Signaling of a Varicelloviral Factor across the Endoplasmic Reticulum Membrane Induces Destruction of the Peptide-loading Complex and Immune Evasion

Abstract

Cytotoxic T lymphocytes eliminate infected cells upon surface display of antigenic peptides on major histocompatibility complex I molecules. To promote immune evasion, UL49.5 of several varicelloviruses interferes with the pathway of major histocompatibility complex I antigen processing. However, the inhibition mechanism has not been elucidated yet. Within the macromolecular peptide-loading complex we identified the transporter associated with antigen processing (TAP1 and TAP2) as the prime target of UL49.5. Moreover, we determined the active oligomeric state and crucial elements of the viral factor. Remarkably, the last two residues of the cytosolic tail of UL49.5 are essential for endoplasmic reticulum (ER)-associated proteasomal degradation of TAP. However, this process strictly requires additional signaling of an upstream regulatory element in the ER lumenal domain of UL49.5. Within this new immune evasion mechanism, we show for the first time that additive elements of a small viral factor and their signaling across the ER membrane are essential for targeted degradation of a multi-subunit membrane complex.

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

“…4, D and F). However, huTAP1 expressed individually in insect cells showed a clear association with UL49.5 (61). Most likely, the relatively high expression levels of the individual proteins in Sf9 insect cells promote interactions between UL49.5 and the TAP subunits.…”

“…The interaction between the ER-exposed domains of UL49.5 and TAP may further obstruct TAP function. Data from our laboratories indicate that UL49.5 occurs in the TAP complex as a disulfide-linked homodimer (61). Therefore, the interference with TAP function described above might be conducted by two UL49.5 molecules simultaneously, with one UL49.5 molecule interacting with TAP1 and one with TAP2.…”

“…This might explain the observation that UL49.5 is able to interact with both TAP1 and TAP2. Interestingly, a UL49.5 recombinant lacking this ER-luminal cysteine appeared to be equally active (61).…”

“…The interaction of the ER-luminal and TM domains of UL49.5 with the 6ϩ6 TM core complex may cause a conformational arrest of the transporter that results in the inhibition of peptide transport (38). Both the TM helix and the ER-luminal domain of UL49.5 are required for this effect (38,61). Insertion of the TM domain of UL49.5 within the core complex of TAP might prevent essential rearrangements of the TAP TM helices and may thus freeze TAP in a translocation-incompetent state.…”

“…High, stable levels of TAP are wellknown advantages of this system. In this study, Sf9 cells were infected with baculoviruses to coexpress UL49.5 with wild-type and recombinant forms of huTAP (61). To investigate whether UL49.5 was able to inhibit the function of the 6ϩ6 TM huTAP core complex, Sf9 cells expressing huTAP1⌬N and huTAP2⌬N FIGURE 7.…”