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© The Rockefeller University Press, 0021-9525/1998//1155 $5.00
The Journal of Cell Biology, Volume 143, Number 5, , 1998 1155-1166

Article

Membrane Fusion Mediated by Baculovirus gp64 Involves Assembly of Stable gp64 Trimers into Multiprotein Aggregates



Ingrid Markovic, Helena Pulyaeva, Alexander Sokoloff, and Leonid V. Chernomordik

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.

Key Words: viral fusion • baculovirus gp64 • fusion protein assembly • fusion inhibitors • thiol/disulfide exchange

Abbreviations used in this paper: Ac, Autographa californica strain of baculovirus; CELISA, cell surface enzyme-linked immunoabsorbent assay; CF, 6-carboxyfluorescein; DTSSP, DTT-cleavable N-hydroxysuccinimide ester; ECL, enhanced chemiluminescence; ECF, enhanced chemifluorescence; gp64 HC, gp64-containing heavy complexes; GSSG, glutathione disulfide; HA, influenza virus hemagglutinin; LPC, lysophosphatidylcholine; m.o.i., multiplicity of infection; MW, molecular weight; NaIA, sodium iodoacetate; Op, Orgyia pseudotsugata strain of baculovirus; RBC, red blood cells.

We greatly acknowledge E. Leikina (National Institute of Child Health and Human Development [NICHD], National Institutes of Health [NIH], Bethesda, MD) for valuable help and insightful discussions. We are grateful to J. Zimmerberg (NICHD, NIH) for continuous and enthusiastic support of the present work and creative discussions. We appreciate G.W. Blissard (Boyce Thompson Institute, Cornell University, Ithaca, NY) for his generosity in supplying us with Op1D cells and antibodies and for valuable discussion. We acknowledge P.S. Backlund, Jr. and A.L. Bailey (both from NICHD, NIH) for critical review of the manuscript and discussions. We thank A. Chrambach (NICHD, NIH) for his support and S.T. Case (University of Mississippi Medical Center, Jackson, MS) for providing us with high MW protein standard. We also thank other members in the Laboratory of Cellular and Molecular Biophysics for their input.


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