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Use of a novel cell-based fusion reporter assay to explore the host range of human respiratory syncytial virus F protein.
Branigan PJ
,
Liu C
,
Day ND
,
Gutshall LL
,
Sarisky RT
,
Del Vecchio AM
.
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Human respiratory syncytial virus (HRSV) is an important respiratory pathogen primarily affecting infants, young children, transplant recipients and the elderly. The F protein is the only virion envelope protein necessary and sufficient for virus replication and fusion of the viral envelope membrane with the target host cell. During natural infection, HRSV replication is limited to respiratory epithelial cells with disseminated infection rarely, if ever, occurring even in immunocompromised patients. However, in vitro infection of multiple human and non-human cell types other than those of pulmonary tract origin has been reported. To better define host cell surface molecules that mediate viral entry and dissect the factors controlling permissivity for HRSV, we explored the host range of HRSV F protein mediated fusion. Using a novel recombinant reporter gene based fusion assay, HRSV F protein was shown to mediate fusion with cells derived from a wide range of vertebrate species including human, feline, equine, canine, bat, rodent, avian, porcine and even amphibian (Xenopus). That finding was extended using a recombinant HRSV engineered to express green fluorescent protein (GFP), to confirm that viral mRNA expression is limited in several cell types. These findings suggest that HRSV F protein interacts with either highly conserved host cell surface molecules or can use multiple mechanisms to enter cells, and that the primary determinants of HRSV host range are at steps post-entry.
Figure 1. A) Syncytia formation by RSV-F DNA in transfected cells. 293T cells were mock transfected or transfected with pHRSVFOptA2 and visualized by light microscopy 48 hours post transfection. The arrow indicates giant multinucleated cell formation. B) Processing of RSV F in transfected cells. 293T cells were either mock transfected (lane 1), transfected with pCMV-β-gal (lane 2), transfected with pHRSVFOptA2 (lane 3), pHRSVFOptB18537 (lane 4), or infected with RSV (Long strain, MOI = 1) for 24 hours followed by metabolic labeling for 6 hours with [35S]-cysteine/methionine. Labeled cell lysates were immunoprecipitated with HRSV F specific mAbs, and immunoprecipitates were resolved by SDS-PAGE as described in methods. C) Cell surface expression of RSV F in transfected cells. 293T cells were either mock transfected or transfected with pHRSVFOptA2 for 24 hours followed by flow cytometry using HRSV F specific monoclonal antibodies as described in methods.
Figure 2. A) Dose dependent fusion activity of HRSV F derived. 293T cells were transfected with either pFR-Luc alone (■), pBD-NFκB alone (▲), co-transfected with pFR-Luc and pBD-NFκB (▼), or co-transfected with pHRSVFOptA2 and pBD-NFκB and mixed 24 hours after transfection in various amounts with cells that had been transfected with pFR-Luc alone (◆). Luciferase activity was measured 24 hours post mixing as described in methods and is reported as relative light units. B) Fusion activity of HRSV F derived from subgroups A and B. 293T cells co-transfected with pBD-NFκB and either pHRSVFOptA2, pHRSVFOptB18537, or vector only (NFκB only). Cells were mixed 24 hours later with a separate population of 293T cells transfected with pFR-Luc, and luciferase activity was measured 24 hours post mixing as described in methods. Luciferase activity is reported as relative light units.
Figure 3. A) Comparison of fusion activity of wild-type and a fusion peptide mutant of HRSV F. 293T cells co-transfected with pBD-NFκB and either pHRSVFOptA2 or pL138R were mixed 24 hours later with a separate population of 293T cells that had been transfected with pFR-Luc, and luciferase activity was measured 24 hours post mixing as described in methods. Luciferase activity is reported as relative light units. B) Processing of the wild-type and L138R mutant of HRSV F was determined by metabolic labeling 293T cells transfected with either pHRSVFOptA2 (lane 1), pL138R (lane 2), pCMV-β-gal (lane 3), or mock transfected (lane 4) for 6 hours with [35S]-cysteine/methionine followed by immunoprecipitation of lysates with HRSV F specific mAbs, and analysis of immunoprecipitates by SDS-PAGE as described in methods. C) Cell surface expression of the wild-type and L138R mutant F proteins in 293T cells transfected with either pHRSVFOptA2 or pL138R was compared by flow cytometry as described in methods.
