title: The cytoplasmic tail of the human respiratory syncytial virus F protein plays critical roles in cellular localization of the F protein and infectious progeny production.
abstract: The importance of the F protein cytoplasmic tail (CT) for replication of human respiratory syncytial virus (HRSV) was examined by monitoring the behavior of viruses expressing F proteins with a modified COOH terminus. The F protein mutant viruses were recovered and amplified under conditions where F protein function was complemented by expression of a heterologous viral envelope protein. The effect of the F protein modifications was then examined in the context of a viral infection in standard cell types (Vero and HEp-2). The F protein modifications consisted of a deletion of the predicted CT or a replacement of the CT with the CT of the vesicular stomatitis virus (VSV) G protein. In addition, engineered HRSVs that lacked all homologous glycoprotein genes (SH, G, and F) and expressed instead either the authentic VSV G protein or a VSV G containing the HRSV F protein CT were examined. We found that deletion or replacement of the F protein CT seriously impaired the production of infectious progeny. Cells infected with viruses bearing CT modifications displayed increased F protein surface expression and increased syncytium formation. The distribution of F protein in the plasma membrane of infected cells was altered, resulting in an F protein that was evenly distributed rather than localized predominantly to virus-induced surface filaments. CT deletion or exchange also abrogated interaction of F protein with Triton-insoluble lipid rafts. Addition of the F protein CT to the VSV G protein, expressed as the only viral glycoprotein in an HRSV genome, had the opposite effects: the number of infectious progeny was higher, the surface distribution was changed from relatively even to localized, and the proportion of VSV G protein associated with lipid rafts was higher. Together, these results show that the HRSV F protein CT plays a critical role in F protein cellular localization and production of infectious virus and suggest that the function provided by the CT is independent of the F protein ectodomain and transmembrane domain and is mediated by F protein-lipid raft interaction.
authors.affiliation: Department of Pathology, University of Virginia, MR5 Building, P.O. Box 800904, Charlottesville, VA 22908-0904, USA.
authors.forename: Antonius G P
authors.initials: AG
authors.lastname: Oomens
authors.forename: Kevin P
authors.initials: KP
authors.lastname: Bevis
authors.forename: Gail W
authors.initials: GW
authors.lastname: Wertz
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D018076:DNA, Complementary / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D006801:Humans; D008561:Membrane Fusion; D021962:Membrane Microdomains / Q000378:metabolism / Q000821:virology; D008969:Molecular Sequence Data; D016296:Mutagenesis; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018113:Respiratory Syncytial Virus, Human / Q000235:genetics / Q000472:pathogenicity / Q000502:physiology*; D014709:Vero Cells; D014760:Viral Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D014779:Virus Replication
chemical_list: D018076:DNA, Complementary; D004279:DNA, Viral; D011994:Recombinant Proteins; D014760:Viral Fusion Proteins