CPXV012 blocks ATP binding to TAP1 and TAP2.

<p>(A) CPXV012 inhibits ATP binding to TAP. TAP1, TAP2, and ^C8CPXV012 were coexpressed in <i>Sf</i>9 cells as indicated. TAP1/TAP2 or TAP1/TAP2/^C8CPXV012 complexes were affinity purified using anti-TAP1 (mAb 148.3) or anti-C8 antibodies, respectively. Dynabead immobilized proteins were pre-incubated with 15 µM 8-azido-ATP[γ]biotin in the presence or absence of 5 mM ATP for 5 min on ice and subsequently subjected to UV irradiation. Dynabead immobilized proteins were then separated by SDS-PAGE and detected by immunoblotting with the antibodies as indicated. Biotinylated proteins were visualized using extravidin-HRP conjugate. In the presence (open bars) and absence of ^C8CPXV012 (filled bars), the amount of ATP cross-linked TAP was normalized to the TAP2 protein expression levels. (B) CPXV012 inhibits ATP binding sites to TAP1 and TAP2. TAP1, ^TsnTAP2, and ^flagCPXV012 were coexpressed and crude membranes were incubated with 8-azido-ATP[γ]biotin (15 µM) in the presence or absence of ATP (5 mM) for 5 min on ice. After UV irradiation, proteins were immunoprecipitated with TAP1 (mAb 148.3) or flag-specific antibodies (IP). The HC10-antibody was used as negative control (mock). Immunoprecipitated samples were then separated by SDS-PAGE and analyzed by immunoblotting with extravidin-HRP or the corresponding antibodies. The amount of TAP photo cross-linked by 8-azido-ATP in the absence (filled bars) or presence (open bars) of ^flagCPXV012 was normalized to TAP1 or ^TsnTAP2 protein expression levels, respectively.</p

CPXV012 evolved a unique ER-lumenal sequence that is essential for TAP inhibition.

<p>(A) Sequence alignment of CPXV012 and its orthologs. Abbreviations and accession numbers are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004554#ppat.1004554.s008" target="_blank">Table S1</a>. 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 BNLF2a^C8 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, BNLF2a^C8, ^C8CPXV012 full-length, and isotype transfection are from the same data set in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004554#ppat-1004554-g001" target="_blank">Fig. 1A</a>. (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 BNLF2a^C8 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 coreTAP1^mVenus-C8 and coreTAP2^{mCerulean-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.</p

The ER-lumenal CPXV012 fragment is sufficient for trans-inhibition of antigen translocation.

<p>(A) CPXV012 sequence and synthetic peptides derived from the C-terminal ER-lumenal end of CPXV012. (B) The C-terminal 10 residues of CPXV012 are sufficient for inhibition of the peptide-stimulated ATP hydrolysis of TAP. Purified coreTAP (0.2 µM) was incubated with ATP (1 mM) and high-affinity substrate peptide R9LQK (1 µM) in the presence and absence of C-terminal CPXV012 peptides (20 µM) as indicated. Release of inorganic phosphate was quantified and normalized to the coreTAP-dependent hydrolysis in the presence of R9LQK. Each data point represents the mean of triplicate measurements. Error bars show S.D. (C) The ATP hydrolysis activity of purified TAP (0.2 µM) was measured in the presence of ATP (1 mM), substrate peptide R9LQK (0.5 µM), and increasing concentrations of CPXV012 10mer-peptide. By fitting of the data, a half-maximum inhibition value (IC₅₀) of 72±20 µM was determined. (D) 10mer CPXV012 fragment blocks peptide translocation <i>in vitro</i>. Transport of RRYC^(F)KSTEL peptide (1 µM; C^(F), fluorescein-labeled cysteine) by reconstituted coreTAP was analyzed in the presence and absence of ATP (3 mM) in combination with 100 µM unlabeled peptide R9LQK or CPXV012 10mer-peptide added to or entrapped in liposomes (external and lumenal, respectively). (E) Inhibition mechanism of CPXV012. TAP transports peptides until a critical concentration inside the ER lumen induces trans-inhibition of the transport complex.</p

HCMV-US6 and EBV-BNLF2a prevent the formation of CPXV012•TAP complexes.

<p>(A) Schematic presentation of the MHC I peptide-loading complex and the viral immune evasin US6 (type I membrane protein), BNLF2a (tail-anchored), and CPXV012 (type II). (B, C) ^flagCPXV012, TAP1, and TAP2 were coexpressed with either US6^myc (B) or BNLF2a^C8 (C) in <i>Sf</i>9 cells. Proteins were affinity-purified with myc-, flag-, or C8-specific antibodies (IP). The HC10-antibody was used as negative control (mock). Samples were analyzed by immunoblotting with the corresponding antibodies. An aliquot (1/20) of the crude membrane input is shown. *, glycosylated protein.</p

CPXV012 evolved a unique ER-lumenal sequence that is essential for TAP inhibition.

<p>(A) Sequence alignment of CPXV012 and its orthologs. Abbreviations and accession numbers are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004554#ppat.1004554.s008" target="_blank">Table S1</a>. 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 BNLF2a^C8 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, BNLF2a^C8, ^C8CPXV012 full-length, and isotype transfection are from the same data set in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004554#ppat-1004554-g001" target="_blank">Fig. 1A</a>. (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 BNLF2a^C8 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 coreTAP1^mVenus-C8 and coreTAP2^{mCerulean-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.</p

CPXV012 inhibits peptide transport but not peptide binding to TAP.

<p>TAP1, TAP2, ^flagCPXV012, and BNLF2a^C8 were coexpressed in <i>Sf</i>9 cells as indicated. (A) CPXV012 inhibits peptide transport of TAP. Crude membranes were incubated with RRYQ<u>NST</u>C^(F)L peptide (C^(F), fluorescein-labeled cysteine; N-core glycosylation site underlined) in the absence or presence of ATP. Translocated and N-core glycosylated peptides were bound to ConA-beads and quantified by fluorescence. Peptide transport by TAP was normalized to 100%. The means of at least three independent experiments are shown. Error bars indicate the S.D. (B) CPXV012 does not inhibit peptide binding to TAP. Crude membranes were incubated with high-affinity peptide RRYC^(F)KSTEL (filled bars). A 100-fold excess of RRYQKSTEL (R9LQK) was used to probe for unspecific binding (open bars). Bound peptides were quantified by fluorescence. The membranes used for (A) and (B) expressed similar amounts of TAP1/2 (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004554#ppat.1004554.s001" target="_blank">S1 Figure</a>). (C) CPXV012 does not interfere with a peptide-induced conformational change of TAP. Membranes were incubated in the presence or absence of the peptide R9LQK. After EGS cross-linking, samples were analyzed by SDS-PAGE (6%) and immunoblotting with TAP1 (mAb 148.3) and TAP2 (mAb 435.3) specific antibodies. (D) CPXV012 binds to coreTAP. ^flagCPXV012 was coexpressed with either full-length TAP1/2 (left) or coreTAP1/2 (right). Proteins were immunoprecipitated with a mixture of polyclonal TAP1 (1p2) and TAP2 specific (2p4) antibodies (IP). The anti-US6 antibody was used a negative control (mock). Samples were analyzed by SDS-PAGE (12%) and subsequent immunoblotting with either anti-flag, monoclonal anti-TAP1 (mAb 148.3), or anti-TAP2 (mAb 435.3) antibodies. An aliquot (1/20) of the crude membrane input is shown.</p

CPXV012 inhibits MHC I antigen presentation by targeting TAP directly.

<p>(A) Down-regulation of MHC I surface expression by CPXV012. HeLa cells were transiently transfected with empty vector, ^flagCPXV012, ^C8CPXV012, BNLF2a^C8, US6^myc or ICP47 in pIRES2-EGFP, respectively. For clarity, US6 and ICP47 are summarized only in panel B. Peptide-loaded MHC I molecules at the cell surface were analyzed by flow cytometry. Only GFP-positive cells were analyzed; isotype control (dotted line). (B) Evaluation of the histograms from A. Mean fluorescence intensity (MFI) was calculated for cells transfected with the indicated constructs. (C–F) CPXV012 binds directly to TAP. ^flagCPXV012 was coexpressed with TAP1/2 (C), TAPL (D), Tsn (E), or TAP1/2 and Tsn (F) in <i>Sf</i>9 cells. Proteins were immunoprecipitated with TAP1 (mAb 148.3), TAP2 (mAb 435.3), or flag-specific antibodies (IP). The HC10-antibody was used as negative control (mock). Samples were analyzed by immunoblotting with the corresponding antibodies. An aliquot (1/20) of the crude membrane input is shown.</p