Enlargement of Influenza Virus Hemagglutinin Cytoplasmic Tail by Tagging with an Enhanced Green Fluorescent Protein Interferes with Hemagglutinin-mediated Membrane Fusion Prior to the Lipid-mixing Step

Abstract

The cytoplasmic tail (CT) of the influenza virus hemagglutinin (HA) consists of an amino acid sequence that is highly conserved among various subtypes of the influenza A virus. To study the effect of CT size on HA-mediated membrane fusion, we constructed a cDNA encoding a chimeric protein in which the size of CT of influenza virus HA (from A/Hong Kong/1/68; subtype H3) was enlarged by tagging with enhanced green fluorescent protein (EGFP). The EGFP-tagged HA (HA-EGFP) or wild-type HA was individually expressed in HeLa cells, and cell membrane fusion activities were examined using dual-labeled chicken red blood cells. The red blood cells were labeled with both lipidic and aqueous dyes to verify the fusion activity of the HA-EGFP at different steps of the fusion process: 1) lipid-mixing, 2) content-mixing and 3) fusion pore formation. Our data showed that the binding activity of HA towards red blood cells was not affected by enlargement of the CT of HA with EGFP. However, its lipid-mixing, subsequent content-mixing, and fusion pore formation activities were drastically impaired by the enlargement. Therefore, we conclude that CT size is crucial for progression from the binding step to the lipid-mixing step in the process of HA-mediated membrane fusion.