The non-ELR CXC chemokine encoded by human cytomegalovirus UL146 genotype 5 contains a C-terminal β-hairpin and induces neutrophil migration as a selective CXCR2 agonist

Human cytomegalovirus (HCMV) is a major pathogen in immunocompromised patients. The UL146 gene exists as 14 diverse genotypes among clinical isolates, which encode 14 different CXC chemokines. One genotype (vCXCL1(GT1)) is a known agonist for CXCR1 and CXCR2, while two others (vCXCL1(GT5) and vCXCL1(GT6)) lack the ELR motif considered crucial for CXCR1 and CXCR2 binding, thus suggesting another receptor targeting profile. To determine the receptor target for vCXCL1(GT5), the chemokine was probed in a G protein signaling assay on all 18 classical human chemokine receptors, where CXCR2 was the only receptor being activated. In addition, vCXCL1(GT5) recruited β-arrestin in a BRET-based assay and induced migration in a chemotaxis assay through CXCR2, but not CXCR1. In contrast, vCXCL1(GT1) stimulated G protein signaling, recruited β-arrestin and induced migration through both CXCR1 and CXCR2. Both vCXCL1(GT1) and vCXCL1(GT5) induced equally potent and efficacious migration of neutrophils, and ELR vCXCL1(GT4) and non-ELR vCXCL1(GT6) activated only CXCR2. In contrast to most human chemokines, the 14 UL146 genotypes have remarkably long C-termini. Comparative modeling using Rosetta showed that each genotype could adopt the classic chemokine core structure, and predicted that the extended C-terminal tail of several genotypes (including vCXCL1(GT1), vCXCL1(GT4), vCXCL1(GT5), and vCXCL1(GT6)) forms a novel β-hairpin not found in human chemokines. Secondary NMR shift and TALOS+ analysis of vCXCL1(GT1) supported the existence of two stable β-strands. C-terminal deletion of vCXCL1(GT1) resulted in a non-functional protein and in a shift to solvent exposure for tryptophan residues likely due to destabilization of the chemokine fold. The results demonstrate that non-ELR chemokines can activate CXCR2 and suggest that the UL146 chemokines have unique C-terminal structures that stabilize the chemokine fold. Increased knowledge of the structure and interaction partners of the chemokine variants encoded by UL146 is key to understanding why circulating HCMV strains sustain 14 stable genotypes.