Structural basis for ligand promiscuity and high signaling activity of Kaposi’s Sarcoma-associated Herpesvirus-encoded GPCR

Kaposi's Sarcoma-associated Herpesvirus encodes ORF74, a viral G protein-coupled receptor homologous to CXCR2, which plays a crucial role in Kaposi's Sarcoma development through its high basal signaling activity. Our cryoEM analysis of ORF74 in ligand-free, BRIL-fused ligand-free, and CXCL1-Gitrimer-bound forms elucidated its ligand-independent signaling activity. The widely open, static extracellular cavity contributes to its ligand promiscuity by facilitating dynamic access and various binding modes. Impairment of CWxP, E/DRY, and NPxxY micro-switches destabilizes the inactive structure of ORF74 to allow ligand-independent activation. Metadynamics simulations demonstrate a highly dynamic equilibrium between inactive and active conformations in ORF74, enabling spontaneous inactive-active transitions. Finally, CXCR2-ORF74 chimeras underscore the functional importance of intracellular loops 2 and 3 in modulating basal and ligand-induced receptor activity. This study reveals the multifactorial structural elements of KSHV ORF74 that are responsible for its promiscuous ligand binding, spontaneous inactive-active conformational transitions, and ligand-independent high basal signaling, explaining the structural basis of ORF74-mediated viral oncogenesis.