Kinetics of HCV envelope proteins’ interaction with CD81 large extracellular loop

BBRC Biochemical and Biophysical Research Communications 328 (2005) 1091–1100 www.elsevier.com/locate/ybbrc

Kinetics of HCV envelope proteinsÕ interaction with CD81 large extracellular loop Hideki Nakajimaa, Laurence Cocquerela, Nobutaka Kiyokawab, Junichiro Fujimotob, Shoshana Levya,* b

a Division of Oncology, Department of Medicine, Stanford University Medical Center, Stanford, CA, USA Department of Developmental Biology and Pathology, National Research Institute for Child Health and Development, 2-10-1 Okura, Setagaya, Tokyo 157-8535, Japan

Received 12 January 2005 Available online 26 January 2005

Abstract We used BIAcore to analyze the kinetics of interactions between CD81 and hepatitis C virus (HCV) envelope proteins. We immobilized different forms of HCV envelope proteins (E1E2, E2, and E2661) on the sensor and monitored their interaction with injected fusion proteins of CD81 large extracellular loop (CD81LEL) and glutathione-S-transferase (CD81LEL-GST) or maltose binding protein (CD81LEL-MBP). The difference between the GST and MBP fusion proteins was their multimeric and monomeric forms, respectively. The association rate constants between CD81LEL-GST or CD81LEL-MBP and the E1E2, E2 or E2661 HCV envelope proteins were similar. However, the dissociation rate constants of CD81LEL-MBP were higher than those of CD81LEL-GST. Interestingly, the dissociation rate constant of CD81LEL-GST from E1E2 was much lower than from E2 or E2661. The interaction between both forms of the CD81LEL fusion proteins and the HCV envelope proteins best-fitted the ‘‘heterogeneous ligand’’ model. This model implies that two kinds of interactions occur between envelope proteins and CD81LEL: one is strong, the other is weak. It also implies that the heterogeneity is likely due to the HCV envelope proteins, which are known to form non-covalently linked heterodimers and disulfide-linked aggregate.
2005 Elsevier Inc. All rights reserved. Keywords: Tetraspanin; CD81; Hepatitis C virus; Kinetics; Surface plasmon resonance

Hepatitis C virus (HCV) is the major etiological agent of posttransfusion non-A, non-B hepatitis, which currently afflicts over 170 million people worldwide [1]. Only a minority of patients mount a successful immune response to clear the virus in the acute phase, while the majority become chronic carriers with a high risk of developing liver cirrhosis and hepatocellular carcinoma [2]. In addition, HCV infection is associated with B cell proliferative diseases such as cryoglobulinemia and lymphoma [3]. HCV is a positive-strand RNA virus of the Flaviviridae family, encoding a single open reading frame

*

Corresponding author. Fax: +1 650 736 1457. E-mail address: [email protected] (S. Levy).

0006-291X/$ - see front matter
2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.01.056

of about 3000 amino acids. This polyprotein is processed co- and posttranslationally by cellular and viral proteases to produce the mature structural core and envelope proteins, and the nonstructural proteins [4]. The envelope proteins, E1 and E2, are type I transmembrane proteins with N-terminal ectodomains and C-terminal hydrophobic anchors and are highly glycosylated. The E1 and E2 glycoproteins have been shown to form non-covalent linked E1E2 complexes and misfolded disulfide-linked aggregates [5]. It is thought that the non-covalent heterodimers play the key role in binding and entry into host cells, whereas the misfolded aggregates are likely deadend products [6]. The characterization of the viral envelope proteins has been hampered by the lack of a cell culture system for efficient viral replication and particle

