INTRODUCTION

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Human herpesvirus 8 (HHV-8) or Kaposi's sarcoma-associated herpesvirus (KSHV) DNA has been detected in the Kaposi's sarcoma (KS) tissues from patients with AIDS-KS, classic KS, Africa-endemic KS, and transplantation-associated KS (12, 44). HHV-8 has a broad in vivo and in vitro cellular tropism. HHV-8 DNA and transcripts have been identified in vivo in human B cells (15), macrophages (4), endothelial cells (5, 52), and epithelial cells (16). In the KS tissues, HHV-8 DNA is present in a latent form in the vascular endothelial and spindle cells (5, 15, 17, 44, 52). In addition, a low percentage of HHV-8 lytic cycle has been detected in the infiltrating inflammatory monocytes (4). HHV-8 DNA has been also detected in primary effusion lymphomas or body-cavity-based lymphomas (BCBL) (9, 17). BCBL cell lines such as BCBL-1 and BC-3 carry HHV-8 in a latent form, and a lytic cycle can be induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) (3, 17, 27, 40, 44, 50).

The in vitro infectious process of HHV-8 differ from many members of alpha-, beta-, and gammaherpesvirus families. HHV-8 has been shown to infect a variety of human and animal cells, such as human B cells, epithelial cells (293), human endothelial cells, human foreskin fibroblast (HFF) cells, human carcinoma cells (bladder, prostate, lung, and squamous), owl monkey kidney cells, and baby hamster kidney (BHK-21) cells (20, 34, 39, 58). If in vitro permissiveness of a cell type is judged by a productive lytic replication of HHV-8 after entry into cells, there is as yet no suitable cell culture system to support a lytic replication of input HHV-8. Only a latent HHV-8 infection is observed in the infected cells (20, 34, 39, 58). However, if in vitro permissiveness is judged by the establishment of HHV-8 latency and the ability to support HHV-8 lytic replication after activation by agents, cells such as HFF, human carcinoma cells and endothelial cells are permissive, as evidenced by the retention of viral genome in a latent form, by the expression of HHV-8 latency-associated open reading frame (ORF) 73 protein and by the ability to support lytic replication upon activation by agents such as TPA or by human cytomegalovirus (HCMV) infection (20, 34, 39, 58).

Since the analysis of in vitro HHV-8 interaction with host cells and quantitation of infection has been hampered by the absence of the lytic replication cycle and a reliable plaque assay, to monitor the HHV-8 binding and entry process BCBL-1 cells carrying a recombinant HHV-8 expressing the green fluorescent protein (GFP-HHV-8) were established (58). In a recent study, using the GFP-HHV-8 in the supernatant of TPA induced BCBL-GFP cells as the inoculum for infection and the [³H]thymidine-labeled purified HHV-8, we demonstrated that the broad cellular tropism of HHV-8 may be in part due to its interaction with the ubiquitous host cell surface HS molecule (2). This conclusion was based on the following findings: (i) HHV-8 infection of HFF cells was inhibited in a dose-dependent manner by soluble heparin, a glycosaminoglycan closely related to HS; (ii) enzymatic removal of HFF cell surface HS with heparinase I and III reduced HHV-8 infection; (iii) soluble heparin inhibited the binding of radiolabeled HHV-8 to human B-cell lines, embryonic kidney epithelial (293) cells, and HFF cells, suggesting interference at the virus attachment stage; (iv) cell surface-adsorbed HHV-8 was displaced by soluble heparin; and (v) radiolabeled HHV-8 also bound to wild-type HS expressing Chinese hamster ovary (CHO-K1) cells. In contrast, binding of virus to mutant CHO cells deficient in HS was significantly reduced. These data suggested that the gamma-2-HHV-8 is adsorbed to cells by binding to cell surface HS-like moieties. In this respect, gamma-2-HHV-8 resembles some members of the alpha (herpes simplex virus type 1 [HSV-1], HSV-2, pseudorabies virus [PRV], bovine herpesvirus 1 [BHV-1])-, beta (HCMV, HHV-7)-, and gamma-2 (BHV-4)-herpesviruses, where the initial virus-cell interaction also involves the binding to the cell surface HS (21, 24, 30, 31, 33, 35, 37, 46, 47, 49, 51, 57).

The identity of HHV-8 envelope glycoprotein(s) involved in the interaction with HS is not known. HHV-8 encodes for more than 80 ORFs, and ORFs 4 to 75 are designated based on the similarity to herpesvirus saimiri (HVS) ORFs (1). HHV-8 unique ORFs are designated with the prefix K (36, 43). HHV-8 has counterparts to other herpesvirus glycoproteins such as gB (ORF 8), gH (ORF 22), gM (ORF 39), and gL (ORF47) (36, 43). In addition, K1 and K8.1 genes encode for glycoproteins unique for HHV-8 (10, 36, 43). We have previously reported the identification of cDNAs originating from the HHV-8 K8.1 gene encoding two ORFs from spliced messages (10). One cDNA encoded for a 228-amino-acid (aa) protein designated gpK8.1.A and contains a signal sequence, transmembrane domain, and four N-glycosylation sites. The splicing event has generated the transmembrane domain in the gpK8.1A ORF not seen in the genomic K8.1 ORF. Another cDNA encoded for an ORF of 167 aa, designated gpK8.1.B. This protein has three N-glycosylation sites and shares similar amino and carboxy termini with ORF K8.1.A but with an in-frame deletion (10). Our studies with human sera demonstrated the immunogenic nature of gpK8.1A and gpK8.1B (10, 61). Using monoclonal antibodies (MAbs), we have also shown that gpK8.1A and gpK8.1B contain N- and O-linked sugars and that gpK8.1A is the predominant form detected within the infected cells and the virion envelopes (60).

