Human and viral chemokines differentially modulate G protein signaling, β-arrestin recruitment and chemotaxis mediated by the viral G protein-coupled receptor ORF74.

backgroundHerpesviruses are DNA viruses that establish lifelong latent infections in their hosts. During host-virus co-evolution, several members of the herpesvirus family acquired genes reminiscent of human chemokines and their G protein-coupled receptors (GPCRs), which contribute to immune evasion, viral latency and virus-induced oncogenesis. Open reading frame 74 (ORF74) is the single viral GPCR encoded in the genome of human herpesvirus 8 (HHV8), also known as Kaposi's sarcoma-associated herpesvirus (KSHV). ORF74 is most closely related to human CXC chemokine receptor 2 (CXCR2), but its signaling properties are quite different given that ORF74 displays high constitutive activity and interacts with both CC and CXC chemokines. Despite many indications that ORF74 plays an important role during KSHV infection and -associated diseases, studies with regard to chemokine induced G protein signaling, β-arrestin recruitment and chemotaxis remain scarce and fragmented, and often include only a limited set of chemokines.

methodsWe conducted a side-by-side comparison of ORF74 chemokine ligands using in vitro cell-based assays. To validate the binding affinity of previously reported ORF74-interacting chemokines, an antibody competition binding assay was performed. To functionally characterize ORF74-binding chemokines, we employed G protein-dependent calcium mobilization and NanoLuc Binary Technology (NanoBiT)-based β-arrestin recruitment assays. Furthermore, to evaluate a hallmark function of chemokine receptors, ORF74-mediated chemokine-induced cell migration was investigated using Transwell assays.

resultsTwelve chemokines (CXCL1-8, CXCL10, CXCL12, CCL1 and vCCL2) demonstrated displacement of anti-ORF74 antibody binding with varying affinities. CXCL1 appeared to be the most potent and efficacious ORF74 agonist in all functional assays whereas the remaining ELR+ chemokines (CXCL2-3, CXCL5-8) also behaved as full or partial agonists. Other chemokines modulated ORF74 activity in a different way: CXCL10 and CXCL12 inhibited CXCL1-induced calcium mobilization and acted as inverse agonists in β-arrestin recruitment. To our knowledge, this is the first study to show that CXCL1-3 and CXCL6-8 induce ORF74-dependent chemotaxis in line with their pharmacological profiles. In addition, ORF74 expression also influenced chemotaxis mediated by the human chemokine receptor CXCR4.

conclusionOur comprehensive profiling of ORF74 chemokine ligands reveals ligand-dependent activation of canonical signaling pathways, including calcium mobilization, β-arrestin recruitment and chemotaxis. We confirm that ORF74 exhibits constitutive β-arrestin recruitment and, upon co-expression with CXCR4, is able to modulate CXCL12-CXCR4-mediated cell migration. These data highlight the complex chemokine-ORF74 interplay and will contribute to a better understanding of KSHV infection and pathology through multiple ORF74-dependent mechanisms.