CX3CR1 Is Expressed in Differentiated Human Ciliated Airway Cells and Co-Localizes with Respiratory Syncytial Virus on Cilia in a G Protein-Dependent Manner.

Kwang-Il Jeong,Joshua M Dinapoli,Michael Kishko,Linong Zhang,Mark Parrington,Rachel P Groppo,Simon Delagrave,Harry Kleanthous,Peter A Piepenhagen,Jeffrey Almond,John S Tregoning

doi:10.1371/journal.pone.0130517

Abstract

Respiratory syncytial virus (RSV) is the principal cause of bronchiolitis in infants and a significant healthcare problem. The RSV Glycoprotein (G) mediates attachment of the virus to the cell membrane, which facilitates interaction of the RSV Fusion (F) protein with nucleolin, thereby triggering fusion of the viral and cellular membranes. However, a host protein ligand for G has not yet been identified. Here we show that CX3CR1 is expressed in the motile cilia of differentiated human airway epithelial (HAE) cells, and that CX3CR1 co-localizes with RSV particles. Upon infection, the distribution of CX3CR1 in these cells is significantly altered. Complete or partial deletion of RSV G results in viruses binding at least 72-fold less efficiently to cells, and reduces virus replication. Moreover, an antibody targeting an epitope near the G protein’s CX3CR1-binding motif significantly inhibits binding of the virus to airway cells. Given previously published evidence of the interaction of G with CX3CR1 in human lymphocytes, these findings suggest a role for G in the interaction of RSV with ciliated lung cells. This interpretation is consistent with past studies showing a protective benefit in immunizing against G in animal models of RSV infection, and would support targeting the CX3CR1-G protein interaction for prophylaxis or therapy. CX3CR1 expression in lung epithelial cells may also have implications for other respiratory diseases such as asthma.

Highlights

Respiratory syncytial virus (RSV) is responsible for more than 500,000 emergency room visits and over 50,000 hospitalizations annually in the U.S alone [1,2,3]
human airway epithelial (HAE) cells are reported not to express heparan sulfate proteoglycans (HSPG) [11] and while it has been hypothesized that G protein mediates attachment of RSV through its interaction with the host fractalkine receptor CX3CR1 [12,13,14], data to support this view were not based on observations in differentiated lung cells
We use differentiated HAE cell cultures, immunofluorescence, confocal microscopy, and molecular virology to show that CX3CR1 is expressed in ciliated cells targeted by RSV and that an antibody to G protein, or deletion of G, inhibits virus binding to HAE cells and reduces viral replication significantly

Summary

Introduction

RSV is responsible for more than 500,000 emergency room visits and over 50,000 hospitalizations annually in the U.S alone [1,2,3]. HAE cells are reported not to express HSPG [11] and while it has been hypothesized that G protein mediates attachment of RSV through its interaction with the host fractalkine receptor CX3CR1 [12,13,14], data to support this view were not based on observations in differentiated lung cells. We use differentiated HAE cell cultures, immunofluorescence, confocal microscopy, and molecular virology to show that CX3CR1 is expressed in ciliated cells targeted by RSV and that an antibody to G protein, or deletion of G, inhibits virus binding to HAE cells and reduces viral replication significantly.

Results

Conclusion