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
. 2003 Mar;77(5):3131-47.
doi: 10.1128/jvi.77.5.3131-3147.2003.

Human herpesvirus 8 envelope glycoprotein B mediates cell adhesion via its RGD sequence

Fu-Zhang Wang  1 , Shaw M Akula, Neelam Sharma-Walia, Ling Zeng, Bala Chandran
Affiliations

Human herpesvirus 8 envelope glycoprotein B mediates cell adhesion via its RGD sequence

Fu-Zhang Wang et al. J Virol. 2003 Mar.

Abstract

Human herpesvirus 8 (HHV-8) or Kaposi's sarcoma-associated herpesvirus, implicated in the pathogenesis of Kaposi's sarcoma, utilizes heparan sulfate-like molecules to bind the target cells via its envelope-associated glycoproteins gB and gpK8.1A. HHV-8-gB possesses the Arg-Gly-Asp (RGD) motif, the minimal peptide region of many proteins known to interact with subsets of host cell surface integrins. HHV-8 utilizes alpha3beta1 integrin as one of the receptors for its entry into the target cells via its gB interaction and induces the activation of focal adhesion kinase (FAK) (S. M. Akula, N. P. Pramod, F.-Z. Wang, and B. Chandran, Cell 108:407-419, 2002). Since FAK activation is the first step in the outside-in signaling necessary for integrin-mediated cytoskeletal rearrangements, cell adhesions, motility, and proliferation, the ability of HHV-8-gB to mediate the target cell adhesion was examined. A truncated form of gB without the transmembrane and carboxyl domains (gBdeltaTM) and a gBdeltaTM mutant (gBdeltaTM-RGA) with a single amino acid mutation (RGD to RGA) were expressed in a baculovirus system and purified. Radiolabeled HHV-8-gBdeltaTM, gBdeltaTM-RGA, and deltaTMgpK8.1A proteins bound to the human foreskin fibroblasts (HFFs), human dermal microvascular endothelial (HMVEC-d) cells, human B (BJAB) cells, and Chinese hamster ovary (CHO-K1) cells with equal efficiency, which was blocked by preincubation of proteins with soluble heparin. Maxisorp plate-bound gBdeltaTM protein induced the adhesion of HFFs and HMVEC-d and monkey kidney epithelial (CV-1) cells in a dose-dependent manner. In contrast, the gBdeltaTM-RGA and DeltaTMgpK8.1A proteins did not mediate adhesion. Adhesion mediated by gBdeltaTM was blocked by the preincubation of target cells with RGD-containing peptides or by the preincubation of plate-bound gBdeltaTM protein with rabbit antibodies against gB peptide containing the RGD sequence. In contrast, adhesion was not blocked by the preincubation of plate-bound gBdeltaTM protein with heparin, suggesting that the adhesion is mediated by the RGD amino acids of gB, which is independent of the heparin-binding domain of gB. Integrin-ligand interaction is dependent on divalent cations. Adhesion induced by the gBdeltaTM was blocked by EDTA, thus suggesting the role of integrins in the observed adhesions. Focal adhesion components such as FAK and paxillin were activated by the binding of gBdeltaTM protein to the target cells but not by gBdeltaTM-RGA protein binding. Inhibition of FAK phosphorylation by genistein blocked gBdeltaTM-induced FAK activation and cell adhesion. These findings suggest that HHV-8-gB could mediate cell adhesion via its RGD motif interaction with the cell surface integrin molecules and indicate the induction of cellular signaling pathways, which may play roles in the infection of target cells and in Kaposi's sarcoma pathogenesis.

