High-affinity ELR+ CXC chemokines display balanced G protein-, GRK- and β-arrestin-associated transducer engagement at the viral G protein-coupled receptor ORF74.

Open reading frame 74 (ORF74) is the single G protein-coupled receptor (GPCR) encoded in the genome of Kaposi's sarcoma-associated herpesvirus (KSHV). Due to its high constitutive activity and broad chemokine binding profile, ORF74 contributes to KSHV-associated diseases through autocrine and paracrine mechanisms that promote cell proliferation, inflammation and angiogenesis. Previous studies showed that ELR+ CXC chemokines (i.e., CXCL1-3 and CXCL5-8), which contain a conserved Glu-Leu-Arg motif, bind ORF74 with high affinity. Among these chemokines, CXCL1-3 and CXCL6-8 act as full or partial agonists in ORF74-mediated G protein signaling and β-arrestin recruitment, whereas CXCL5 behaves as a neutral ligand. However, whether these chemokines preferentially engage specific intracellular transducers remains unclear. We therefore applied a unified NanoLuc Binary Technology (NanoBiT) platform in HEK293A cells to systematically compare how ELR+ CXC chemokines modulate ORF74-transducer proximity across the G protein-GRK-β-arrestin axis. CXCL1-3 and CXCL6-8, but not CXCL5, induced concentration-dependent ORF74-specific responses involving miniGi recruitment, Gβ1 proximity, GRK2/3/5/6 proximity and β-arrestin1/2 recruitment. Using CXCL1 as the reference ligand, bias analysis showed that the active ELR+ CXC chemokines engage ORF74 in a largely balanced manner across these proximal transducer readouts.