CX3CR1 is an important surface molecule for respiratory syncytial virus infection in human airway epithelial cells

Abstract

Respiratory syncytial virus (RSV) is a major cause of severe pneumonia and bronchiolitis in infants and young children, and causes disease throughout life. Understanding the biology of infection, including virus binding to the cell surface, should help develop antiviral drugs or vaccines. The RSV F and G glycoproteins bind cell surface heparin sulfate proteoglycans (HSPGs) through heparin-binding domains. The G protein also has a CX3C chemokine motif which binds to the fractalkine receptor CX3CR1. G protein binding to CX3CR1 is not important for infection of immortalized cell lines, but reportedly is so for primary human airway epithelial cells (HAECs), the primary site for human infection. We studied the role of CX3CR1 in RSV infection with CX3CR1-transfected cell lines and HAECs with variable percentages of CX3CR1-expressing cells, and the effect of anti-CX3CR1 antibodies or a mutation in the RSV CX3C motif. Immortalized cells lacking HSPGs had low RSV binding and infection, which was increased markedly by CX3CR1 transfection. CX3CR1 was expressed primarily on ciliated cells, and ,50 % of RSV-infected cells in HAECs were CX3CR1 + . HAECs with more CX3CR1-expressing cells had a proportional increase in RSV infection. Blocking G binding to CX3CR1 with anti-CX3CR1 antibody or a mutation in the CX3C motif significantly decreased RSV infection in HAECs. The kinetics of cytokine production suggested that the RSV/CX3CR1 interaction induced RANTES (regulated on activation normal T-cell expressed and secreted protein), IL-8 and fractalkine production, whilst it downregulated IL-15, IL1-RA and monocyte chemotactic protein-1. Thus, the RSV G protein/CX3CR1 interaction is likely important in infection and infection-induced responses of the airway epithelium, the primary site of human infection.

Broadly Reactive Anti-Respiratory Syncytial Virus G Antibodies from Exposed Individuals Effectively Inhibit Infection of Primary Airway Epithelial Cells

“…Tripp and colleagues (11,12) have shown that the G protein can influence immune signaling by interaction with the fractalkine receptor (CX3CR1), a receptor present on leukocytes (13), and that blocking this interaction abrogates inflammation and viral replication in mice. Recent reports support the hypothesis that CX3CR1 is a cellular receptor for RSV in primary human epithelial cell cultures (14)(15)(16).…”

“…In addition, it was shown that genetic ablation of the G protein reduces RSV infectivity in mice and cotton rats (7,8), indicating that viral entry of airway epithelial cells might be hampered without a functional G protein. The G protein expresses a CX3C motif which can bind to the CX3CR1 receptor and, hence, can facilitate viral entry in vivo in airway epithelial cells and in vitro in HAE cell cultures (14)(15)(16). Inhibition of this interaction by G-specific antibodies may thus lead to reduced viral infection in HAE cell cultures.…”

Mutating the CX3C Motif in the G Protein Should Make a Live Respiratory Syncytial Virus Vaccine Safer and More Effective

“…Since G binding to CX3CR1 through the CX3C motif in G is important to RSV disease, mutations to the motif that prevent G binding to CX3CR1 might prevent disease. The finding that CX3CR1 is an important receptor for primary human airway epithelial cells (hAECs), though not for cell lines usually used to study RSV (24,25,27), suggests the CX3C motif is more important to human RSV disease than studies to date suggest. Consequently, we chose to investigate the potential benefit of mutating the wild-type (wt) CX3C motif in G to CX4C, which does not bind to CX3CR1 (24).…”

“…Importantly, the G protein contains a CX3C chemokine motif in its central conserved region, and through this motif, RSV binds to the CX3C receptor, CX3CR1 (23)(24)(25). We have previously shown that G binding to CX3CR1 induces leukocyte migration in vitro similar to the one CX3C chemokine, fractalkine (23), and, in mouse studies, explains G-associated altered migration of T cells to RSV-infected lungs (26), depressed respiratory rates (22), and FI-RSV vaccine-induced ERD (20).…”

Respiratory syncytial virus infection of newborn CX3CR1-deficent mice induces a pathogenic pulmonary innate immune response

“…The CX3C motif is present in the chemokine CX3CL1 (also known as fractalkine), which is the ligand of CX3CR1 (16). CX3CR1 expression on human AECs was shown to facilitate virus entry (17)(18)(19). However, deletion or knockdown of CX3CR1 expression in human AECs or in mice only partially reduces infection (19).…”