HHV-8 K8.1 gene is positionally colinear to the glycoprotein genes in the members of gammaherpesvirus group such as the Epstein-Barr virus (EBV) gene encoding the major envelope glycoproteins gp350 and gp220 (22), gp150 of murine gammaherpesvirus 68 (MHV-68) (53), HVS ORF 51 gene (1), and the BORFD1 gene of BHV-4 (36, 43). EBV gp350/gp220 glycoprotein has been studied extensively and shown to be involved in the binding of the virus to the target cells (22). HHV-8 gpK8.1A shows several similarities to the EBV glycoproteins. Like EBV gp350/gp220, HHV-8 gpK8.1A elicits a strong human humoral immune response (61) and is a virion envelope-and infected cell membrane-associated glycoprotein containing both N- and O-linked sugars. Because of these similarities to EBV gp350/gp220, we examined the ability of HHV-8 gpK8.1A to interact with the target cells. We expressed gpK8.1A without the transmembrane and carboxyl domains (TMgpK8.1A) in the baculovirus system and purified the protein. Using radiolabeled purified TMgpK8.1A, we show that gpK8.1A interaction with target cells involves cell surface HS-like moieties. These results suggest that HHV-8 interaction with HS could be in part mediated by virion envelope gpK8.1A.

	MATERIALS AND METHODS

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Cells. HFF cells, 293 cells, CHO-K1 cells (ATCC CCL-61), HS-deficient CHO derivative pgsD-677 cells (ATCC CRL-2244), HS-and chondroitin sulfate-deficient CHO derivative pgsA-745 cells (ATCC CRL-2242) (18, 32), BCBL-1 cells (HHV-8⁺ human B cells) (40, 50), and BJAB cells (HHV-8 human B cells) were used in this study. HFF and 293 monolayer cells were grown in Dulbecco modified Eagle medium (DMEM; Gibco-BRL, Grand Island, N.Y.) supplemented with 2 mM glutamine, 10% fetal bovine serum (FBS), and antibiotics. Suspension cultures of 293 cells were grown in 293-SF medium (Gibco-BRL). Adult human dermal microvascular endothelial cells (HMVEC-d Ad; CC-2543; Clonetics, San Diego, Calif.) were grown in endothelial growth medium (EGM; CC-3202; Clonetics). Monolayers of CHO-K1, pgsD-677, and pgsA-745 cells were grown in Ham's F-12K medium (Gibco-BRL) supplemented with 10% FBS and antibiotics. Suspension cultures of BCBL-1 and BJAB cells were grown in RPMI 1640 medium with glutaMAX I (Gibco-BRL) supplemented with 10% FBS and antibiotics. Spodoptera frugiperda ovarian cells (Sf9) were grown in TNM-FH insect medium (PharMingen, San Diego, Calif.).

Antibodies. The production and characterization of MAbs against gpK8.1A and ORF 59 have been described previously (11, 60). High-titer-antibody-containing ascitic fluids were made by injecting hybridoma cells intraperitoneally into pristane-primed BALB/c mice. Immunoglobulin G (IgG) antibodies from the ascitic fluid and normal mouse sera were purified on protein A-Sepharose columns (Amersham Pharmacia Biotech AB, Uppsala, Sweden). Protein concentrations were adjusted to 1 mg/ml with phosphate-buffered saline (PBS; pH 7.4), and aliquots were stored at 20°C. Rabbit polyclonal antibodies raised against the baculovirus-expressed purified glutathione S-transferase-HHV-8 latency-associated ORF 73 protein (34, 58, 61) were used as a control.

Construction, expression, and purification of recombinant HHV-8 TMgpK8.1A. The 576-bp TMgpK8.1A gene region encoding aa 1 to 192 lacking the transmembrane and the carboxyl domains was amplified from the full-length HHV-8 gpK8.1A cDNA (10) using the primers TMgpK8.1A forward (5'-TTC CGC GTG AGC TCA TGA GTT CCA CAC AGA-3' with a SacI site) and TMgpK8.1A reverse (5'-GAT GGG TCG GTA CCT CTG CAT TGT AGT-3' with a KpnI site) (Advantage cDNA PCR Kit; Clontech, Palo Alto, Calif.). The TMgpK8.1A PCR product was cloned into the pAcHLT-A baculovirus transfer vector (PharMingen) and verified by sequencing. To generate the recombinant baculovirus, TMgpK8.1A-pAcHLT-A plasmid was cotransfected with BaculoGold DNA (PharMingen) into Sf9 insect cells. Recombinant viruses were passaged three times before use.

Western blot assays. Samples were boiled in sample buffer with 2-mercaptoethanol (2-ME), subjected to SDS-PAGE, and electrophoretically transferred onto nitrocellulose membranes. Standard prestained molecular weight markers (Gibco-BRL) were included in parallel lanes. The membranes were soaked in blocking solution (10 mM Tris-HCl, pH 7.2; 150 mM NaCl; 5% skim milk or 5% bovine serum albumin [BSA]; 0.02% NaN₃) at 4°C overnight and then reacted with antibodies for 3 h at room temperature. The membranes were washed five times with washing buffer (10 mM Tris-HCl, pH 7.2; 150 mM NaCl; 0.3% Tween 20) and incubated for 1 h with alkaline phosphatase (AP)-conjugated secondary antibodies (KPL, Gaithersburg, Md.). Bound enzyme-labeled antibodies were detected by evaluating the color reaction of AP with nitroblue tetrazolium and BCIP (5-bromo-4-chloro-3-indolyphosphate) substrates (Sigma). The reactions were stopped by washing the membranes in distilled water.

Surface immunofluorescence assay (SIFA). To detect the binding of gpK8.1A and TMgpK8.1 to the cell surface, BJAB and 293 suspension cells or HFF and 293 cell monolayers in chamber slides were used (2, 60). A suspension of cells (10⁷) was washed once with RPMI 1640, resuspended in 10 ml of ice-cold 0.1% paraformaldehyde in PBS (pH 7.4), and centrifuged at 125 × g for 10 min. The cells were washed twice, the concentration was adjusted to 10⁶ cells per ml, the cells were centrifuged for 10 min at 125 × g and the supernatants were discarded. Dilutions of purified gpK8.1A (1 µg/ml) and TMgpK8.1A (1 µg/ml) in 200 µl of RPMI 1640 with 10% FBS were added to the cell pellets, mixed, and incubated for 30 min at 37°C. These cells were washed five times with RPMI 1640 with 0.01% NaN₃ and then incubated with prestandardized dilutions of gpK8.1A MAb or control antibodies for 30 min at 37°C. Cells were washed five times with RPMI 1640 with 0.01% NaN₃ and incubated for 30 min at 37°C with prestandardized fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse or anti-rabbit IgG antibodies. Cells were washed and mounted on glass slides, and cell-bound gpK8.1A was examined under a fluorescence microscope. For binding with monolayers, cells were treated with 0.1% paraformaldehyde, washed five times with RPMI 1640, incubated with dilutions of purified proteins, and processed as described above.

