter cell assay with activating Ly49L-, Ly49P-, and Ly49D2-RCs coincubated with BALB/c (H-2d; A and B) and BALB.K (H-2k; C) MEFs. (A) Representative histograms showing activation of Ly49L-RCs after coincubation with BALB/c MEFs infected with indicated viruses at 1.5 PFU/cell. Activation of Ly49-RCs was measured after 24 h of cocultivation by measuring the frequency of GFP expressing Ly49-RCs. Both m04 and MATp1 were necessary for the proper activation Ly49L-, Ly49P-, and Ly49D2-RCs. All the experiments were performed at least three times, except two times with Ly49D2-RCs. One-way ANOVA with post hoc test was used for statistical analysis. Graphs show mean with SEM as error bars. ****, P ≤ 0.0001; ***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05. (D) Ly49L receptor specifically recognizes altered-self H-2Dd in an MHC I– and m04-dependent manner. Primary BALB/c MEFs were infected with indicated viruses and then incubated with 10 µg of indicated antibody for 1 h at 4°C before the addition of RCs. Ly49L-RCs were cocultivated with targets for 24 h, as described previously. Addition of α-H-2Dd antibody (34-5-8S) completely abrogated recognition of either infected or mock-infected cells by Ly49A-RCs, demonstrating that they indeed recognize H-2Dd. Incubation with α-m04.16 antibody partially blocked Ly49L-RC activation, reducing it to the level of activation seen when Ly49-RCs are incubated with mock-infected MEFs. α-MATp1 antibody was unable to block interaction between MCMV-infected cells and Ly49L-RCs. Perpendicular dashed lines denote MFI of Ly49A-RCs after incubation with WT MCMV–infected MEFs. Representative histograms are shown; experiment was performed three times with equal results. (E) MATp1 ORF is highly variable among different field isolates. Whole-genome sequences of MCMV strains Smith (gB acc. no. NC_004065.1), G4 (gB acc. no. EU579859), K181 (gB acc. no. AM886412), C4C (gB acc. no. HE610453), C4D (gB acc. no. HE610456), and WP15B (gB acc. no. EU579860) were aligned in software Geneious, with Smith MCMV sequence used as a reference. MATp1 ORF is the only variable part of the MAT transcript, and sequence similarity is <30% throughout the whole MATp1 ORF, while the rest of the MAT transcript is conserved (Corbett et al., 2007; Smith et al., 2008). (F and G) Reporter cell assay with Ly49-RCs coincubated with BALB/c MEFs infected with MCMV field isolates as described in A. Bar charts of Ly49L-, Ly49P-, and Ly49A-RCs incubated with BALB/c MEFs infected with indicated viruses that represent three or more independent experiments with indicated RCs. Representative plots are shown in Fig. S5 C. One-way ANOVA with post hoc test was used for statistical analysis. Graphs show mean with SEM as error bars. ****, P ≤ 0.0001; ***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05. norm., normalized.

MATp1 is necessary for infected cell recognition by activating Ly49 receptors, which led to its high variability among field MCMV isolates and loss of recognition. (A–C) Reporter cell assay with activating Ly49L-, Ly49P-, and Ly49D2-RCs coincubated with BALB/c (H-2d; A and B) and BALB.K (H-2k; C) MEFs. (A) Representative histograms showing activation of Ly49L-RCs after coincubation with BALB/c MEFs infected with indicated viruses at 1.5 PFU/cell. Activation of Ly49-RCs was measured after 24 h of cocultivation by measuring the frequency of GFP expressing Ly49-RCs. Both m04 and MATp1 were necessary for the proper activation Ly49L-, Ly49P-, and Ly49D2-RCs. All the experiments were performed at least three times, except two times with Ly49D2-RCs. One-way ANOVA with post hoc test was used for statistical analysis. Graphs show mean with SEM as error bars. ****, P ≤ 0.0001; ***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05. (D) Ly49L receptor specifically recognizes altered-self H-2Dd in an MHC I– and m04-dependent manner. Primary BALB/c MEFs were infected with indicated viruses and then incubated with 10 µg of indicated antibody for 1 h at 4°C before the addition of RCs. Ly49L-RCs were cocultivated with targets for 24 h, as described previously. Addition of α-H-2Dd antibody (34-5-8S) completely abrogated recognition of either infected or mock-infected cells by Ly49A-RCs, demonstrating that they indeed recognize H-2Dd. Incubation with α-m04.16 antibody partially blocked Ly49L-RC activation, reducing it to the level of activation seen when Ly49-RCs are incubated with mock-infected MEFs. α-MATp1 antibody was unable to block interaction between MCMV-infected cells and Ly49L-RCs. Perpendicular dashed lines denote MFI of Ly49A-RCs after incubation with WT MCMV–infected MEFs. Representative histograms are shown; experiment was performed three times with equal results. (E) MATp1 ORF is highly variable among different field isolates. Whole-genome sequences of MCMV strains Smith (gB acc. no. NC_004065.1), G4 (gB acc. no. EU579859), K181 (gB acc. no. AM886412), C4C (gB acc. no. HE610453), C4D (gB acc. no. HE610456), and WP15B (gB acc. no. EU579860) were aligned in software Geneious, with Smith MCMV sequence used as a reference. MATp1 ORF is the only variable part of the MAT transcript, and sequence similarity is <30% throughout the whole MATp1 ORF, while the rest of the MAT transcript is conserved (Corbett et al., 2007; Smith et al., 2008). (F and G) Reporter cell assay with Ly49-RCs coincubated with BALB/c MEFs infected with MCMV field isolates as described in A. Bar charts of Ly49L-, Ly49P-, and Ly49A-RCs incubated with BALB/c MEFs infected with indicated viruses that represent three or more independent experiments with indicated RCs. Representative plots are shown in Fig. S5 C. One-way ANOVA with post hoc test was used for statistical analysis. Graphs show mean with SEM as error bars. ****, P ≤ 0.0001; ***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05. norm., normalized.

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