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
(A) Construction of HHV-8-gBΔTM ORF without the transmembrane and carboxyl domains. The top line shows the schematic diagram of HHV-8 genome and the locations of encoded glycoprotein ORFs. HHV-8-gB ORF (ORF 8) is 845 aa long with a signal sequence (SS, aa 1 to 23) and a transmembrane domain (TM, aa 710 to 729). The locations of RGD motif, putative cleavage site, and the four synthetic gB peptides (N1, aa 27 to 47; N2, aa 167 to 191; N3, aa 573 to 593; and C, aa 828 to 845) used for raising rabbit antibodies are indicated. gBΔTM was constructed by using primers amplifying aa 1 to 702 with the signal sequence but lacking the transmembrane and the carboxyl domains and with a His tag at the carboxyl terminus. The gBΔTM/pAcGP67-B plasmid was generated by cloning the amplified fragment (2,124 bp) into the BamHI-EcoRI sites of the pAcGP67-B vector. The gBΔTM-RGA mutant was generated by mutating the RGD amino acids in the gBΔTM/pAcGP67-B plasmid to RGA by site-directed mutagenesis. (B) Expression and purification of gBΔTM protein in the baculovirus system. High-5 cells were infected with baculovirus-gBΔTM for 5 to 7 days, and His-tagged gBΔTM protein from cell pellets was purified by use of a nickel column. Protein purity was analyzed by SDS-10% PAGE gels and Western blotting with anti-gB and anti-gB-peptide rabbit IgG antibodies. Samples were boiled in sample buffer with 2-ME (lanes 1 to 6) or without 2-ME (lane 7). Lane 1, Coomassie stain of purified gBΔTM; lane 2, purified gBΔTM in Western blot reactions with rabbit anti-gB antibodies; lane 3, purified gBΔTM in Western blot reactions with rabbit anti-gB-N1 antibodies; lane 4, purified gBΔTM in Western blot reactions with rabbit anti-gB-N2 antibodies; lane 5, purified gBΔTM in Western blot reactions with rabbit anti-gB-N3 antibodies; lane 6, purified gBΔTM in Western blot reactions with rabbit anti-gB-C antibodies; and lane 7, purified gBΔTM run under nonreducing conditions in Western blot reactions with rabbit anti-gB antibodies. The numbers on the left indicate the molecular masses (in kilodaltons) of the standard protein markers run in parallel lanes. The numbers on the right indicate the approximate molecular masses (in kilodaltons) of the purified gBΔTM protein. The asterisks represent the multimer forms of gBΔTM protein with approximate molecular masses of >180 kDa.
FIG. 2.
FIG. 2.
HHV-8-gBΔTM binds to the target cells. (A) Binding of purified gBΔTM protein detected by surface immunofluorescence assay. Panel 1: HFFs in chamber slides were incubated with 16 μg of purified gBΔTM/ml for 90 min at 4°C, washed, fixed with 4% paraformaldehyde for 10 min at room temperature, washed, and incubated with rabbit anti-HHV-8-gB IgG antibodies for 60 min at 4°C. Cells were washed, incubated for 60 min at 4°C with FITC-conjugated goat anti-rabbit IgG antibodies, washed, mounted, and examined under a fluorescence microscope. Panel 2: BJAB cells fixed with 0.1% paraformaldehyde were incubated with 33 μg of purified gBΔTM/ml for 90 min at 4°C, washed, and incubated with rabbit anti-HHV-8-gB IgG antibodies and processed as described for panel 1. Panel 3: BJAB cells incubated with purified gBΔTM, rabbit anti-HHV-8 gpK8.1A IgG antibodies, and FITC-anti-rabbit antibodies. Panel 4: BJAB cells incubated with DMEM, rabbit anti-HHV-8-gB IgG antibodies, and FITC-anti-rabbit antibodies. (B) Morphological changes induced by binding of gBΔTM protein to the target cells. Confluent HFFs in chamber slides were incubated with different concentrations of recombinant gBΔTM, gBΔTM-RGA, or ΔTMgpK8.1A protein (150 μl/well) diluted in serum-free DMEM for 30 min at room temperature, and slides were observed.
FIG. 3.
FIG. 3.
Quantitation of HHV-8-gBΔTM binding to the target cells. (A and B) Binding of radiolabeled proteins to target cells. Different concentrations of [35S]methionine-labeled purified gBΔTM, gBΔTM-RGA, or ΔTM gpK8.1A protein were incubated for 90 min at 4°C with HFFs (A) or HMVEC-d (B) or CHO-K1 (B) cells in 24-well plates. After incubation, cells were washed five times and lysed with 1% SDS and 1% Triton X-100, and the cell-bound protein radioactivity was counted. Each reaction was done in triplicate, and each point represents the average plus or minus standard deviation of three experiments. (C) Inhibition of [35S]methionine-labeled purified proteins binding to HFFs by heparin. [125I]-labeled EGF lacking heparin-binding activity was used as the negative control. A predetermined quantity of 125I-labeled EGF (259,272 cpm/ng) or [35S]methionine-labeled purified gBΔTM, gBΔTM-RGA, or ΔTM gpK8.1A protein within the linear range of the dose response-curve (6 μg) (A and B) was mixed with medium alone or with different concentrations of heparin or CS-A and was incubated for 60 min at 4°C. These were then incubated with HFFs for 90 min at 4°C, washed, and lysed with 1% SDS and 1% Triton X-100, and the cell-bound protein radioactivity was counted. The cell-associated protein counts per minute in the presence or absence of heparin and the percentage of inhibition of protein binding were calculated. Each reaction was done in triplicate, and each point represents the average plus or minus standard deviation of three experiments.
