Fusion activity of transmembrane and cytoplasmic domain chimeras of the influenza virus glycoprotein hemagglutinin

J Virol. 1998 Jan;72(1):133-41. doi: 10.1128/JVI.72.1.133-141.1998.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Cell Line
  • DNA Primers / genetics
  • Erythrocytes / physiology
  • Fluorescent Dyes
  • Gene Expression
  • Hemagglutinin Glycoproteins, Influenza Virus / chemistry
  • Hemagglutinin Glycoproteins, Influenza Virus / genetics
  • Hemagglutinin Glycoproteins, Influenza Virus / physiology*
  • Humans
  • In Vitro Techniques
  • Kinetics
  • Lipid Bilayers / chemistry
  • Membrane Fusion / physiology*
  • Molecular Sequence Data
  • Orthomyxoviridae / genetics
  • Orthomyxoviridae / pathogenicity
  • Orthomyxoviridae / physiology
  • Polymerase Chain Reaction
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / physiology

Substances

  • DNA Primers
  • Fluorescent Dyes
  • Hemagglutinin Glycoproteins, Influenza Virus
  • Lipid Bilayers
  • Recombinant Fusion Proteins