FIG. 2.
The EG-EA repeat of ORF73 inhibits presentation of CD8+ T-cell peptides. (A) Schematic representation of ORF73 with the H2-Kb-restricted SIINFEKL peptide inserted 5′ and 3′ of the EG-EA repeat (73-S-B and 73-S-Bx) and complementary constructs with the EG-EA repeat deleted (73Δ-S-B and 73Δ-S-Bx). All constructs were tagged with GFP at the C terminus. (B) Immunoblot analysis for expression of the ORF73-SIINFEKL chimeras shown in panel A. 293 cells were transfected with the indicated plasmids, and total protein was extracted at 48 h posttransfection. Twenty μg of protein was resolved on 10% SDS-PAGE gels and transferred to nitrocellulose. Duplicate blots were probed as described for Fig. 1. (C) Presentation of the SIINFEKL peptide to B3Z T cells by cells expressing either 73-S-B, 73Δ-S-B, 73-S-Bx, or 73Δ-S-Bx. 293-Kb cells were transfected with p73-S-B, p73Δ-S-B, p73-S-Bx, or p73Δ-S-Bx, and 24 h later these target cells were cocultured with B3Z cells for 18 to 20 h. B3Z T-cell recognition of target cells was monitored by assaying release of IL-2. Control transfections included the empty vector, pcDNA3, or positive control pNP-S-GFP. (D) Presentation of SIINFEKL to B3Z T cells after coculture with 293-Kb cells transfected with various amounts of plasmids p73-S-B, p73Δ-S-B, p73-S-Bx, and p73Δ-S-Bx. Results of T-cell assays represent mean values of IL-2 release into the supernatant measured from triplicate cultures (+ the standard deviation). T-cell data are from one representative experiment out of three.