A Negative Feedback Modulator of Antigen Processing Evolved from a Frameshift in the Cowpox Virus Genome
Figure 5
CPXV012 evolved a unique ER-lumenal sequence that is essential for TAP inhibition.
(A) Sequence alignment of CPXV012 and its orthologs. Abbreviations and accession numbers are shown in Table S1. GER91: The first 97 amino acids of the protein are aligned. The CPXV012 sequence of Brighton Red strain was used in this study (red box). The N- and C-terminal deletion constructs C8NΔ6-CPXV012 and C8CPXV012-CΔ5 are indicated as blue and green bars. (B) The last C-terminal, ER-lumenal residues of CPXV012 are essential for TAP inhibition. HeLa cells were transiently transfected with empty vector, full-length C8CPXV012, C8CPXV012-CΔ5, C8NΔ6-CPXV012, or BNLF2aC8 in pIRES2-EGFP, respectively. MHC I surface expression was analyzed by flow cytometry. Only GFP-positive cells were analyzed. The dotted histogram represents the isotype control. Histograms for mock, BNLF2aC8, C8CPXV012 full-length, and isotype transfection are from the same data set in Fig. 1A. (C) Evaluation of the histograms from B. Mean fluorescence intensity (MFI) was calculated for cells transfected with the indicated constructs. (D) Similar expression levels of the C8CPXV012 constructs and BNLF2aC8 in cells analyzed by flow cytometry were confirmed by anti-C8 and anti-actin immunoblotting. (E) Inactive C8CPXV012-CΔ5 still binds to coreTAP1/2 heterodimers. Human coreTAP1mVenus-C8 and coreTAP2mCerulean-StrepII were coexpressed in HEK293T cells together with the C8CPXV012 variants as indicated. TAP1/2 heterodimeric complexes were tandem-affinity purified using streptavidin and anti-TAP1 (mAb 148.3) matrices. The HC10-antibody was used as negative control (mock). Input (solubilizate, 1/30 aliquot) and affinity purified complexes were analyzed by immunoblotting using C8- or TAP2-specific (mAb 435.3) antibodies, respectively. #, partially unfolded mCerulean.