Membrane fusion mediated by baculovirus gp64 involves assembly of stable gp64 trimers into multiprotein aggregates.
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.
Markovic
I
I
Laboratory of Cellular and Molecular Biophysics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-1855, USA.
Pulyaeva
H
H
Sokoloff
A
A
Chernomordik
L V
LV
D000818:Animals; D016367:Baculoviridae / Q000472:pathogenicity* / Q000502:physiology*; D002459:Cell Fusion / Q000502:physiology; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry; D003432:Cross-Linking Reagents; D004220:Disulfides / Q000737:chemistry; D004229:Dithiothreitol; D004912:Erythrocytes / Q000502:physiology; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008561:Membrane Fusion / Q000502:physiology*; D008958:Models, Molecular; D008970:Molecular Weight; D011487:Protein Conformation; D017510:Protein Folding; D018411:Spodoptera; D014760:Viral Fusion Proteins / Q000737:chemistry* / Q000502:physiology*
D003432:Cross-Linking Reagents; D004220:Disulfides; D046911:Macromolecular Substances; D014760:Viral Fusion Proteins; D004229:Dithiothreitol