Radiolabeled TMgpK8.1A binding assays. A protein binding assay was performed according to the method described previously (6), with minor modifications. Briefly, confluent monolayers of HFF, 293, HMVEC-d, CHO-K1, and CHO mutant derivative cells in 96- or 24-well plates were washed and blocked for 30 min at 4°C with PBS containing 1% FBS, 5 mM albumin, and 0.1 mM CaCl₂. Cells were incubated with different concentrations of purified TMgpK8.1A (7,666 cpm/µg of protein) or purified ORF 73 protein (14,672 cpm) in DMEM with 10% FBS and 0.01% NaN₃ for 90 min at 4°C. After incubation, cells were washed five times with DMEM and lysed with 1% SDS and 1% Triton X-100 in distilled water, and the cell-bound TMgpK8.1A radioactivity was counted. All experiments were done in triplicate and were repeated three times.

Blocking HHV-8 binding by purified TMgpK8.1A. Radiolabeled HHV-8 binding assay was performed using HFF cells as per methods described previously (2). Briefly, HFF cells were incubated with increasing concentrations of purified unlabeled TMgpK8.1A for 90 min at 4°C, followed by the addition of a fixed quantity of [³H]thymidine-labeled purified HHV-8 (2,684 cpm) (2). For a control, a fixed quantity of [³H]thymidine-labeled purified HHV-8 (2,684 cpm) was mixed with 10 µg of heparin per ml for 90 min at 4°C and then added to HFF cells. After incubation for 90 min at 4°C with the virus, cells were washed five times and lysed with 1% SDS and 1% Triton X-100, and the radioactivity was precipitated with trichloroacetic acid (TCA) and counted. The cell-associated virus cpm in the absence or presence of unlabeled TMgpK8.1A or heparin and the percentage of inhibition of virus binding were calculated. All reactions were done in triplicate and repeated three times.

TMgpK8.1A binding with heparin-agarose. Purified TMgpK8.1A (3.5 µg) was preincubated with 350 µg of various glycosaminoglycans (GAGs) such as heparin, HS, chondroitin sulfate A (CS-A), chondroitin sulfate B (CS-B), chondroitin sulfate C (CS-C), N-acetyl heparin, and de-N-sulfated heparin (Sigma). After being mixed for 1 h at 4°C, 100 µl of a 50% slurry of heparin-agarose beads (Sigma) equilibrated in radioimmunoprecipitation assay (RIPA) lysis buffer (0.05 M Tris hydrochloride, pH 7.5; 0.15 M NaCl; 1% sodium deoxycholate; 1% Triton X-100, 100 U of aprotinin per ml; 0.1 mM phenylmethylsulfonyl fluoride) (60) was added and further mixed for 2 h at 4°C. The heparin-agarose beads were washed five times in RIPA buffer. The bound material was eluted by boiling the beads in sample buffer with 2-ME, resolved by SDS-12%PAGE, Western blotted, and analyzed with anti-gpK8.1A MAb.

Virion envelope-associated gpK8.1A binding with heparin-agarose. HHV-8 from TPA-induced BCBL-1 cells was purified by two cycles on a sucrose density gradient as per method described before (13). Purified virus was labeled with biotin according to the manufacturer's recommendations (Gibco-BRL), and the free biotin was removed by extensive dialysis against 0.5 M sodium carbonate buffer (pH 9.0) and then against PBS (pH 7.4). The cell-binding activity of biotin-labeled virus was tested by SIFA as described above, and bound virus was detected by use of gpK8.1A MAb or FITC-labeled streptavidin. To test the binding activity with heparin-agarose, biotin-labeled purified HHV-8 was lysed with RIPA buffer, sonicated, and centrifuged at 100,000 × g for 1 h at 4°C. The resulting soluble biotinylated envelope protein supernatant was mixed with 100 µl of 50% slurry of heparin-agarose or agarose beads in RIPA buffer and mixed for 2 h at 4°C. The beads were washed five times in RIPA buffer, boiled in sample buffer with 2-ME, resolved by SDS-10% PAGE, Western blotted, and analyzed with anti-gpK8.1A MAb or with AP-conjugated streptavidin (Dako, Carpinteria, Calif.).

Purification of HHV-8 full-length gpK8.1A. Full-length gpK8.1A was purified using methods previously described (60). Briefly, TPA (Sigma, St. Louis, Mo.)-induced BCBL-1 cells were lysed on ice for 1 h with lysis buffer (10 mM Tris-HCl, pH 8.0; 140 mM NaCl; 0.025% NaN₃; 2% Triton X-100; 1% sodium deoxycholate; 0.2 U of aprotinin per ml; 1 mM phenylmethylsulfonyl fluoride). Cell lysates were passed over a column of Sepharose 4B covalently coupled with gpK8.1A-specific MAb 4D6 at 4°C. The unbound proteins were removed by extensive washing with lysis buffer. The bound gpK8.1A was eluted with low-pH buffer (50 mM glycine-HCl [pH 2.5] in 150 mM NaCl and 0.1% NP-40) and immediately neutralized with a 1/10 volume of 1 M Tris-HCl (pH 8.0). The peak fractions were pooled, dialyzed against PBS (pH 7.0), and stored at 70°C.

