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

Abstract

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 (vCXCL1GT1) is a known agonist for CXCR1 and CXCR2, while two others (vCXCL1GT5 and vCXCL1GT6) lack the ELR motif considered crucial for CXCR1 and CXCR2 binding, thus suggesting another receptor targeting profile. To determine the receptor target for vCXCL1GT5, 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, vCXCL1GT5 recruited β-arrestin in a BRET-based assay and induced migration in a chemotaxis assay through CXCR2, but not CXCR1. In contrast, vCXCL1GT1 stimulated G protein signaling, recruited β-arrestin and induced migration through both CXCR1 and CXCR2. Both vCXCL1GT1 and vCXCL1GT5 induced equally potent and efficacious migration of neutrophils, and ELR vCXCL1GT4 and non-ELR vCXCL1GT6 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 vCXCL1GT1, vCXCL1GT4, vCXCL1GT5, and vCXCL1GT6) forms a novel β-hairpin not found in human chemokines. Secondary NMR shift and TALOS+ analysis of vCXCL1GT1 supported the existence of two stable β-strands. C-terminal deletion of vCXCL1GT1 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.

Identification of a Salt Bridge That Is Functionally Important for Chemokine Receptor CXCR1 but not CXCR2

“…As all the human chemokine ligands for CXCR1/2 (CXCL1/2/3/5/6/7/8) contain a Glu-Leu-Arg (ELR) motif in the N-terminus, it has long been believed that this motif was a structural requirement for activation of CXCR1 and CXCR2; however, Berg et al have recently shown that the ELR-motif is not strictly required for activation of CXCR2. 27 Liu et al have recently proposed a structural explanation for the CXCR1/2 selectivity of the chemokine ligands, attributing it to the complementarity between the CXCR N-terminus and CXCL N-loop, i.e., CRS1 interactions. 20 The present study focuses on positions 3.32 and 7.39 that are part of CRS2, which is the receptor region typically targeted by small-molecule ligands.…”

Changes in chemokine and growth factor levels may be useful biomarkers for monitoring disease severity in COVID-19 patients; a pilot study

“…Both chemokines belong to the CX subfamily and the non-ELR subgroup ( 36 , 37 ). Non-ELR chemokines lack the ELR (Glu-Leu-Arg) motif, and they act as chemoattractants and activators of monocytes, dendritic cells, lymphocytes (T, B, and NK cells), basophils and eosinophils, without angiogenic potential ( 38 , 39 ). IP-10 and MIG bind to chemokine receptor 3 (CXCR3), and they play a particularly important role in the Th14 immune response by recruiting leukocytes to the site of inflammation ( 40 ).…”

Therapeutic targeting of HCMV-encoded chemokine receptor US28: Progress and challenges

“…HCMV has a surprisingly diverse genome for a DNA virus displaying a high degree of sequence variability across many different genes including major immune modulators ( 67 ). For example, the chemokine-encoding UL146 gene is subject to extensive inter-strain diversity ( 68 ) that leads to structural and functional changes of the chemokine ( 69 , 70 ). The US28 gene in contrast is quite conserved, strengthening its position as a therapeutic target ( 67 ).…”