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

Abstract

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.

Three SNARE complexes cooperate to mediate membrane fusion

“…The hemagglutinin fusion protein of influenza is proposed to function as a trimer (16). The gp64 viral fusion protein of baculovirus also oligomerizes into multimers to mediate fusion (17). Although it has been suggested that SNARE complexes may form a ring around the fusion site, there are little or no data in support of this hypothesis.…”

A Discrete Stage of Baculovirus GP64-mediated Membrane Fusion

“…Electrophysiological experiments on baculovirus-infected Sf9 cells led Plonsky and Zimmerberg (1996) to propose that a ring of five to seven GP64 trimers may form a fusion machine that facilitates opening of the fusion pore within this ring. Furthermore, using polypeptide cross-linking agents and sulfhydryl group reagents, Markovic et al (1998) demonstrated that GP64 forms transient, higher-order complexes of trimers upon application of low pH buffer (only in the presence of target membrane) at the same time that fusion is catalyzed. The ring hypothesis is further supported by work with influenza HA, which suggests that HA trimers function cooperatively and inhibit lipid dye transmigration during the initial fusion phases (Ellens et al, 1990;Clague et al, 1991;Tse et al, 1993;Zimmerberg et al, 1994;Blumenthal et al, 1996;Danieli et al, 1996;Chernomordik et al, 1998).…”

“…These transient ring structures are proposed to facilitate conversion of kinetic energy, released during protein conformational change, into work on the target and host membranes required for membrane fusion. Indeed, biochemical evidence for baculovirus GP64 aggregates during membrane fusion has recently been reported (Markovic et al 1998). Whether and how viral fusion proteins might function in a coordinated and cooperative manner is a major question in the field of membrane fusion.…”

“…GP64 is temporally ‡ Corresponding author. E-mail address: joshz@helix.nih.gov.© 1999 by The American Society for Cell Biology 4191 expressed during both the early and late phases of viral infection (Blissard and Rohrmann, 1989;Jarvis and Garcia, 1994;Kogan et al, 1994;Oomens et al, 1995;Garrity et al, 1997) and oligomerizes to form two electrophoretically distinct trimers (Oomens et al, 1995;Markovic et al, 1998). This protein is both necessary and sufficient for mediating pH-dependent membrane fusion (Blissard and Wenz, 1992), is essential for cell-to-cell transmission of the enveloped virion (Monsma et al, 1996), and has host cell receptor-binding activity (Hefferon et al, 1999).…”

Prm1 Functions as a Disulfide-linked Complex in Yeast Mating

“…The gp64 fusion protein of baculovirus has intermolecular disulfide bonds linking the three subunits of a trimer. Reduction of these bonds with DTT inhibits membrane fusion and prevents gp64 from assembling into a higher order fusion complex (23). Disulfide bonds within the human immunodeficiency virus fusion protein must be rearranged by protein disulfide isomerase isoforms present on the cell surface to permit a conformational shift required for membrane merger (24 -26).…”