	RESULTS

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HHV-8 envelope glycoprotein gpK8.1A binds to the target cells. The HHV-8 K8.1 gene encodes two ORFs, gpK8.1A and gpK8.1B, which are derived from spliced mRNAs (10). The gpK8.1A ORF is 228 aa long with a signal sequence and a transmembrane domain, consisting of a 167-aa region identical to gpK8.1B and a unique 61-aa region (Fig. 1A). The amino-terminal 142-aa region of gpK8.1A is identical to the 197-aa genomic K8.1 ORF with the splicing event generating the gpK8.1A ORF transmembrane domain absent in the genomic K8.1 ORF (Fig. 1A) (10). HHV-8 gpK8.1A is a virion envelope-associated immunogenic glycoprotein containing both N- and O-linked sugars (60). MAbs against gpK8.1A recognized multiple proteins with molecular masses ranging from 34 to 72 kDa from BCBL-1 cells and 68- to 72-kDa proteins from the virion particles (60). These multiple proteins represent the precursor and glycosylated forms of gpK8.1A (60).

View larger version (26K): [in this window] [in a new window]   FIG. 1.   (A) Construction of TMgpK8.1A without the transmembrane and carboxyl domains. The top line shows the schematic diagram of HHV-8 genome and the location of encoded glycoprotein ORFs. The genomic K8.1 ORF is 197 aa long with a signal sequence (SS) and without the transmembrane (TM) sequence. The 228-aa gpK8.1A ORF with signal and transmembrane sequences is derived from a spliced mRNA (10). The TMgpK8.1A was constructed by using primers amplifying aa 1 to 192 with the signal sequence but lacking the transmembrane and the carboxyl domains. (B) Expression and purification of TMgpK8.1A in the baculovirus expression system. Sf9 insect cells were infected with TMgpK8.1A-baculovirus for 2 days and labeled with [35S]methionine for 20 h. His-tagged TMgpK8.1A protein from the cell pellet was purified by use of a nickel column. Protein purity was analyzed by SDS-12% PAGE gels, Western blots with anti-gpK8.1 MAb, and autoradiography. Lane 1, full-length gpK8.1A affinity purified from HHV-8-infected BCBL-1 cells detected by Western blot reaction with anti-gpK8.1A MAb; lane 2, TMgpK8.1A-expressing Sf9 insect cell lysate in Western blot reactions with anti-gpK8.1A MAb; lane 3, TMgpK8.1A-expressing Sf9 cells culture supernatant in Western blot reactions with anti-gpK8.1A MAb; lane 4, [35S]methionine-labeled purified TMgpK8.1A in Western blot reactions with anti-gpK8.1A MAb; lane 5, autoradiography of [35S]methionine-labeled purified TMgpK8.1A. The numbers on the left indicate the molecular masses (in kilodaltons) of the standard protein markers run in parallel lanes. The glycosylated forms of TMgpK8.1A are marked on the right.

View larger version (9K): [in this window] [in a new window]   FIG. 2.   HHV-8 gpK8.1A binds to the target cells. Binding of purified full-length gpK8.1A and TMgpK8.1A to the target cells was detected by surface immunofluorescence assay. Paraformaldehyde-treated BJAB, 293, HFF, or HMVEC-d cells were incubated with medium alone (controls) or medium with purified proteins for 30 min at 37°C. After cells were washed, anti-gpK8.1A-specific MAb or anti-HHV-8 ORF 59 MAb (11) or rabbit anti-HHV-8 ORF 73 antibodies (34) were added, incubated for 30 min at 37°C, washed, and incubated for an additional 30 min at 37°C with FITC-conjugated goat anti-mouse or anti-rabbit IgG antibodies. Cells were washed, mounted, and examined under a fluorescence microscope. (A and B) BJAB and 293 cells, respectively, incubated with the full-length affinity-purified gpK8.1A and anti-gpK8.1A MAb. (C) BJAB cells incubated with anti-gpK8.1A MAb alone. (D) BJAB cells incubated with the purified His-tagged TMgpK8.1A and anti-gpK8.1A MAb. Fluorescence signals detected on the surface of cells indicate the cell-bound gpK8.1A and TMgpK8.1A.

Expression and purification of HHV-8 TMgpK8.1A without transmembrane and cytoplasmic domains. Since only about 20% of TPA-induced BCBL-1 cells expressed HHV-8 lytic-cycle proteins, the yield of purified gpK8.1A by affinity chromatography was insufficient for binding studies. Hence, a 576-bp TMgpK8.1A gene region encoding aa 1 to 192 lacking the transmembrane and the carboxyl domains was amplified by PCR (Fig. 1A), cloned, and expressed in the baculovirus system. The TMgpK8.1A-baculovirus-infected Sf9 cell pellets and the culture supernatant were analyzed in Western blot reactions with gpK8.1A-specific MAbs. The predicted molecular mass of unglycosylated TMgpK8.1A is about 21 kDa. MAbs recognized proteins ranging from 29 to 42 kDa from the TMgpK8.1A-baculovirus-infected Sf9 cell pellets and culture supernatant (Fig. 1B, lanes 2 and 3). These proteins represent the different glycosylated forms of TMgpK8.1A. The molecular masses of baculovirus-expressed TMgpK8.1A proteins (Fig. 1B, lanes 2 and 3) were smaller than the gpK8.1A from the BCBL-1 cells (Fig. 1B, lane 1). This could be due to the absence of 36 aa in TMgpK8.1A, as well as to the differences in the efficiency of N and O glycosylation between insect and mammalian cells (60, 61). HHV-8 ORF 59 MAbs or normal mouse IgG did not react with TMgpK8.1A protein in Western blot reactions (data not shown).

HHV-8 TMgpK8.1A binds to the target cells. To determine whether TMgpK8.1A binds to the target cells, unlabeled purified TMgpK8.1A was allowed to bind the paraformaldehyde-treated BJAB, 293, HFF, or HMVEC-d cells, which were washed and tested with anti-gpK8.1A MAbs in SIFAs. Bright-ring-type fluorescence was observed only on cells incubated with TMgpK8.1A, and the results with BJAB cells are shown in Fig. 2D. Binding was not detected when cells were incubated with purified His-tagged ORF 73 protein (data not shown). These data further confirm the interaction of gpK8.1A with the cell surface and show that the extracellular domains of gpK8.1A mediate this binding.