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assembly. Thus, truncated forms of E2, deleting the hydrophobic transmembrane domain [7,8], liposomes embedding E1E2 [9], and virus like particles expressed in insect cells [10–12] have been used as surrogates for native virus particles. Most recently, pseudotype viral particles expressing both HCV E1E2 envelope proteins, but not E1 or E2 alone, were shown to enter hepatocytes in a CD81-dependent manner [13–17]. CD81 was originally identified as the putative receptor for the virus by a truncated form of E2 [8]. CD81 is a membrane-associated protein belonging to the tetraspanin family [18]. Tetraspanins tend to associate with membrane and signaling proteins in the ‘‘tetraspanin web’’ [19–21] and are likely to function as organizers of signaling complexes [18–20,22]. Tetraspanins contain four transmembrane domains, a small extracellular loop (SEL), a large extracellular loop (LEL), and short cytoplasmic tails. Signatures of conserved amino acid residues differentiate them from other four transmembrane proteins. In particular, their LELs contain conserved cysteine residues that facilitate the proper folding of this domain [19,23,24]. Soluble CD81LEL was shown to bind viral HCV particles in plasma and to inhibit the binding of HCV envelope proteins to cells [8]. The binding epitope within CD81LEL has been mapped and was subsequently shown to be exposed in a ‘‘mushroom-like’’ structure in the crystallized CD81LEL [25,26]. The dissociation constant (KD) between CD81LEL and truncated E2 was previously determined at 1.8 nM by quenching the fluorescence of intrinsic E2 tryptophans [27]. However, real time measurement of both association and dissociation rate constants may better describe ligand–cell interactions [28]. Moreover, it is likely that truncated E2 may not fully mimic the surface of HCV, which is more likely to be composed of E1E2 heterodimers [13,14,29]. To better understand the interaction between HCV envelope proteins and CD81, we studied the kinetics of association and dissociation in real time using a biosensor. A major advantage of this approach is the ability to measure the association and dissociation between proteins whose concentration is known, as is the case for the CD81LEL, with proteins whose concentration cannot be determined accurately as is the case for the HCV envelope proteins. Materials and methods Materials. Anti-hCD81 monoclonal antibody (mAb) 5A6 was described previously [30]. Anti-HCV E2 mAbs H53 and 3/11 [31,32] were kindly provided by Dr. Dubuisson. CD81LEL fusion protein preparation. A recombinant carboxylterminal fusion protein containing the large extracellular loop (LEL) of human (h) CD81 or African green monkey (agm) CD81 fused to glutathione-S-transferase (CD81LEL-GST) was prepared as described previously [33]. To construct a fusion of hCD81LEL and maltose binding protein (MBP) in pMAL (New England BioLabs, Beverly, MA), we used the primers, (forward: 5 0 -AGGAATTCGGCATCTGG

GGCTTTGTCAA-3 0 , reverse: 5 0 -AGCTGCAGCTACAGCTTCCCG GAGAAGAGGTCATCG-3 0 ) and the pCDM8-CD81 template. The amplified hCD81LEL was digested by EcoRI and PstI, and inserted into the respective sites in pMAL-c2x and the nucleotide sequence was confirmed. Transformed Escherichia coli BL21 were induced to express the hCD81LEL-MBP protein, which was purified using Amylose resin (New England Biolabs). The protein was further purified using HiPrep 26/60 Sephacryl S-200 HR (Amersham Biosciences). HCV envelope protein. COS7 cells were infected with adenovirus (Adv) encoding the HCV envelope protein AdvE2661 (amino acid 384– 661), AdvE2 full (amino acid 384–746), and AdvE1E2p7NS2 (amino acid 171–1026) or a control LacZ (AdvLacZ). The recombinant adenoviruses were produced by the Adeno-X expression system (BD Biosciences Clontech, Palo Alto, CA) and described previously [34]. COS7 cells were infected for 48 h with 25 PFU per cell, washed twice with ice-cold DulbeccoÕs phosphate-buffered saline (D-PBS), and lysed in 1% Triton X-100 in D-PBS containing complete mini protease inhibitor cocktail (Roche) for 30 min, and cell lysates were kept at
80 C until use. Cell lysates were centrifuged (10,000g for 30 min) prior to injection to the BIAcore sensor. About 1.4 · 107 cells expressing E2 or E1E2 were lysed in 1 ml and 30 ll of the lysate was used to immobilize onto the sensorchip as described below. E2661 was used as a culture supernatant of the AdvE2661 infected COS7 cells. The culture supernatant was concentrated 5-fold using Centriplus (Millipore) before injection to the BIAcore sensor. Immunoprecipitation. Cell lysates expressing the HCV envelope protein were incubated overnight at 4 C with 60 ll (1:1 slurry) of H53 captured on protein A–Sepharose beads (Sigma) via rabbit anti-mouse Ig (Dako). Immunoprecipitates were collected by centrifugation at 5000 rpm for 5 min at 4 C, washed at least three times with 1% Triton X-100 in PBS, and eluted by boiling in NuPAGE LDS sample buffer (Invitrogen) with or without reducing reagent. Gel electrophoresis and immunoblotting. The samples were separated by 4–12% NuPAGE (Invitrogen) and electrotransferred onto polyvinylidene difluoride membranes. HCV envelope proteins were detected by incubating the membranes with 3/11 (anti-HCV E2). Membranes were washed in PBS with 0.1% Tween 20 and incubated with goat antirat Ig HRP-linked secondary Abs (Southern Biotechnology Associates). Blots were visualized by chemiluminescence detection (ECL; Amersham) following instructions of the manufacturer. Surface plasmon resonance (SPR) analysis. All analyses of interactions between HCV envelope proteins and CD81LEL fusion proteins were performed in HBS-EP buffer (BIAcore, Uppsala, Sweden) at 25 C and at a flow rate of 10 ll per minute. In this report, immobilized HCV envelope proteins and cell surface receptor CD81LEL will be referred to as ‘‘ligand’’ and ‘‘analyte,’’ respectively. SPR and molecular mass on the sensor were measured with a BIAcore 1000 or 2000 system using the sensor chip CM5 (BIAcore). CM5 contains a dextran matrix and immobilizes the protein by chemical conjugation of their amino groups using an amine coupling kit (BIAcore). To capture the envelope proteins on the sensorchip, we used a conformation-dependent antiHCV E2 antibody, H53, which does not inhibit the interaction between E2 and CD81 [35]. About 10770.9–11950.8 resonance units (RU) of H53 was immobilized per flow cell corresponding to 107.7–119.5 ng of this mAb. Following immobilization of H53, cell lysates containing HCV envelope protein were injected at a flow rate of 1 ll/min for 30 min, captured values being indicated in each experiment. Nonspecific binding to the cell lysates of AdvLacZ infected cells was subtracted. Capture of the envelope protein on the sensor was followed by injection of 1% Triton X-100 in D-PBS to remove non-specific binding. The CD81LEL fusion proteins were then injected and the interactions between the HCV envelope proteins and CD81LEL were monitored. No typical mass-transport effect was observed at 10 ll/min. Following binding of CD81LEL fusion protein, the sensor was regenerated. This was done in one of two ways: to remove bound analyte we injected 0.2 mg/ml 5A6 (an anti-CD81 mAb) at a flow rate of 1 ll/min for 30 min. To remove the analyte–ligand complex from immobilized H53,

H. Nakajima et al. / Biochemical and Biophysical Research Communications 328 (2005) 1091–1100 we injected 10 mM glycine (pH 2.5) for 1 min. After removal of the analyte–ligand by 10 mM glycine (pH 2.5), the sensor was regenerated by injection of cell lysates expressing the HCV envelope or control LacZ proteins. Both regeneration procedures yielded similar results. These cycles were repeated using increasing concentrations of CD81LEL fusion protein to calculate the kinetics. Results show representative data obtained at least in three independent experiments. Kinetic analysis of sensorgram data. Kinetic constants were calculated from the sensorgrams with BIAevaluation software, version 3.1 (BIAcore), according to the global fitting model. Response curves were prepared for fitting after subtracting the signal generated simultaneously on the control flow cell injected with cell lysate derived from AdvLacZ infected cells. The response curves using all the analyte concentrations tested were globally fitted to several binding models provided with the BIAevaluation 3.1 software. They included a simple 1:1 (Langmuir) binding model (A + B M AB), a bivalent analyte (first step: A + B M AB; second step: AB + B M AB2) model, a two-state reaction (conformation change) model (A + B M AB M ABx), and a heterogeneous ligand (interaction one: A + B1 M AB1; interaction two: A + B2 M AB2) model. Apparent rate constants are presented from the best fit heterogeneous ligand model. Rate constants of interaction one (kon1, koff1) and interaction two (kon2, koff2) are described by the following equations, where A = CD81LEL, B = HCV envelope glycoprotein, and Rmax = maximum binding capacity. B1½0 ¼ Rmax1; dB1=dt ¼
½k on 1  A  B1
k off 1  AB1 B2½0 ¼ Rmax2; dB2=dt ¼
½k on 2  A  B2
k off 2  AB2 AB1½0 ¼ 0; dAB1=dt ¼ ½k on 1  A  B1
k off 1  AB1 AB2½0 ¼ 0; dAB2=dt ¼ ½k on 2  A  B2
k off 2  AB2

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Fig. 1. Specific binding of human (h) CD81LEL-GST fusion protein to HCV E1E2 envelope proteins. A sensorchip immobilized with HCV E1E2 (130.8 RU) was injected with 100 nM hCD81LEL-GST or with agmCD81LEL-GST. a, Start injection of the CD81LEL-GST fusion protein; b, stop injection by change to the running buffer. The association phase is from a to b, the dissociation phase begins at b.

ð1Þ

ð2Þ

ð3Þ

ð4Þ

Determination of molecular mass. Molecular mass analysis of the CD81LEL-fusion protein by size exclusion chromatography was car˚ (Alltech, ried out at room temperature on a Macrosphere column 100 A Deerfield, IL) equilibrated with D-PBS. The flow rate (0.1 ml/min) was controlled using a SYSTEM GOLD HPLC (Beckman Coulter, Fullerton, CA) and the signal was detected using PhotoDiodeArray on that system. We used Gel Filtration LMW Calibration Kit (Amersham) as a standard of molecular mass on size exclusion chromatography. Aliquots of each fraction were dot-blotted onto nitrocellulose membrane and detected by anti-CD81 mAb 5A6. Sedimentation equilibrium analysis was performed using a Beckman Coulter model XLA analytical ultracentrifuge. Briefly, equilibrium sedimentation of 9.6 lM CD81LEL-MBP in D-PBS was analyzed in a 1.2 cm pathlength doublesector quartz cell and measured at 280 nm. Centrifugation was performed at 20 C in an An-55Ti rotor at 2 h intervals until equilibrium was reached. Data were collected after 22 h at 8000 rpm and after 20 h of centrifugation at 10,000 rpm and analyzed by ORIGIN software provided by the manufacturer (Beckman Coulter, Fullerton, CA).

Results Specific binding of human CD81LEL to HCV envelope protein A cell lysate containing HCV E1E2 envelope proteins was immobilized onto a BIAcore sensorchip as detailed in ‘‘Materials and methods, Surface plasmon resonance

