title: Respiratory syncytial virus fusion protein-induced toll-like receptor 4 (TLR4) signaling is inhibited by the TLR4 antagonists Rhodobacter sphaeroides lipopolysaccharide and eritoran (E5564) and requires direct interaction with MD-2.
abstract: Respiratory syncytial virus (RSV) is a leading cause of infant mortality worldwide. Toll-like receptor 4 (TLR4), a signaling receptor for structurally diverse microbe-associated molecular patterns, is activated by the RSV fusion (F) protein and by bacterial lipopolysaccharide (LPS) in a CD14-dependent manner. TLR4 signaling by LPS also requires the presence of an additional protein, MD-2. Thus, it is possible that F protein-mediated TLR4 activation relies on MD-2 as well, although this hypothesis has not been formally tested. LPS-free RSV F protein was found to activate NF-κB in HEK293T transfectants that express wild-type (WT) TLR4 and CD14, but only when MD-2 was coexpressed. These findings were confirmed by measuring F-protein-induced interleukin 1β (IL-1β) mRNA in WT versus MD-2(-/-) macrophages, where MD-2(-/-) macrophages failed to show IL-1β expression upon F-protein treatment, in contrast to the WT. Both Rhodobacter sphaeroides LPS and synthetic E5564 (eritoran), LPS antagonists that inhibit TLR4 signaling by binding a hydrophobic pocket in MD-2, significantly reduced RSV F-protein-mediated TLR4 activity in HEK293T-TLR4-CD14-MD-2 transfectants in a dose-dependent manner, while TLR4-independent NF-κB activation by tumor necrosis factor alpha (TNF-α) was unaffected. In vitro coimmunoprecipitation studies confirmed a physical interaction between native RSV F protein and MD-2. Further, we demonstrated that the N-terminal domain of the F1 segment of RSV F protein interacts with MD-2. These data provide new insights into the importance of MD-2 in RSV F-protein-mediated TLR4 activation. Thus, targeting the interaction between MD-2 and RSV F protein may potentially lead to novel therapeutic approaches to help control RSV-induced inflammation and pathology.
  
  
  OBJECTIVE
  This study shows for the first time that the fusion (F) protein of respiratory syncytial virus (RSV), a major cause of bronchiolitis and death, particularly in infants and young children, physically interacts with the Toll-like receptor 4 (TLR4) coreceptor, MD-2, through its N-terminal domain. We show that F protein-induced TLR4 activation can be blocked by lipid A analog antagonists. This observation provides a strong experimental rationale for testing such antagonists in animal models of RSV infection for potential use in people.
authors.affiliation: Department of Microbiology and Immunology, University of Maryland, Baltimore, Maryland, USA.
authors.forename: Prasad
authors.initials: P
authors.lastname: Rallabhandi
authors.forename: Rachel L
authors.initials: RL
authors.lastname: Phillips
authors.forename: Marina S
authors.initials: MS
authors.lastname: Boukhvalova
authors.forename: Lioubov M
authors.initials: LM
authors.lastname: Pletneva
authors.forename: Kari Ann
authors.initials: KA
authors.lastname: Shirey
authors.forename: Theresa L
authors.initials: TL
authors.lastname: Gioannini
authors.forename: Jerrold P
authors.initials: JP
authors.lastname: Weiss
authors.forename: Jesse C
authors.initials: JC
authors.lastname: Chow
authors.forename: Lynn D
authors.initials: LD
authors.lastname: Hawkins
authors.forename: Stefanie N
authors.initials: SN
authors.lastname: Vogel
authors.forename: Jorge C G
authors.initials: JC
authors.lastname: Blanco
mesh_terms: D000818:Animals; D002460:Cell Line; D015536:Down-Regulation; D006801:Humans; D008050:Lipid A / Q000031:analogs & derivatives* / Q000378:metabolism; D008070:Lipopolysaccharides / Q000378:metabolism; D051218:Lymphocyte Antigen 96 / Q000235:genetics / Q000378:metabolism*; D017737:Macrophages, Peritoneal / Q000276:immunology / Q000382:microbiology; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D011485:Protein Binding; D018357:Respiratory Syncytial Virus Infections / Q000235:genetics / Q000276:immunology / Q000378:metabolism* / Q000382:microbiology; D012136:Respiratory Syncytial Viruses / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D015398:Signal Transduction; D051197:Toll-Like Receptor 4 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D014760:Viral Fusion Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: C404742:E5564; C495377:LY96 protein, human; D008050:Lipid A; D008070:Lipopolysaccharides; D051218:Lymphocyte Antigen 96; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D014760:Viral Fusion Proteins