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J. Cell Biol.,
Volume 143, Number 5, November 30, 1998 1155-1166
Laboratory of Cellular and Molecular Biophysics, National Institute of Child Health and Human Development, National
Institutes of Health, Bethesda, Maryland 20892-1855
The baculovirus fusogenic activity depends
on the low pH conformation of virally-encoded trimeric
glycoprotein, gp64. We used two experimental approaches to investigate whether monomers, trimers, and/or higher order oligomers are functionally involved
in gp64 fusion machine. First, dithiothreitol (DTT)-
based reduction of intersubunit disulfides was found to
reversibly inhibit fusion, as assayed by fluorescent
probe redistribution between gp64-expressing and target cells (i.e., erythrocytes or Sf9 cells). This inhibition correlates with disappearance of gp64 trimers and appearance of dimers and monomers in SDS-PAGE.
Thus, stable (i.e., with intact intersubunit disulfides)
gp64 trimers, rather than independent monomers, drive
fusion. Second, we established that merger of membranes is preceded by formation of large (greater than 2 MDa), short-lived gp64 complexes. These complexes
were stabilized by cell-surface cross-linking and characterized by glycerol density gradient ultracentrifugation. The basic structural unit of the complexes is stable
gp64 trimer. Although DTT-destabilized trimers were
still capable of assuming the low pH conformation, they
failed to form multimeric complexes. The fact that formation of these complexes correlated with fusion in
timing, and was dependent on (a) low pH application,
(b) stable gp64 trimers, and (c) cell-cell contacts, suggests that such multimeric complexes represent a fusion
machine.
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