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.

Christian Berg,Lasse H Hansen,Brian F Volkman,Hans R Lüttichau,Francis C Peterson,Motiejus Melynis,Roman R Schlimgen,Michael J Wedemeyer,Mette M Rosenkilde,Jon Våbenø,Robert F Kalejta

doi:10.1371/journal.ppat.1010355

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.

Highlights

Human cytomegalovirus (HCMV) is a highly prevalent human herpesvirus that causes severe disease in immunocompromised patients
A common feature for herpesviruses is that a large part of their immunomodulatory genes are somehow connected to the chemokine system, as these viruses either encode chemokine receptors or chemokine ligands used to manipulate the host immune system, or they modulate the expression of host-encoded chemokine receptors [2,3]
We have previously demonstrated that UL146 genotype 1 induces chemotaxis through its agonistic activity on G protein signaling of the neutrophil chemokine receptors CXCR1 and CXCR2 [9]

Summary

Introduction

Human cytomegalovirus (HCMV) is a highly prevalent human herpesvirus that causes severe disease in immunocompromised patients. Both primary infection and reactivation of latent infection is a significant concern among HIV/AIDS patients, organ transplant recipients, in chemotherapy, and during pregnancy due to the risk of mother-to-child transmission, as it can lead to disability and death [1]. The 235 kb genome of HCMV encodes a large number of immune modulators that are used for immune evasion, viral dissemination, and latency. Previous studies have observed a high degree of genetic diversity among different clinical HCMV isolates, including disruptive mutations to many immunomodulatory genes [4,5]. One of the most diverse genes, UL146, encodes the chemokine vCXCL1 [6] of which 14 distinct genotypes have been identified from clinical isolates [7,8]

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Conclusion