Glycoprotein D of herpes simplex virus (HSV) binds directly to HVEM, a member of the tumor necrosis factor receptor superfamily and a mediator of HSV entry

Abstract

Glycoprotein D (gD) is a structural component of the herpes simplex virus (HSV) envelope which is essential for virus entry into host cells. Chinese hamster ovary (CHO-K1) cells are one of the few cell types which are nonpermissive for the entry of many HSV strains. However, when these cells are transformed with the gene for the herpesvirus entry mediator (HVEM), the resulting cells, CHO-HVEM12, are permissive for many HSV strains, such as HSV-1(KOS). By virtue of its four cysteine-rich pseudorepeats, HVEM is a member of the tumor necrosis factor receptor superfamily of proteins. Recombinant forms of gD and HVEM, gD-1(306t) and HVEM(200t), respectively, were used to demonstrate a specific physical interaction between these two proteins. This interaction was dependent on native gD conformation but independent of its N-linked oligosaccharides, as expected from previous structure-function studies. Recombinant forms of gD derived from HSV-1(KOS)rid1 and HSV-1(ANG) did not bind to HVEM(200t), explaining the inability of these viruses to infect CHO-HVEM12 cells. A variant gD protein, gD-1(⌬290-299t), showed enhanced binding to HVEM(200t) relative to the binding of gD-1(306t). Competition studies showed that gD-1(⌬290-299t) and gD-1(306t) bound to the same region of HVEM(200t), suggesting that the differences in binding to HVEM are due to differences in affinity. These differences were also reflected in the ability of gD-1(⌬290-299t) but not gD-1(306t) to block HSV type 1 infection of CHO-HVEM12 cells. By gel filtration chromatography, the complex between gD-1(⌬290-299t) and HVEM(200t) had a molecular mass of 113 kDa and a molar ratio of 1:2. We conclude that HVEM interacts directly with gD, suggesting that HVEM is a receptor for virion gD and that the interaction between these proteins is a step in HSV entry into HVEM-expressing cells.

Antibody-Induced Conformational Changes in Herpes Simplex Virus Glycoprotein gD Reveal New Targets for Virus Neutralization

“…The methods for production and DL6 affinity purification of gD1(306t), gD2(306t), and gD2(285t) have been described previously (29,41,48). Production of HveA(200t) (HVEM) and HveC(346t) (nectin-1) by nickel-agarose purification has also been described previously (28,59). Recombinant baculovirus expressing gD2(s322) was obtained by first cloning the 0.3-kbp ApaI (blunted with mung bean nuclease)-BamHI fragment from plasmid D2-H20 (11) (which contains an insertion of charged amino acids into the middle of the transmembrane region) into plasmid pAN243 (41) (which had been digested with EcoRI and blunted with Klenow fragment) and BamHI.…”

“…Enhancement of receptor binding by MAbs. ELISA plates were coated overnight with soluble receptors [5 g/ml of HVEM(200t), 10 g/ml of nectin-1(346t)] (28,59) and then blocked with 5% milk-0.1% Tween-PBS for 1 h. Concurrently, 0.5 M each MAb was incubated with dilutions of gD2(306t) or gD2(285t) starting at 0.5 M for 1 h at RT in 5% milk-0.1% Tween-PBS. The MAb-gD mixture was then added to the receptor-coated plates for 1 h at RT.…”

HVEM/LIGHT/BTLA/CD160 cosignaling pathways as targets for immune regulation

“…HVEM (TNFRSF14) is a protein of the TNFR SF that serves as a shared ligand for the costimulatory and coinhibitory receptors discussed in this review. HVEM got its name based on the ability of Types 1 and 2 herpesvirus gD to bind to it for entry into the cell [22][23][24]. HVEM not only interacts with viral gD proteins but also binds receptors in the Ig SF (BTLA and CD160 [11,14,25]) and receptors in the TNF SF (LIGHT and LT␣ [9,26,27]).…”

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

“…Our working hypothesis is that the requirements for HSV association with a host cell membrane during virus entry can be studied in vitro using purified virions, soluble gD receptors, and liposomes as target membranes. The soluble receptors HVEMt and nectin-1t bind to gD (18,39) as well as to purified HSV (24), whereas nectin-2t fails to bind gD of wild-type HSV strains (38) and was therefore used as a negative control. In the first experiment ( Fig.…”