Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2005 Sep;79(18):11588-97.
doi: 10.1128/JVI.79.18.11588-11597.2005.

Herpes simplex virus glycoprotein B binds to cell surfaces independently of heparan sulfate and blocks virus entry

Florent C Bender  1 , J Charles Whitbeck, Huan Lou, Gary H Cohen, Roselyn J Eisenberg
Affiliations

Herpes simplex virus glycoprotein B binds to cell surfaces independently of heparan sulfate and blocks virus entry

Florent C Bender et al. J Virol. 2005 Sep.

Abstract

Virion glycoproteins gB, gD, and gH/gL play essential roles for herpes simplex virus (HSV) entry. The function of gD is to interact with a cognate receptor, and soluble forms of gD block HSV entry by tying up cell surface receptors. Both gB and the nonessential gC interact with cell surface heparan sulfate proteoglycan (HSPG), promoting viral attachment. However, cells deficient in proteoglycan synthesis can still be infected by HSV. This suggests another function for gB. We found that a soluble truncated form of gB bound saturably to the surface of Vero, A431, HeLa, and BSC-1 cells, L-cells, and a mouse melanoma cell line expressing the gD receptor nectin-1. The HSPG analog heparin completely blocked attachment of the gC ectodomain to Vero cells. In contrast, heparin only partially blocked attachment of soluble gB, leaving 20% of the input gB still bound even at high concentrations of inhibitor. Moreover, heparin treatment removed soluble gC but not gB from the cell surface. These data suggest that a portion of gB binds to cells independently of HSPG. In addition, gB bound to two HSPG-deficient cell lines derived from L-cells. Gro2C cells are deficient in HSPG, and Sog9 cells are deficient in HSPG, as well as chondroitin sulfate proteoglycan (CSPG). To identify particular gB epitopes responsible for HSPG-independent binding, we used a panel of monoclonal antibodies (MAbs) to gB to block gB binding. Only those gB MAbs that neutralized virus blocked binding of soluble gB to the cells. HSV entry into Gro2C and Sog9 cells was reduced but still detectable relative to the parental L-cells, as previously reported. Importantly, entry into Gro2C cells was blocked by purified forms of either the gD or gB ectodomain. On a molar basis, the extent of inhibition by gB was similar to that seen with gD. Together, these results suggest that soluble gB binds specifically to the surface of different cell types independently of HSPG and CSPG and that by doing so, the protein inhibits entry. The results provide evidence for the existence of a cellular entry receptor for gB.

