Unlocking viral evasion: Luminal charge interactions in BoHV-1 UL49.5 allosterically control TAP degradation

Abstract

The UL49.5 protein of bovine alphaherpesvirus 1 (BoHV-1) is known to inhibit the transporter associated with antigen processing (TAP) and interfere with antigen presentation, in part by promoting TAP degradation. However, the role of electrostatic interactions within the N-terminal luminal domain in controlling these processes remains unclear. Here, we combined circular dichroism (CD), solution nuclear magnetic resonance spectroscopy (NMR), all-atom molecular dynamics simulations, and cell-based assays to define the structural and functional contribution of charged residues within the N-terminal luminal domain of UL49.5. Two N-terminal variants of UL49.5, UL49.522-56RR(30-31)DD and UL49.522-56D36K, with substitutions of charged-reversal amino acid residues, were designed. These two mutants formed membrane-induced -helical structures but showed altered helix stability and interaction patterns. Molecular dynamics simulations of the UL49.5-TAP complexes revealed that wild-type UL49.5 forms a stable electrostatic interface with TAP, particularly in the unkinked conformation, while charge-reversal mutations remodel salt-bridge networks, destabilize the luminal helix, and alter the positioning and dynamics of the transmembrane and cytoplasmic C-terminal regions. The structural changes within the N-terminus alter the exposure of the C-terminal degron required for KLHDC3-dependent degradation. Consistent with these findings, the mutants did not induce proteasomal degradation of TAP, despite maintaining near wild-type levels of downregulation of MHC class I. Together, these results identify N-terminal electrostatic interactions as allosteric determinants of UL49.5-driven TAP degradation and demonstrate that TAP degradation can be mechanically uncoupled from downregulation of MHC class I. This study improves our understanding of viral immune evasion strategies and potential therapeutic targets.