View larger version (14K): [in this window] [in a new window]   FIG. 3.   (A) Binding of radiolabeled TMgpK8.1A to HFF cells. Different concentrations of [35S]methionine-labeled purified TMgpK8.1A (7,666 cpm/µg of protein) or ORF 73 (14,672 cpm/µg of protein) proteins were incubated for 90 min at 4°C with HFF cells in 96- or 24-well plates. After incubation, cells were washed five times and lysed with 1% SDS and 1% Triton X-100, and the cell-bound TMgpK8.1A radioactivity was counted. Each reaction was done in triplicate and each point represents the average ± the standard deviation (SD) of three experiments. Similar results were seen with cells in 96- and 24-well plates, and the results with the 96-well plates are shown here. (B) Inhibition of labeled TMK8.1A binding to cells by unlabeled TMK8.1A protein. HFF cells were preincubated with the indicated concentrations of nonlabeled TMgpK8.1A for 15 min and then incubated with 3.5 µg (for cells in the 96-well plate) or 15 µg (for cells in the 24-well plate) of 35S-labeled TMgpK8.1A (7,666 cpm/µg of protein) for 90 min at 4°C. Cells were washed five times and lysed with 1% SDS and 1% Trition X-100, and the cell-bound TMgpK8.1A radioactivity was counted. The cell-associated radiolabeled TMgpK8.1A cpm in the presence or absence of unlabeled protein was calculated. In the absence of unlabeled TMgpK8.1A protein, about 30% of the input labeled TMgpK8.1A (1.1 and 4.5 µg for cells in the 96-well and 24-well plates, respectively) became associated with the cells. Each reaction was done in triplicate, and each point represents the average ± the SD of three experiments.

Purified TMgpK8.1A blocks HHV-8 binding to the target cells. To determine if the interaction of gpK8.1A with the cell surfaces is biologically relevant, the ability of purified nonradiolabeled TMgpK8.1A to complete with [³H]thymidine-labeled HHV-8 binding to HFF cells was examined. HFF cells were preincubated with increasing concentrations of purified unlabeled TMK8.1A for 90 min at 4°C, followed by a constant quantity of [³H]thymidine-labeled purified HHV-8 (2,684 cpm), which is within the linear range of the dose-response curve (2). As a control for these experiments, a constant quantity of [³H]thymidine-labeled purified HHV-8 (2,684 cpm) was incubated with 10 µg of heparin per ml for 90 min at 4°C and then added to HFF cells. In the absence of heparin or unlabeled TMgpK8.1A protein, approximately 21% of the input HHV-8 radioactivity became associated with the cells. As in to our earlier observation (2), 10 µg of heparin blocked approximately 90% of HHV-8 attachment to the cells (data not shown). HHV-8 adsorption was also blocked by the unlabeled purified TMgpK8.1A in a dose-dependent fashion (Fig. 4). HHV-8 binding was diminished by approximately 70% compared to the untreated control (Fig. 4). Treatment of cells with identical concentrations of BSA or ORF 73 protein had no effect on virus binding, suggesting that the block in HHV-8 binding was probably to the engagement of a necessary HHV-8 cellular receptor by gpK8.1A and was not simply due to protein-protein interference. This suggested that gpK8.1A occupied cell surface molecule(s) that functions as an HHV-8 attachment receptor.

View larger version (15K): [in this window] [in a new window]   FIG. 4.   Nonradiolabeled TMgpK8.1A blocks HHV-8 attachment. HFF cells were incubated with increasing concentrations of purified unlabeled TMgpK8.1A for 90 min at 4°C, followed by the addition of a fixed quantity of [3H]thymidine-labeled purified HHV-8 (2,684 cpm) (2). For a control, a fixed quantity of [3H]thymidine-labeled purified HHV-8 (2,684 cpm) was mixed with 10 µg of heparin per ml for 90 min at 4°C and then added to HFF cells. After incubation for 90 min at 4°C with the virus, cells were washed five times and lysed with 1% SDS and 1% Triton X-100, and the radioactivity was precipitated with TCA and counted. The cell-associated virus cpm in the absence or presence of unlabeled TMgpK8.1A and heparin and the percentage of inhibition of virus binding were calculated. In the absence of heparin or TMgpK8.1A, approximately 21% of the input HHV-8 radioactivity (552 cpm) became associated with the cells. Approximately 90% of HHV-8 attachment to the cells was blocked by heparin. Each reaction was done in triplicate, and each point represents the average ± the SD of three experiments.

Heparin blocks HHV-8 TMgpK8.1A binding to the target cells. Heparin is closely related to HS, and inhibition of virus infectivity by heparin treatment has been considered as an evidence for alpha-, beta-, and gamma-2-herpesvirus interaction with cell surface HS molecules (21, 24, 30, 31, 33, 35, 37, 46-47, 49, 51, 57). Our recent studies showed that HHV-8 interaction with host cell surface involved HS and soluble heparin prevented HHV-8 infectivity (2). To determine whether heparin inhibits TMgpK8.1A binding, a constant quantity of purified radiolabeled TMgpK8.1A within the linear range of the dose-response curve (3.5 µg for cells in 96-well plates or 15 µg for cells in 24-well plates) (Fig. 3A) was mixed with medium alone or medium with different concentrations of heparin and incubated at 4°C for 90 min. These were then added to the paraformaldehyde-treated target cells and incubated at 37°C for 90 min or to the untreated target cells and incubated at 4°C for 90 min. After incubation, cells were washed five times and cell-associated TMgpK8.1A cpm values were counted. Similar results were observed when binding assays were performed at 4 or at 37°C, and results with untreated cells 4°C are shown in Fig. 5A.

View larger version (23K): [in this window] [in a new window]   FIG. 5.   Inhibition of [35S]methionine-labeled purified TMgpK8.1A binding to target cells by heparin. (A) A constant quantity of purified labeled TMgpK8.1A (7,666 cpm/µg of protein) within the linear range of the dose-response curve (3.5 µg for cells in the 96-well plate or 15 µg for cells in the 24-well plate) (Fig. 3A) was mixed with medium alone or with different concentrations of heparin or CS-A, CS-B, or CS-C and then incubated for 90 min at 4°C. These mixtures were then incubated with HFF or adult HMVEC-d (Endo) for 90 min at 4°C and washed five times. Cells were lysed with 1% SDS-1% Triton X-100 and counted. The cell-associated TMgpK8.1A cpm in the presence or absence of heparin and the percentage of inhibition of TMgpK8.1A binding were calculated. In the absence of heparin, approximately 30% of the input TMgpK8.1A radioactivity (1.1 and 4.5 µg for cells in the 96-well and 24-well plates, respectively) became associated with the cells. Each reaction was done in triplicate and each point represents the average ± the SD of three experiments. (B) Displacement of adsorbed TMgpK8.1A from the HFF cell surface by heparin. HFF cell. monolayers in 96-well plates were incubated with a constant quantity (3.5 µg) of purified labeled TMgpK8.1A (7,666 cpm/µg of protein). At the indicated time points, cells were incubated with medium (controls) or with medium containing 10 µg of heparin or CS-A, CS-B, or CS-C per ml. Cells were further incubated for a total of 90 min at 4°C, washed five times, and then counted. The cell-associated TMgpK8.1A cpm in the presence or absence of heparin and the percentage of inhibition of TMgpK8.1A binding were calculated. In the absence of heparin, approximately 30% of the input TMgpK8.1A radioactivity (1.1 µg) became associated with the cells. Each reaction was done in triplicate, and each point represents the average ± the SD of three experiments.

Displacement of cell surface adsorbed HHV-8 TMgpK8.1A by heparin. To determine the specificity of HHV-8 TMgpK8.1A interaction with cell surface HS and the inhibition by heparin, labeled TMgpK8.1 was first allowed to adsorb to the HFF cells and at different times postadsorption, heparin or CS-A, -B, or -C were added to a final concentration of 10 µg/ml. Cells were further incubated for a total period of 90 min, and the cell-associated radioactivity was counted. Similar results were observed when binding assays were performed with paraformaldehyde-treated cells at 37°C or with untreated cells at 4°C. The results with untreated cells at 4°C are shown in Fig. 5. Pretreatment of HFF cells with heparin did not affect TMgpK8.1 binding (data not shown). In the absence of heparin, about 30% of the input labeled TMgpK8.1A (1.1 µg) became associated with the cells. In contrast, when heparin was added to the TMgpK8.1A protein-cell mixture, it was capable of displacing already-adsorbed TMgpK81A even when added 40 min after the protein addition to the cells (Fig. 5B). The partial reversal of binding by the addition of heparin after 50 min of protein-cell interaction (Fig. 5B) could be due to the onset of interactions between gpK8.1A and cellular receptors other than HS molecules. Reversal of TMgpK8.1A binding to the cells by heparin demonstrated the specificity of HS interaction with HHV-8 gpK8.1A. Specificity was also shown by the absence of any significant inhibition by the same amount (10 µg/ml) of CS-A, -B, and -C (Fig. 5B).

HHV-8 TMgpK8.1A binds to the HS-expressing CHO-K1 cell line but not to cells lacking HS. To verify the role of HS in the attachment of TMgpK8.1A to the target cells, binding assays were done with wild-type CHO-K1 cell line expressing HS and its two mutant cell lines, pgsD-677 cells (deficient in HS but not in chondroitin sulfate) and pgsA-745 cells (deficient in both HS and chondroitin sulfate). Radiolabeled TMgpK8.1A (3,310 cpm/µg of protein) bound readily to the wild-type CHO-K1 cells to the same extent as HFF cells. About 4 µg or 26% of the input labeled TMgpK8.1A became associated with the CHO-K1 cells (Fig. 6). In contrast, TMgpK8.1A binding to the mutant cells was significantly impaired, and about fivefold-less binding was detected with the pgsD-677 and pgsA-745 cells (Fig. 6). These results confirmed the interaction of HHV-8 TMgpK8.1A with the cell surface HS. The low percentage of TMgpK8.1A binding to the cells lacking HS further supported the notion that gpK8.1A also probably binds other host cell molecules.

View larger version (14K): [in this window] [in a new window]   FIG. 6.   Binding of radiolabeled TMgpK8.1A to CHO-K1 cells. Confluent monolayers of wild-type CHO-K1 cells and of two CHO mutants, pgsD-677 (lacking HS but not chondroitin sulfate) and pgsA-745 (lacking both HS and chondroitin sulfate), in 24-well plates were incubated with 15 µg of [35S]methionine-labeled purified TMgpK8.1A (3,310 cpm/µg of protein) for 90 min at 4°C. The cells were washed five times and lysed in 1% SDS-1% Triton X-100, and the cell-associated radioactivity was counted. About 4 µg or 26% of the input TMgpK8.1A radioactivity bound to CHO-K1 cells. The results are expressed as the percentage of radioactivity bound to the wild-type CHO-K1 cells. Each reaction was done in triplicate, and each point represents the average ± the SD of three experiments.

HHV-8 TMgpK8.1A specifically binds to heparin. To verify the specificity of gpK8.1A binding to HS, the ability of TMgpK8.1A to bind the heparin-agarose beads was tested. Purified TMgpK8.1A, HHV-8 gL, or HHV-8 ORF 73 (2.5 µg) protein was incubated with heparin-agarose beads. After an extensive washing, the beads were boiled in sample buffer. The eluted proteins were detected by immunoblot using anti-gpK8.1A MAb, rabbit anti-gL antibodies, and rabbit anti-ORF 73 antibodies. Representative results are presented in Fig. 7. HHV-8 gL and ORF 73 proteins were not precipitated by heparin-agarose beads (data not shown). In contrast, heparin-agarose beads precipitated the various glycosylated forms of TMgpK8.1A (Fig. 7A, lane 1). To determine the specificity of this reaction, various GAGs were tested to compete with the heparin-agarose binding activity of TMgpK8.1A. Heparin-agarose binding activity of TMgpK8.1A was competitively inhibited by preincubating the protein with 350 µg of heparin (Fig. 7A, lane 2) or 350 µg of HS (Fig. 7A, lane 3). In contrast, 350 µg of CS-A, -B, and -C, N-acetyl heparin, and de-N-sulfated heparin did not inhibit the TMgpK8.1 interaction with the heparin-agarose beads (Fig. 7A, lanes 4 to 8). No reactivity was seen with agarose beads alone (Fig. 7A, lane 9), thus demonstrating the specificity of these reactions. These results confirmed the interaction of gpK8.1A with HS and heparin.

View larger version (25K): [in this window] [in a new window]   FIG. 7.   (A) HHV-8 TMgpK8.1A binding to heparin-agarose beads. Purified TMgpK8.1A (2.5 µg) was incubated with or without 350 µg of various GAGs for 1 h at 4°C and then with heparin-agarose beads for 2 h at 4°C. These mixtures were washed five times, and bound material was eluted by boiling in sample buffer, analyzed by SDS-12% PAGE gels, and tested with anti-gpK8.1A MAb in Western blot reactions. Lane 1, purified TMgpK8.1A with heparin-agarose beads; lanes 2 to 8, purified TMgpK8.1A preincubated with heparin (lane 2), HS (lane 3), CS-A (lane 4), CS-B (lane 5), CS-C (lane 6), N-acetyl heparin (lane 7), and de-N-sulfated heparin (lane 8) before the addition of heparin-agarose beads; lane 9, purified TMgpK8.1A with agarose beads. The numbers on the left indicate the molecular masses (in kilodaltons) of the standard protein markers run in parallel lanes. The glycosylated forms of TMgpK8.1A are marked on the right. (B) Dose-response results of heparin blocking HHV-8 TMgpK8.1A binding to heparin-agarose beads. Purified TMgpK8.1A (2.5 µg) was preincubated with different concentrations of heparin for 1 h at 4°C and then incubated with heparin-agarose beads for 2 h at 4°C. These mixtures were washed five times, and bound material was eluted by boiling the beads in sample buffer and then analyzed by SDS-12% PAGE gels and in Western blot reactions with anti-gpK8.1A MAb. Lane 1, purified TMgpK8.1A with heparin-agarose beads; lanes 2 to 7, purified TMgpK8.1A preincubated with 300 µg (lane 2), 150 µg (lane 3), 75 µg (lane 4), 38 µg (lane 5), or 19 µg (lane 6) of heparin before the addition of heparin-agarose beads; lane 7, purified TMgpK8.1A with agarose beads. The numbers on the left indicate the molecular masses (in kilodaltons) of the standard protein markers run in parallel lanes. The glycosylated forms of TMgpK8.1A are marked on the right.

Virion envelope-associated gpK8.1A binds heparin-agarose. To determine whether virion envelope associated gpK8.1A binds heparin-agarose, density gradient-purified HHV-8 was labeled with biotin. The biotinylated virus bound to the target cells in the surface immunofluorescence assay (data not shown). Similar to our earlier findings (60), gpK8.1A MAbs recognized the 68- to 72-kDa protein in Western blot reactions with purified HHV-8 (Fig. 8, lane 1). Biotinylated purified virus was lysed with RIPA buffer, sonicated, and centrifuged at 100,000 × g. The resulting supernatant containing the soluble biotinylated envelope proteins was mixed with heparin-agarose or agarose beads for 2 h at 4°C and washed. The bound material was eluted by boiling in sample buffer, resolved by SDS-PAGE, Western blotted, and analyzed with AP-labeled streptavidin and substrate. Polypeptides of about 30 to 45 kDa precipitated both by agarose and by heparin-agarose were considered nonspecific bands (Fig. 8, lanes 2 and 3). Polypeptides of ca. 75, 72, and 54 kDa were specifically precipitated by heparin-agarose beads only (Fig. 8, lane 3). Preincubation of soluble biotinylated proteins with 350 µg of heparin or HS prevented the interaction of these specific proteins with heparin-agarose beads (data not shown). When the proteins precipitated by heparin-agarose beads were reacted with anti-gpK8.1A MAbs in the Western blots, only the 72-kDa protein was specifically recognized (Fig. 8, lane 4), and no reactivity was seen with agarose bead-precipitated proteins (data not shown). No reaction was seen when proteins precipitated by heparin-agarose beads were reacted with rabbit anti-gL IgG antibodies (Fig. 8, lane 5). The identity of the heparin-agarose interacting 75- and 54-kDa HHV-8 envelope glycoproteins is under investigation. These results demonstrated the ability of virion envelope-associated gpK8.1A to interact with heparin-agarose and thus HS.

View larger version (39K): [in this window] [in a new window]   FIG. 8.   Virion envelope-associated gpK8.1A binding with heparin-agarose. Biotin-labeled purified HHV-8 was lysed with RIPA buffer, sonicated, and centrifuged at 100,000 × g for 1 h at 4°C. The resulting supernatant containing soluble biotinylated envelope proteins was mixed with heparin-agarose or agarose beads, mixed for 2 h at 4°C, and washed five times in RIPA buffer. The bound material was eluted by boiling the beads in sample buffer with 2-ME, resolved by SDS-12% PAGE, Western blotted, and analyzed. Lane 1, purified virus solubilized by sample buffer in Western blot reactions with anti-gpK8.1A MAb; lane 2, biotinylated proteins eluted from the agarose beads reacted with AP-labeled streptavidin and substrate; lane 3, biotinylated proteins eluted from the heparin-agarose beads reacted with AP-labeled streptavidin and substrate; lane 4, biotinylated proteins eluted from the heparin-agarose beads in Western blot reactions with anti-gpK8.1A MAb; lane 5, biotinylated proteins eluted from the heparin-agarose beads in Western blot reactions with rabbit anti-HHV-8 gL IgG antibodies. The numbers on the left indicate the molecular masses (in kilodaltons) of the standard protein markers run in parallel lanes.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Proteoglycans are found abundantly in the extracellular matrices or cell surfaces of animal cells and mediate many fundamental cellular processes, including cell-to-cell and cell-to-matrix adhesion, motility, growth, and signaling (28). A proteoglycan is formed by the linkage of glycosaminoglycans such as HS or chondroitin sulfate to a protein core. HS is the initial binding target of many microorganisms, including parasites, bacteria, and viruses (23, 42, 54). Several alphaherpesviruses, such as HSV-1, HSV-2, PRV, and BHV-1 (21, 24, 30, 31, 33, 46, 47, 51, 59), betaherpesviruses, such as HCMV and HHV-7 (35, 37, 45, 49), and gamma-2-herpesviruses, such as BHV-4 (57), interact specifically with HS-like moieties. HS is also recognized by a wide spectrum of other viruses such as human immunodeficiency virus type 1 (38, 41), vaccinia virus (14), Sindbis virus (7), foot-and-mouth-disease virus (26), respiratory syncytial virus (19, 29), and adeno-associated virus (55).