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
Construction and expression of a soluble form of HSV-1 gB. (A) A schematic representation of full-length gB indicates the position of the 30-amino-acid signal sequence (hatched box) and the two short hydrophobic sequences (gray boxes) preceding the transmembrane region (TMR) (black box) (45). In the baculovirus-expressed gB construct [gB(730t)], the extracellular domain between the signal peptide and the first hydrophobic sequence was fused to the melittin signal sequence. (B) Purified gB(730t) was electrophoresed under denaturing (D) or nondenaturing “native” (N) conditions on SDS-PAGE gels, either silver stained or Western blotted, and probed with MAb SS10 or DL16. Migration positions of molecular mass markers are indicated (on the left, in kilodaltons) along with the expected position of the gB(730t) monomer and multimer (arrows).
FIG. 2.
FIG. 2.
A soluble form of gB binds saturably to the surface of different cell types. (A) C10 cells were seeded in 96-well plates and incubated at 4°C with increasing concentrations of gB(730t) or gD(306t). After 1 h, cells were washed, and CELISA was used to quantitate the amounts of the glycoproteins associated with the cell surface using R69 or R7 for gB and gD, respectively. A value of 100% is defined by the binding of one of the different glycoproteins at the maximal concentration [1 μM and 0.4 μM for gB(730t) and gD(306t), respectively] used in the experiment. Values were plotted after subtraction of background generated by individual polyclonal antibodies. The concentration of gB required to saturate 50% of the cell surface was estimated graphically (insert). Results are representative of at least three independent experiments run in duplicate. (B) Vero cells, L-cells, or A431 cells were seeded in 96-well plates and incubated at 4°C with increasing concentrations of gB(730t). After 1 h, cells were washed and amounts of glycoproteins associated with the cell surface were measured by CELISA with R69. (C) C10 or B78 cells were seeded in 12-well plates were incubated with 0, 0.1, 0.2, or 0.5 μM gB(730t) (reading from left to right) or 0.1 μM of gD(285t) for 1 h at 4°C. Cells were washed; proteins were extracted, resolved by SDS-PAGE, and transferred to membranes; and the amounts of gB or gD associated with the cell extract were measured by Western blotting with polyclonal antibody R69 or R7, respectively. Nectin-1 expression was revealed using MAb CK6. Migration positions of gB(730t), gD(285t), and nectin-1 are indicated.
FIG. 3.
FIG. 3.
Heparin only partially removes gB or competes with attachment of gB to cells. (A) Vero cells seeded in 96-well plates were incubated with 10 nM gB(730t) or 6 nM gC(457t). After 1 h at 4°C, glycoprotein-containing medium was removed and replaced by fresh medium together with various concentrations of heparin. Cells were incubated for 1 h, washed, and fixed and the level of glycoprotein associated with the cell surface was measured by CELISA with polyclonal antibodies R47 and R69 to gC and gB, respectively. (B) gB(730t) or gC(457t) was incubated for 1 h in the presence of various concentrations of heparin. Glycoprotein-plus-heparin mixtures were added to Vero cells seeded in 96-well plates, and incubation continued for another hour. Cells were then washed and fixed, and glycoproteins associated with the cell surface were revealed by CELISA as described in the legend to panel A.
FIG. 4.
FIG. 4.
Soluble gB binds saturably to the surface of cells deficient in HSPG. (A) L-cells, Gro2C cells, or Sog9 cells seeded in 96-well plates were incubated with increasing concentrations of gB(730t) for 1 h at 4°C. Cells were washed and fixed, and binding of gB was quantified by CELISA using anti-gB polyclonal antibody R69. A value of 100% is defined by the binding of gB(730t) to L-cells at the maximal concentration (1 μM) used in the experiment. The experiment shown is representative of three independent experiments. (B) L-cells, Gro2C cells, or Sog9 cells seeded in 12-well plates were incubated for 1 h at 4°C with 1 μM gB(730t). After being washed, total cell proteins were extracted, resolved by SDS-PAGE, transferred to nitrocellulose membranes, and probed with polyclonal antibody R69. Migration positions of molecular mass markers are indicated (on the left, in kilodaltons) along with the expected position of gB(730t) (arrow).
FIG. 5.
FIG. 5.
Monoclonal antibodies to gB block binding of soluble gB to the cells. (A) gB(730t) at a final concentration of 0.5 μM was incubated in the presence of MAb SS48, SS55, or SS10 at a final concentration of 300, 30, or 3 μg/ml (reading from left to right) or with no antibody (no Ab). The mixture was then added to C10 cells seeded into 12-well plates and incubated for 1 h. After being washed, total cell proteins were extracted, resolved by SDS-PAGE, transferred to membranes, and probed against gB with R69. (B) gB(730t) at a final concentration of 0.5 μM was incubated in the presence of MAb SS10 at a final concentration of 500, 50, or 5 μg/ml (reading from left to right) or DL16 at a final concentration of 500 μg/ml. The mixture was then added to Gro2C cells seeded into 12-well plates and incubated for 1 h. Cells were then washed and fixed, and the level of glycoprotein associated with the cell surface was measured by CELISA using R69.
FIG. 6.
FIG. 6.
Monoclonal antibodies to gB neutralize entry into L-cells and Gro2C. (A) HSV-1 KOS/tk12 was incubated for 1 h with increasing concentrations of MAb to gB (SS10, SS48, and SS55) or anti-myc antibody (control). The virus-antibody mixture was then used to infect C10 cells seeded in 96-well plates at an MOI of 10 PFU/cell. β-Galactosidase activity was assayed at 6 h p.i. as a measure of entry. (B and C) HSV-1 KOS/tk12 was incubated for 1 h with increasing concentrations of MAbs to gB (SS10 and SS55) or anti-myc antibody (control). Virus at a multiplicity of infection of 10 PFU/cell was then used to infect L-cells (B) and Gro2C cells (C) seeded in 96-well plates. β-Galactosidase activity was assayed at 6 h postinfection.
FIG. 7.
FIG. 7.
Soluble gB bound to cells deficient in HSPG inhibits HSV entry. Vero cells (A), L-cells (B), and Gro2C cells (C and D) seeded in 96-well plates were incubated with increasing concentrations of gB(730t) (A to D) or gD(306t) (A to C). After 1 h at 4°C, glycoprotein-containing medium was removed and cells were either directly infected or washed three times with DFH and then infected with HSV-1 KOS/tk12 at an MOI of 10 PFU per cell. Cells were transferred to 37°C, and β-galactosidase activity was assayed at 6 h postinfection. Concentrations of gB(730t) required to inhibit 50% of entry are estimated graphically for each cell line.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Akula, S. M., N. P. Pramod, F. Z. Wang, and B. Chandran. 2002. Integrin α3β1 (CD 49c/29) is a cellular receptor for Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) entry into the target cells. Cell 108:407-419. - PubMed
    1. Banfield, B. W., Y. Leduc, L. Esford, K. Schubert, and F. Tufaro. 1995. Sequential isolation of proteoglycan synthesis mutants by using herpes simplex virus as a selective agent: evidence for a proteoglycan-independent virus entry pathway. J. Virol. 69:3290-3298. - PMC - PubMed
    1. Banfield, B. W., Y. Leduc, L. Esford, R. J. Visalli, C. R. Brandt, and F. Tufaro. 1995. Evidence for an interaction of herpes simplex virus with chondroitin sulfate proteoglycans during infection. Virology 208:531-539. - PubMed
    1. Bender, F. C., J. C. Whitbeck, M. Ponce de Leon, H. Lou, R. J. Eisenberg, and G. H. Cohen. 2003. Specific association of glycoprotein B with lipid rafts during herpes simplex virus entry. J. Virol. 77:9542-9552. - PMC - PubMed
    1. Boehme, K. W., J. Singh, S. T. Perry, and T. Compton. 2004. Human cytomegalovirus elicits a coordinated cellular antiviral response via envelope glycoprotein B. J. Virol. 78:1202-1211. - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources