The Varicellovirus UL49.5 Protein Blocks the Transporter Associated with Antigen Processing (TAP) by Inhibiting Essential Conformational Transitions in the 6+6 Transmembrane TAP Core Complex

Abstract

TAP translocates virus-derived peptides from the cytosol into the endoplasmic reticulum, where the peptides are loaded onto MHC class I molecules. This process is crucial for the detection of virus-infected cells by CTL that recognize the MHC class I-peptide complexes at the cell surface. The varicellovirus bovine herpesvirus 1 encodes a protein, UL49.5, that acts as a potent inhibitor of TAP. UL49.5 acts in two ways, as follows: 1) by blocking conformational changes of TAP required for the translocation of peptides into the endoplasmic reticulum, and 2) by targeting TAP1 and TAP2 for proteasomal degradation. At present, it is unknown whether UL49.5 interacts with TAP1, TAP2, or both. The contribution of other members of the peptide-loading complex has not been established. Using TAP-deficient cells reconstituted with wild-type and recombinant forms of TAP1 and TAP2, TAP was defined as the prime target of UL49.5 within the peptide-loading complex. The presence of TAP1 and TAP2 was required for efficient interaction with UL49.5. Using deletion mutants of TAP1 and TAP2, the 6+6 transmembrane core complex of TAP was shown to be sufficient for UL49.5 to interact with TAP and block its function. However, UL49.5-induced inhibition of peptide transport was most efficient in cells expressing full-length TAP1 and TAP2. Inhibition of TAP by UL49.5 appeared to be independent of the presence of other peptide-loading complex components, including tapasin. These results demonstrate that UL49.5 acts directly on the 6+6 transmembrane TAP core complex of TAP by blocking essential conformational transitions required for peptide transport.

Hydrophobic Interactions Are Key To Drive the Association of Tapasin with Peptide Transporter Subunit TAP2

“…TAP variants lacking the N-domain of TAP2 (e.g., 1-2DN) (7) display altered PLC formation properties with reduced complex stability as well as quality control in MHC I loading (7,83). This makes it very difficult to determine the structural requirements of the N-domain of TAP1 for TPN interaction as it was performed for the N-domain of TAP2 in the present study.…”

CD8+ T Cell Responses against TAP-Inhibited Cells Are Readily Detected in the Human Population

“…This system has been reported recently (32), and the viral TAP inhibitors UL49.5 (from BHV-1), ICP47, US6, and BNLF2a were used, which were described previously (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18). Cells were sorted on GFP expression by FACS to ensure homogenous and high expression of the various TAP inhibitors.…”

“…UL49.5 is a protein encoded by herpesviruses belonging to the genus Varicellovirus. In this study, the bovine herpesvirus-1 (BHV-1) variant of UL49.5 is used, which efficiently inactivates TAP by arresting it into a translocation-incompetent state; moreover, it promotes the degradation of TAP (8)(9)(10). The TAP inhibitor ICP47 is found in HSV type I and II, and it binds in a stable fashion to the cytosolic site of the TAP complex, thereby acting as a high-affinity competitor for peptide binding (11)(12)(13)(14).…”

“…Depending on the protein, either 16.5% Tricine-PAGE (for UL49.5) or 10% SDS page gels (for b-actin and TAP1) were made (9). The gels were loaded, and BenchMark prestained ladder (Invitrogen) was used as marker.…”

“…To ensure inhibition of TAP, a peptide transport assay was done as described previously (9,10). Cells were permeabilized and incubated with the fluorescein-conjugated synthetic peptide CVNKTERAY in the presence or absence of 10 mM ATP.…”