Our recent studies show that the gamma-2-HHV-8, like some members of the alpha-, beta-, and gamma-2-herpesviruses, adsorbs to cells by binding to cell surface HS-like moieties (2). Studies here examined the role of HHV-8 envelope glycoprotein gpK8.1A in the interaction with target cells. Comparison with the human or animal herpesvirus sequences to date show that the gpK8.1A gene is unique for HHV-8. The location of the gpK8.1A gene in the genome clearly suggests an important role of gpK8.1A in the biology of HHV-8. The gpK8.1A gene is positionally colinear to the gamma-1-EBV gp350/gp220 gene (22), the gamma-2-MHV-68 gp150 gene (53), and the gamma-2-HVS ORF 51 gene (1). HHV-8 gpK8.1A shows several similarities with these proteins. Like EBV gp350/gp220 (56) and MHV-68 gp150 (53), HHV-8 gpK8.1A is a virion envelope- and infected cell membrane-associated glycoprotein (60). Antibodies against gp350/220 of EBV and gp150 MHV-68 neutralized the respective virus infectivities (53, 56). Binding of gpK8.1A to the target cells and the gpK8.1A blocking the radiolabeled HHV-8 binding shown here suggest that gpK8.1A plays an important role in the initial events of HHV-8 entry into susceptible cells. Our ongoing studies show that anti-gpK8.1A MAbs neutralize HHV-8 infectivity (data not shown). Inhibition of TMgpK8.1A binding by heparin, binding of TMgpK8.1A to the HS-expressing CHO-K1 cells, limited binding to the mutant derivatives of CHO cell lines lacking HS, specific binding of TMgpK8.1A to HS but not to other GAGs, and the binding of virion gpK8.1A with heparin clearly demonstrate that gpK8.1A is involved in the interaction with HS. Even though heparin lowered the level of TMgpK8.1A binding, the absence of complete inhibition suggests the interaction with other cell surface molecules. The low percentage of binding of TMgpK8.1A to the CHO mutant cells lacking HS also reinforces this suggestion. Our results indicate that TMgpK8.1 interaction with HS is the first important set of ligand-receptor interaction which may lead to the binding of one or more second receptor(s) essential for the subsequent viral entry process (23). The putative second receptor for gpK8.1A needs to be identified.

Inspection of the structure of heparin and/or HS and sequence analysis of the heparin-binding domain (HBD) of several proteins suggested that the negatively charged sulfate or carboxylate groups on heparin could interact via electrostatic interactions to positively charged cationic residues in a protein or peptide (28, 42, 54). HBDs are enriched with positively charged basic amino acids (lysine, arginine, and histidine). Two typical heparin motifs (XBBXBX and XBBBXXBX) have been identified, where "B" is a basic residue and "X" can be any other residue but is usually a hydrophobic residue (8). Analysis of amino acid sequence of gpK8.1A revealed two possible, although atypical heparin-binding motifs: gpK8.1A-H1 (¹⁵⁰SRTTRIRV¹⁵⁷, XBXXBXBX) and gpK8.1A-H2 (¹⁸²TRGRDAHY¹⁸⁹, XBXBXXBX). Whether these gpK8.1A putative HBDs play a role in the interaction with HS requires further investigation. It is also possible that several other weak and/or high-affinity HBDs may appear in HHV-8 gpK8.1A in its native quaternary structure, since the basic amino acids separated apart may lie juxtaposed, forming a typical HBD.

Among the eight HHVs, HS has been shown to mediate the attachment of HSV-1, HSV-2, HCMV, HHV-7, and HHV-8 (2, 21, 24, 30, 31, 33, 35, 37, 45-47, 49, 51, 59). In alphaherpesviruses, the glycoproteins gB and gC are known to bind cell surface HS (21, 24, 30, 31, 33, 51). The gC homologue of alphaherpesviruses is absent in the beta- and gammaherpesviruses, and the gBs of HCMV (betaherpesvirus) and BHV-4 (gammaherpesvirus) have been shown to mediate the HS binding of these viruses (35, 45, 57). Predictive analysis of HHV-8 sequence revealed the presence of putative HBD in HHV-8 gB. Ongoing studies show that HHV-8 envelope-associated gB also binds HS and the 75- and 54-kDa proteins precipitated by heparin-agarose from the biotinylated virus (Fig. 8, lane 2) represent the two cleaved-disulfide linked forms of HHV-8 gB (S. M. Akula et al. unpublished results). The presence of two or more heparin-binding glycoproteins within a single virus is not unexpected, since all well-studied human alpha- and betaherpesviruses contain at least two HS binding glycoproteins, e.g., gC and gB for HSV-1 and HSV-2, gB and gCII for HCMV, and gB and gp65 for HHV-7 (30, 31, 35, 37, 45, 49). The presence of two-HS binding proteins within the same virus indicates the importance of cell surface HS as receptors for viral attachment. HSV-1 gC and gB exhibit differences in their relative affinities for distinct cell surface HS proteoglycans (30). Whether HHV-8 gpK8.1A and gB also exhibit such differences needs to be studied.