Host membrane cholesterol constrains constitutive signaling of the oncogenic KSHV GPCR ORF74

Cholesterol is a major structural component of the plasma membrane and a key allosteric regulator of G protein-coupled receptors (GPCRs), yet its role in controlling constitutive receptor activity remains poorly understood. Virally encoded GPCRs provide an ideal system to address this question because many exhibit constitutive signaling that promotes viral persistence and pathogenesis, although how excessive receptor activation is restrained remains unknown. Here, we identify a previously unrecognized cholesterol-dependent allosteric mechanism by which the oncogenic Kaposi’s sarcoma-associated herpesvirus (KSHV) GPCR ORF74 constrains its constitutive activity. CryoEM structural analysis reveals a cholesterol-binding pocket formed by transmembrane helices 3, 5, and 6 that is present only in the inactive receptor. Cholesterol binding restrains the outward movement of transmembrane helix 6, stabilizes the inactive conformation, and suppresses spontaneous receptor activation, as supported by molecular dynamics simulations. Mechanistically, replacement of the canonical DRY motif with a non-canonical VRY motif exposes the conserved R143 ^3.50 residue, creating a membrane-facing cholesterol-binding interface that couples membrane cholesterol to receptor conformational control. Consistent with this model, cellular cholesterol depletion enhances ORF74 signaling, whereas disruption of cholesterol binding impairs receptor stabilization. Together, our findings uncover a previously unrecognized mechanism by which a viral GPCR exploits host membrane cholesterol to regulate its constitutive activity, suggesting that persistent viruses optimize constitutive signaling by coupling receptor activity to host lipid-dependent allosteric regulation.