FIG. 4.
FIG. 4.
HHV-8-gBΔTM bound to the plates retains heparin-binding activity. (A) Binding of purified proteins to the Maxisorp plates. Plates were coated with different concentrations of unlabeled purified gBΔTM, gBΔTM-RGA, and ΔTMgpK8.1A proteins in PBS (4 μg/ml in PBS, 100 μl/well) overnight at 4°C. Plates were washed, blocked with 10% nonfat milk for 1 h at 37°C, washed, and incubated with rabbit anti-gB or anti-gpK8.1A IgG antibodies for 1 h at 37°C, followed by goat anti-rabbit antibodies coupled to horseradish peroxidase. After reacting with substrate, the reaction was stopped by 2 N H2SO4 and the optical density was read at 450 nm (OD450). Each reaction was done in triplicate, and each point represents the average plus or minus standard deviation of three experiments. (B) Heparin binds to the gBΔTM protein-coated plates. Maxisorp plates were coated with BSA, fibronectin, gBΔTM, gBΔTM-RGA, or ΔTMgpK8.1A protein (4 μg/ml, 100 μl/well) overnight at 4°C. Plates were washed, blocked with 3% BSA in PBS for 2 h at 4°C, washed, and incubated with different concentrations of biotin-labeled heparin (100 μl/well) for 1 h at 4°C. Plates were washed and incubated with streptavidin horseradish peroxidase (0.01 μg/well) for 1 h at room temperature. The reaction was stopped by 2 N H2SO4 and read at 450 nm. Each reaction was done in triplicate, and each point represents the average plus or minus the standard deviation of three experiments.
FIG. 5.
FIG. 5.
HHV-8-gBΔTM induces the adhesion of target cells. Maxisorp plates were coated overnight at 4°C with different concentrations of purified gBΔTM, ΔTMgpK8.1A, fibronectin, vitronectin, collagen I, and gelatin in PBS (100 μl/well). Plates were washed, blocked with 1% BSA in PBS for 2 h at 4°C, washed, and incubated with fresh suspensions of target cells in 0.1% BSA-serum-free DMEM at a concentration of 2 × 104 cells/well for 45 min to 1 h. Cells were washed, and adherent cells were fixed with 4% paraformaldehyde, washed, and stained with 0.5% crystal violet-methanol. The dye was extracted with 0.1 M sodium citrate, and the optical density at 595 nm (OD 595) was quantitated. Each reaction was done in triplicate, and each point represents the average plus or minus standard deviation of three experiments.
FIG. 6.
FIG. 6.
The peptide with RGD amino acids inhibits the cell adhesion induced by the gBΔTM protein. Maxisorp plates were coated with 4 μg of purified gBΔTM protein/ml (100 μl/well) overnight at 4°C, washed, and blocked with 1% BSA-PBS. For testing the effect of RGD amino acids, target cells were preincubated with different concentrations of GRGDSPL or GRADSPL peptides for 30 min at 4°C and were seeded to the protein-coated plates. For testing the effect of heparin, protein-coated plates were incubated with different concentrations of heparin in DMEM for 1 h at 4°C before addition of the target cells. The adhesion assay was carried out as described in the Fig. 3 legend. Data are presented as percentage of inhibition of cell adhesion obtained when cells were preincubated with DMEM only. Each reaction was done in triplicate, and each point represents the average plus or minus standard deviation of three experiments.
FIG. 7.
FIG. 7.
Antibodies against gB peptide containing RGD amino acids inhibit the cell adhesion induced by the gBΔTM protein. Maxisorp plates were coated with 4 μg of purified gBΔTM protein/ml (100 μl/well) overnight at 4°C, washed, and incubated for 1 h at 4°C with rabbit IgG antibodies against HHV-8-gB (anti-gB) or against HHV-8-gB peptide with (anti-gB-N1) and without RGD amino acids (anti-gB-C) or against HHV-gpK8.1A (anti-gpK8.1A) in 0.1% BSA-DMEM. HFFs were seeded into the wells, and cell adhesion assays were carried out as described in the Fig. 3 legend. Data are presented as percentage of inhibition of cell adhesion obtained when gBΔTM was preincubated with DMEM only. Each reaction was done in triplicate, and each point represents the average plus or minus standard deviation of three experiments.
FIG. 8.
FIG. 8.
