Bimolecular complementation reveals that glycoproteins gB and gH/gL of herpes simplex virus interact with each other during cell fusion.
Herpes simplex virus entry into cells requires four glycoproteins, gB, gD, gH, and gL. Binding of gD to one of its receptors triggers steps requiring the core fusion proteins, gB and the gH/gL heterodimer. There is evidence that gH/gL initiates hemifusion of cells, but whether this complex interacts physically with gB to cause complete fusion is unknown. We used bimolecular complementation (BiMC) of enhanced yellow fluorescent protein (EYFP) to detect glycoprotein interactions during cell-cell fusion. The N- or C-terminal half of EYFP was fused to the C terminus of gD, gB, and gH to form six chimeric proteins (Dn, Dc, Bn, Bc, Hn, and Hc). BiMC was detected by confocal microscopy. Receptor-bearing (C10) cells cotransfected with Dn and Bc or Dn, Hc, and untagged gL exhibited EYFP fluorescence, indicative of interactions between gD and gB and between gD and gH/gL. EYFP complementation did not occur in cells transfected with gL, Bc, and Hn. However, when gD was coexpressed with these other three proteins, cell-cell fusion occurred and the syncytia exhibited bright EYFP fluorescence. To separate glycoprotein expression from fusion, we transfected C10 cells with gL, Bc, and Hn for 20 h and then added soluble gD to trigger fusion. We detected fluorescent syncytia within 10 min, and both their number and size increased with exposure time to gD. Thus, when gD binds its receptor, the core fusion machinery is triggered to form a multiprotein complex as a step in fusion and possibly virus entry.
Atanasiu
Doina
D
Department of Microbiology, School of Dental Medicine, and Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104.
Whitbeck
J Charles
JC
Cairns
Tina M
TM
Reilly
Brigid
B
Cohen
Gary H
GH
Eisenberg
Roselyn J
RJ
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D017930:Genes, Reporter / Q000235:genetics; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism*; D008561:Membrane Fusion; D051379:Mice; D011485:Protein Binding; D018139:Simplexvirus / Q000235:genetics / Q000378:metabolism*; D012995:Solubility; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D006023:Glycoproteins; D014764:Viral Proteins