Figure 4. Fusion activity of HRSV F with cell lines derived from various species. Cell lines derived from various species (target cells) were either transfected with pFR-Luc and mixed 24 hours later with 293T cells that had been co-transfected for 24 hours with pHRSVFOptA2 or pVPack-VSV-G and pBD-NFκB (Figs. 4A and 4B), or the target cells were transfected with pBD-NFκB and mixed 24 hours later with 293T cells that had been co-transfected for 24 hours with pHRSVFOptA2 or pVPack-VSV-G together with pFR-Luc (Figs. 4C and 4D). Cell lines derived from various species were transfected with either pFR-Luc or pBD-NFκB only as negative controls. Luciferase activity was measured 24 hours post mixing of the cell populations as described in methods and is reported as relative light units.
Figure 5. Infection of various cell lines by rg224(RSV). Cell lines derived from various species were infected with rgRSV(224) at an MOI = 0.1 and GFP-expressing cells were visualized at 20, 48, and 120 hours post infection by fluorescent microscopy by monitoring fluorescence at 488 nm.
Barenfanger,
R-Mix cells are faster, at least as sensitive and marginally more costly than conventional cell lines for the detection of respiratory viruses.
2001, Pubmed
Barenfanger,
R-Mix cells are faster, at least as sensitive and marginally more costly than conventional cell lines for the detection of respiratory viruses.
2001,
Pubmed
Bolt,
Cleavage of the respiratory syncytial virus fusion protein is required for its surface expression: role of furin.
2000,
Pubmed
Bossart,
Membrane fusion tropism and heterotypic functional activities of the Nipah virus and Hendra virus envelope glycoproteins.
2002,
Pubmed
Bossert,
Respiratory syncytial virus (RSV) nonstructural (NS) proteins as host range determinants: a chimeric bovine RSV with NS genes from human RSV is attenuated in interferon-competent bovine cells.
2002,
Pubmed
Bourgeois,
Heparin-like structures on respiratory syncytial virus are involved in its infectivity in vitro.
1998,
Pubmed
Bukreyev,
Recombinant respiratory syncytial virus from which the entire SH gene has been deleted grows efficiently in cell culture and exhibits site-specific attenuation in the respiratory tract of the mouse.
1997,
Pubmed
Byrd,
Animal models of respiratory syncytial virus infection.
1997,
Pubmed
Chang,
Respiratory syncytial virus infection suppresses lung CD8+ T-cell effector activity and peripheral CD8+ T-cell memory in the respiratory tract.
2002,
Pubmed
Cirino,
Restricted replication of respiratory syncytial virus in human alveolar macrophages.
1993,
Pubmed
Collins,
Production of infectious human respiratory syncytial virus from cloned cDNA confirms an essential role for the transcription elongation factor from the 5' proximal open reading frame of the M2 mRNA in gene expression and provides a capability for vaccine development.
1995,
Pubmed
Collins,
Post-translational processing and oligomerization of the fusion glycoprotein of human respiratory syncytial virus.
1991,
Pubmed
Devincenzo,
Natural infection of infants with respiratory syncytial virus subgroups A and B: a study of frequency, disease severity, and viral load.
2004,
Pubmed
Dowell,
Respiratory syncytial virus is an important cause of community-acquired lower respiratory infection among hospitalized adults.
1996,
Pubmed
Falsey,
Respiratory syncytial virus infection in adults.
2000,
Pubmed
Feldman,
The fusion glycoprotein of human respiratory syncytial virus facilitates virus attachment and infectivity via an interaction with cellular heparan sulfate.
2000,
Pubmed
Ghildyal,
Surfactant protein A binds to the fusion glycoprotein of respiratory syncytial virus and neutralizes virion infectivity.
1999,
Pubmed
Glezen,
Risk of primary infection and reinfection with respiratory syncytial virus.
1986,
Pubmed
González-Reyes,
Cleavage of the human respiratory syncytial virus fusion protein at two distinct sites is required for activation of membrane fusion.
2001,
Pubmed
Hallak,
Iduronic acid-containing glycosaminoglycans on target cells are required for efficient respiratory syncytial virus infection.
2000,
Pubmed
Hallak,
Glycosaminoglycan sulfation requirements for respiratory syncytial virus infection.
2000,
Pubmed
Han,
Respiratory syncytial virus pneumonia among the elderly: an assessment of disease burden.
1999,
Pubmed
Haynes,
Involvement of toll-like receptor 4 in innate immunity to respiratory syncytial virus.
2001,
Pubmed
Heminway,
Analysis of respiratory syncytial virus F, G, and SH proteins in cell fusion.
1994,
Pubmed
Hickling,
A recombinant trimeric surfactant protein D carbohydrate recognition domain inhibits respiratory syncytial virus infection in vitro and in vivo.
1999,
Pubmed
Hierholzer,
Sensitivity of NCI-H292 human lung mucoepidermoid cells for respiratory and other human viruses.
1993,
Pubmed
Huang,
Mink lung cells and mixed mink lung and A549 cells for rapid detection of influenza virus and other respiratory viruses.
2000,
Pubmed
Ison,
Viral infections in immunocompromised patients: what's new with respiratory viruses?
2002,
Pubmed
Jin,
Recombinant respiratory syncytial viruses with deletions in the NS1, NS2, SH, and M2-2 genes are attenuated in vitro and in vivo.
2000,
Pubmed
Karron,
Respiratory syncytial virus (RSV) SH and G proteins are not essential for viral replication in vitro: clinical evaluation and molecular characterization of a cold-passaged, attenuated RSV subgroup B mutant.
1997,
Pubmed
Krusat,
Heparin-dependent attachment of respiratory syncytial virus (RSV) to host cells.
1997,
Pubmed
Kurt-Jones,
Pattern recognition receptors TLR4 and CD14 mediate response to respiratory syncytial virus.
2000,
Pubmed
Lawless-Delmedico,
Heptad-repeat regions of respiratory syncytial virus F1 protein form a six-membered coiled-coil complex.
2000,
Pubmed
Martínez,
Binding of human respiratory syncytial virus to cells: implication of sulfated cell surface proteoglycans.
2000,
Pubmed
Matthews,
The core of the respiratory syncytial virus fusion protein is a trimeric coiled coil.
2000,
Pubmed
Morton,
Structural characterization of respiratory syncytial virus fusion inhibitor escape mutants: homology model of the F protein and a syncytium formation assay.
2003,
Pubmed
Parry,
Pneumoviruses: the cell surface of lytically and persistently infected cells.
1979,
Pubmed
Ray,
Immunoregulatory role of secreted glycoprotein G from respiratory syncytial virus.
2001,
Pubmed
Rixon,
Multiple glycosylated forms of the respiratory syncytial virus fusion protein are expressed in virus-infected cells.
2002,
Pubmed
Sarmiento,
Characteristics of a respiratory syncytial virus persistently infected macrophage-like culture.
2002,
Pubmed
Schlender,
Respiratory syncytial virus (RSV) fusion protein subunit F2, not attachment protein G, determines the specificity of RSV infection.
2003,
Pubmed
Shay,
Bronchiolitis-associated hospitalizations among US children, 1980-1996.
1999,
Pubmed
Shay,
Bronchiolitis-associated mortality and estimates of respiratory syncytial virus-associated deaths among US children, 1979-1997.
2001,
Pubmed
Spann,
Suppression of the induction of alpha, beta, and lambda interferons by the NS1 and NS2 proteins of human respiratory syncytial virus in human epithelial cells and macrophages [corrected].
2004,
Pubmed
Sugrue,
Furin cleavage of the respiratory syncytial virus fusion protein is not a requirement for its transport to the surface of virus-infected cells.
2001,
Pubmed
Techaarpornkul,
Functional analysis of recombinant respiratory syncytial virus deletion mutants lacking the small hydrophobic and/or attachment glycoprotein gene.
2001,
Pubmed
Techaarpornkul,
Respiratory syncytial virus with the fusion protein as its only viral glycoprotein is less dependent on cellular glycosaminoglycans for attachment than complete virus.
2002,
Pubmed
Teng,
Identification of the respiratory syncytial virus proteins required for formation and passage of helper-dependent infectious particles.
1998,
Pubmed
Teng,
Contribution of the respiratory syncytial virus G glycoprotein and its secreted and membrane-bound forms to virus replication in vitro and in vivo.
2001,
Pubmed
Tripp,
Respiratory syncytial virus G and/or SH glycoproteins modify CC and CXC chemokine mRNA expression in the BALB/c mouse.
2000,
Pubmed
Viuff,
Sites of replication of bovine respiratory syncytial virus in naturally infected calves as determined by in situ hybridization.
1996,
Pubmed
Whitehead,
Recombinant respiratory syncytial virus bearing a deletion of either the NS2 or SH gene is attenuated in chimpanzees.
1999,
Pubmed
Wright,
Temperature-sensitive mutants of respiratory syncytial virus: in-vivo studies in hamsters.
1970,
Pubmed
Yui,
Detection of human respiratory syncytial virus sequences in peripheral blood mononuclear cells.
2003,
Pubmed
Zhang,
Respiratory syncytial virus infection of human airway epithelial cells is polarized, specific to ciliated cells, and without obvious cytopathology.
2002,
Pubmed
Zhao,
Structural characterization of the human respiratory syncytial virus fusion protein core.
2000,
Pubmed
