Fusion of influenza virus with cells is triggered by a pH-dependent conformational change in the
viral envelope protein,
hemagglutinin, which results in exposure of the fusion
peptide and its insertion into the target membrane. We have investigated the association of
hemagglutinin with erythrocyte membranes by photosensitized labeling with [125I]
iodonaphthylazide. This technique relies on the collisional energy transfer from a photosensitizing chromophore to [125I]
iodonaphthylazide, which selectively labels
proteins in the vicinity of the chromophore. Incubation of influenza virus with erythrocyte membranes containing chromophore and [125I]
iodonaphthylazide results in labeling of
hemagglutinin under fusogenic conditions (pH 5 and 37 degrees C). We also examined photosensitized labeling of
hemagglutinin upon incubation of the X31 strain of influenza virus with labeled erythrocyte membranes in a pre-fusion state (pH 5 and 4 degrees C). There was little
hemagglutinin labeling under these conditions, although incubation of
bromelain-cleaved
hemagglutinin, which lacks the transmembrane region, resulted in rapid labeling.
Hemagglutinin was also labeled by [125I]
iodonaphthylazide photosensitized by a fluorescent substrate transported through the erythrocyte band 3
sialoglycoprotein.
Hemagglutinin labeling decreased after an initial rapid rise, suggesting that the fusion site is close to the
sialoglycoprotein and that [125I]
iodonaphthylazide photosensitized labeling may be used to assay
protein movement during fusion.