Fusion activity of transmembrane and cytoplasmic domain chimeras of the influenza virus glycoprotein hemagglutinin.
The role of the sequence of transmembrane and cytoplasmic/intraviral domains of influenza virus hemagglutinin (HA, subtype H7) for HA-mediated membrane fusion was explored. To analyze the influence of the two domains on the fusogenic properties of HA, we designed HA-chimeras in which the cytoplasmic tail and/or transmembrane domain of HA was replaced with the corresponding domains of the fusogenic glycoprotein F of Sendai virus. These chimeras, as well as constructs of HA in which the cytoplasmic tail was replaced by peptides of human neurofibromin type 1 (NF1) or c-Raf-1, NF78 (residues 1441 to 1518), and Raf81 (residues 51 to 131), respectively, were expressed in CV-1 cells by using the vaccinia virus-T7 polymerase transient-expression system. Wild-type and chimeric HA were cleaved properly into two subunits and expressed as trimers. Membrane fusion between CV-1 cells and bound human erythrocytes (RBCs) mediated by parental or chimeric HA proteins was studied by a lipid-mixing assay with the lipid-like fluorophore octadecyl rhodamine B chloride (R18). No profound differences in either extent or kinetics could be observed. After the pH was lowered, the above proteins also induced a flow of the aqueous fluorophore calcein from preloaded RBCs into the cytoplasm of the protein-expressing CV-1 cells, indicating that membrane fusion involves both leaflets of the lipid bilayers and leads to formation of an aqueous fusion pore. We conclude that neither HA-specific sequences in the transmembrane and cytoplasmic domains nor their length is crucial for HA-induced membrane fusion activity.
Schroth-Diez
B
B
Institut für Biologie/Biophysik, Mathematisch-Naturwissenschaftliche Fakultät I, Humboldt-Universität zu Berlin, Germany.
Ponimaskin
E
E
Reverey
H
H
Schmidt
M F
MF
Herrmann
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D004912:Erythrocytes / Q000502:physiology; D005456:Fluorescent Dyes; D015870:Gene Expression; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry; D008561:Membrane Fusion / Q000502:physiology*; D008969:Molecular Sequence Data; D009975:Orthomyxoviridae / Q000235:genetics / Q000472:pathogenicity / Q000502:physiology; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology
D017931:DNA Primers; D005456:Fluorescent Dyes; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D008051:Lipid Bilayers; D011993:Recombinant Fusion Proteins