Oligomeric structure of glycoproteins in herpes simplex virus type 1

Abstract

A number of herpes simplex virus (HSV) glycoproteins are found in oligomeric states: glycoprotein E (gE)-gI and gH-gL form heterodimers, and both gB and gC have been detected as homodimers. We have further explored the organization of glycoproteins in the virion envelope by using both purified virions to quantitate glycoprotein amounts and proportions and chemical cross-linkers to detect oligomers. We purified gB, gC, gD, and gH from cells infected with HSV type 1 and used these as immunological standards. Glycoproteins present in sucrose gradient-purified preparations of two strains of HSV type 1, KOS and NS, were detected with antibodies to each of the purified proteins. From these data, glycoprotein molar ratios of 1:2:11:16 and 1:1:14:9 were calculated for gB/gC/gD/gH in KOS and NS, respectively. gL was also detected in virions, although we lacked a purified gL standard for quantitation. We then asked whether complexes of these glycoproteins could be identified, and if they existed as homo-or hetero-oligomers. Purified KOS was incubated at 4؇C with bis(sulfosuccinimidyl) suberate (BS 3 ), an 11.4 Å (1Å ‫؍‬ 0.1 mm) noncleavable, water-soluble cross-linker. Virus extracts were examined by Western blotting (immunoblotting), or immunoprecipitation followed by Western blotting, to assay for homo-and hetero-oligomers. Homodimers of gB, gC, and gD were detected, and hetero-oligomers containing gB cross-linked to gC, gC to gD, and gD to gB were also identified. gH and gL were detected as a hetero-oligomeric pair and could be cross-linked to gD or gC but not to gB. We conclude that these glycoproteins are capable of forming associations with one another. These studies suggest that glycoproteins are closely associated in virions and have the potential to function as oligomeric complexes.

Surface Plasmon Resonance Reveals Direct Binding of Herpes Simplex Virus Glycoproteins gH/gL to gD and Locates a gH/gL Binding Site on gD

“…In support, we have shown that gD is structurally divided into two "faces": one for receptor binding and the other for its presumed interaction with gH/gL. However, despite numerous studies, both from our laboratory and others (19)(20)(21)(22)(23)(24)(25), that indirectly support the ability of gD to interact with gH/gL, before now we have been unable to show that gD and gH/gL bind to each other directly.The critical requirement for an interaction between gD and gH/gL in viral fusion is supported by several studies. First, in Epstein-Barr virus (EBV), cryoelectron microscopy and crystallography have revealed the structure of a gp42-gH/gL complex (26,27).…”

“…gH/gL binds to gD when gD is immobilized via certain anti-gD MAbs. Since we could only detect a direct interaction between gD and gH/gL by either cross-linking the proteins on the virion surface (24) or by stabilizing the endodomains of gD and gH/gL through bimolecular complementation (30), we postulated that the gD-gH/gL interaction was transient, as suggested by Heldwein (32). Therefore, we decided to use SPR, which monitors binding of proteins in real time and is sensitive enough to detect weak or transient interactions.…”

Stable Association of Herpes Simplex Virus with Target Membranes Is Triggered by Low pH in the Presence of the gD Receptor, HVEM

“…HSV-1(KOS) as well as the ␤-galactosidase reporter virus, HSV-1(KOS)tk12 (38), was propagated, and the titers of the virus were determined on Vero cells. Sucrose gradient-purified HSV (16) was used throughout the studies reported here.…”

Oligomeric self-association of basic fibroblast growth factor in the absence of heparin-like glycosaminoglycans

“…It was important to ensure that the analysis of chemical covalent cross-linking would be an accurate reflection of specific FGF-2 associations and not an artifact of adventitious cross-linking or the cross-linking conditions. BS$ was chosen for chemical cross-linking for reasons that included aqueous solubility, homobifunctional specificity for lysine residues, moderate spacer length (11.4 A / ) and a variety of published uses for studying protein associations [27,28]. Control experiments were completed to ensure the specificity of the reaction.…”