(A) HHV-8-gBΔTM protein-induced cell adhesion was abolished by a single amino acid substitution of the RGD sequence. Maxisorp plates were coated with different concentrations of purified gBΔTM or gBΔTM-RGA mutant proteins overnight at 4°C and washed, and cell adhesion assays were carried out as described in the Fig. 3 legend. Each reaction was done in triplicate, and each point represents the average plus or minus the standard deviation of three experiments. (B) EDTA blocks target cell adhesion induced by HHV-8-gBΔTM. Maxisorp plates were coated with 4 μg of purified HHV-8-gBΔTM/ml (100 μl/well) overnight at 4°C, washed, and blocked with 1% BSA-PBS. Target cells were resuspended in medium with 0.1% BSA and were incubated with different concentrations of EDTA for 15 min on ice before seeding to plates. Adhesion assays were carried out as described in the Fig. 3 legend. Data are presented as percentage of inhibition of cell adhesion obtained when cells were preincubated with DMEM without EDTA. Each reaction was done in triplicate, and each point represents the average plus or minus the standard deviation of three experiments.
FIG. 9.
FIG. 9.
HHV-8-gBΔTM activates the integrin-dependent FAK components. Serum-starved HFFs in chamber slides were incubated with 200 μl of serum-free DMEM (mock) or LPA (20 ng/ml) or gBΔTM protein (50 ng/ml) in serum-free DMEM or gBΔTM-RGA (50 ng/ml) in serum-free DMEM. After incubation for 5 min at 37°C, cells were washed, fixed, permeabilized, and reacted with rabbit anti-p125FAK and mouse MAb to paxillin, followed by anti-rabbit-tetramethyl rhodamine isothiocyanate and anti-mouse-FITC antibodies. Stained cells were examined under a fluorescence microscope with appropriate filters. The arrowheads indicate representative areas of FAK-paxillin colocalization. Magnification, ×1,000.
FIG. 10.
FIG. 10.
FAK activation is critical for the cell adhesion induced by HHV-8-gBΔTM. (A) Induction of FAK phosphorylation by HHV-8-gBΔTM protein and its inhibition by genistein. Top panel: Serum-starved HFFs were uninduced (lane 1) and induced with 1 μg of gBΔTM-RGA protein/ml for 15 min at 37°C (lane 2), with 20 ng of LPA/ml for 5 min at 37°C (lane 3), and with 1 μg of gBΔTM protein/ml for 15 min at 37°C (lane 4) or were pretreated with 100, 50, and 25 μM concentrations of genistein for 1 h at 37°C and were then incubated with 1 μg of gBΔTM protein/ml for 15 min at 37°C in the presence of similar concentrations of genistein (lanes 5 to 7, respectively). Cells were lysed, and equivalent amounts of lysates were subjected to SDS-PAGE, transferred to nitrocellulose membranes, reacted with anti-phospho-p125FAK antibodies, and developed using chemiluminescent reagents. Lower panel: after reactions, membranes were stripped and reprobed with MAb to actin. The bands were scanned, and the band intensities were assessed. Data are presented as percentage of inhibition of FAK phosphorylation obtained when the cells were incubated with the gBΔTM protein only. (B) Kinase inhibitor genistein inhibits HHV-8-gBΔTM mediated cell adhesion. Maxisorp plates were coated with 4 μg of gBΔTM protein (100 μl/well) per ml overnight at 4°C, washed, and blocked with 1% BSA-PBS. Serum-starved HFFs were incubated with different concentrations of genistein in serum-free DMEM for 1 h at 37°C, collected by trypsin, washed, suspended in serum-free DMEM-0.1% BSA with different concentrations of genistein, and added to the protein-coated plates. Adhesion assays were carried out as described in the Fig. 3 legend. Each reaction was done in triplicate, and each point represents the average plus or minus the standard deviation of three experiments. Data are presented as percentage of inhibition of cell adhesion when cells were incubated with DMEM without genistein.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Akiyama, T., J. Ishida, S. Nakagawa, H. Ogawara, S. Watanabe, N. Itoh, M. Shibuya, and Y. Fukami. 1987. Genistein, a specific inhibitor of tyrosine-specific protein kinases. J. Biol. Chem. 262:5592-5595. - PubMed
    1. Akula, S. M., N. P. Pramod, F. Z. Wang, and B. Chandran. 2001. Human herpesvirus 8 envelope-associated glycoprotein B interacts with heparan sulfate-like moieties. Virology 284:235-249. - PubMed
    1. Akula, S. M., F. Z. Wang, J. Vieira, and B. Chandran. 2001. Human herpesvirus 8 interaction with target cells involves heparan sulfate. Virology 282:245-255. - PubMed
    1. Akula, S. M., N. P. Pramod, F. Z. Wang, and B. Chandran. 2002. Integrin alpha3beta1 (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. Anderson, H. A., Y. Chen, and L. C. Norkin. 1996. Bound simian virus 40 translocates to caveolin-enriched membrane domains, and its entry is inhibited by drugs that selectively disrupt caveolae. Mol. Biol. Cell 7:1825-1834. - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources