title: Modification of the cytoplasmic domain of influenza virus hemagglutinin affects enlargement of the fusion pore.
abstract: The fusion activity of chimeras of influenza virus hemagglutinin (HA) (from A/fpv/Rostock/34; subtype H7) with the transmembrane domain (TM) and/or cytoplasmic tail (CT) either from the nonviral, nonfusogenic T-cell surface protein CD4 or from the fusogenic Sendai virus F-protein was studied. Wild-type or chimeric HA was expressed in CV-1 cells by the transient T7-RNA-polymerase vaccinia virus expression system. Subsequently, the fusion activity of the expression products was monitored with red blood cells or ghosts as target cells. To assess the different steps of fusion, target cells were labeled with the fluorescent membrane label octadecyl rhodamine B-chloride (R18) (membrane fusion) and with the cytoplasmic fluorophores calcein (molecular weight [MW], 623; formation of small aqueous fusion pore) and tetramethylrhodamine-dextran (MW, 10,000; enlargement of fusion pore). All chimeric HA/F-proteins, as well as the chimera with the TM of CD4 and the CT of HA, were able to mediate the different steps of fusion very similarly to wild-type HA. Quite differently, chimeric proteins with the CT of CD4 were strongly impaired in mediating pore enlargement. However, membrane fusion and formation of small pores were similar to those of wild-type HA, indicating that the conformational change of the ectodomain and earlier fusion steps were not inhibited. Various properties of the CT which may affect pore enlargement are considered. We surmise that the hydrophobicity of the sequence adjacent to the transmembrane domain is important for pore dilation.
authors.affiliation: Institut für Biologie/Biophysik, Mathematisch-Naturwissenschaftliche Fakultät I, Humboldt-Universität zu Berlin, D-10115 Berlin, Germany.
authors.forename: C
authors.initials: C
authors.lastname: Kozerski
authors.forename: E
authors.initials: E
authors.lastname: Ponimaskin
authors.forename: B
authors.initials: B
authors.lastname: Schroth-Diez
authors.forename: M F
authors.initials: MF
authors.lastname: Schmidt
authors.forename: A
authors.initials: A
authors.lastname: Herrmann
mesh_terms: D000818:Animals; D015704:CD4 Antigens / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology*; D004910:Erythrocyte Membrane / Q000378:metabolism; D004912:Erythrocytes / Q000378:metabolism; D005452:Fluoresceins / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009980:Influenza A virus / Q000378:metabolism*; D008561:Membrane Fusion / Q000502:physiology*; D011498:Protein Precursors / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014616:Vaccinia virus / Q000502:physiology; D014759:Viral Envelope Proteins / Q000378:metabolism
chemical_list: D015704:CD4 Antigens; C099057:F0 protein, Sendai virus; D005452:Fluoresceins; D005456:Fluorescent Dyes; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; C007740:fluorexon