(SPR) analysis.’’ We then injected 100 nM of the human CD81LEL (hCD81LEL-GST) or African green monkey CD81LEL (agmCD81LEL-GST) GST fusion proteins to the sensorchip. These two proteins differ only by four amino acids, but only the human CD81 protein binds the HCV envelope proteins [33]. Human CD81LEL bound to the immobilized HCV E1E2 envelope proteins on the sensorchip as seen by the increased mass (RU values) during the association phase of the sensorgram (indicating in Fig. 1, between a and b). The hCD81LEL remained bound to the sensor after the injection was stopped, dissociating slowly after the injection of the running buffer (Fig. 1, b arrow). In contrast to hCD81LEL, no binding was seen with the agmCD81LEL. Binding kinetics of hCD81LEL-GST to HCV envelope proteins To calculate the association and dissociation rate constants, sensorgrams were obtained by injecting increasing concentrations of the hCD81LEL-GST fusion protein onto the HCV E1E2 envelope protein-captured sensorchip. Increased concentrations of hCD81LEL-GST resulted in increased signals as seen by the sensorgrams (Fig. 2A). In this experiment, we also tested the kinetics of binding to the HCV E2 protein, which was captured on the sensorchip at almost twice the mass of E1E2 proteins, because the concentration of E2 in the cell lysate is approximately twice that of E1E2. Consequently, the signal strength on the E2 immobilized flow cell (Fig. 2B) was about twice that of the E1E2 immobilized flow cell. While the association phase of hCD81LEL-GST with E1E2 and E2 had simi-

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hCD81LEL-GST and the various HCV envelope proteins are similar to each other. In contrast, the dissociation rate constant (koff) between hCD81LEL-GST and E1E2 is lower than that measured for its interaction with E2. The half-life of E1E2-hCD81LEL-GST complex (41.3 h) is four times longer than that of E2 (10.5 h). In this experiment, we also analyzed the kinetics of binding of the truncated form E2661 (data not shown). The kinetic constants for the truncated form E2661 are similar to those for E2 (Table 1). Binding kinetics of hCD81LEL-MBP to HCV envelope proteins

Fig. 2. Kinetics of interaction between hCD81LEL-GST and HCV envelope proteins. Increasing concentrations of hCD81LEL-GST at 62.5, 125, 250, 500, 1000, and 2000 nM were injected into E1E2captured (316 RU) (A) or E2-captured (624 RU) (B) sensorchip at a flow rate of 10 ll/min.

lar slopes, the dissociation phase of E2 had a steeper slope than that of the E1E2 proteins. The kinetics of association and dissociation of hCD81LEL-GST to E1E2 or E2 were calculated from the sensorgram using the (1:1) Langmuir fitting model (Table 1). The calculated association rate constants (kon) between Table 1 Kinetic constant of interaction between CD81LEL-GST and HCV envelope protein obtained by (1:1) Langmuir fitting model Analyte

Ligand

kon (1/Ms)

koff (1/s)

KD (M)

4


4

hCD81LEL-GST

E1E2 E2 E2661

0.89 · 10 1.15 · 104 1.53 · 104

0.47 · 10 1.83 · 10
4 1.72 · 10
4

0.52 · 10
8 1.60 · 10
8 1.12 · 10
8

agmCD81LEL-GST

E1E2 E2

0.72 · 103 1.25 · 103

1.24 · 10
3 1.29 · 10
3

1.71 · 10
6 1.03 · 10
6

We initially tested the interaction with the hCD81LEL-GST protein because it was used in numerous studies to inhibit binding of the HCV envelope proteins to cell-surface-expressed CD81 [13,14,36,37]. However, hCD81LEL-GST tends to form dimers and multimers, which react with both heterodimeric and misfolded E1E2 [38]. We therefore repeated the analysis using a fusion protein of hCD81LEL and MBP because this fusion protein is less prone to multimer formation [39,40]. We first compared the molecular mass of hCD81LEL-GST and hCD81LEL-MBP in solution using size exclusion chromatography. Fractionation of hCD81LEL-GST showed a minor peak of CD81-positive fraction near the void volume and a major peak at the theoretical dimer position (gray lines in Fig. 3A), implying that this protein forms dimers and higher molecular weight multimers. Fractionation of hCD81LEL-MBP yielded a single CD81-positive peak (Fig. 3B) eluted between bovine serum albumin (BSA) (69 kDa) and ovalbumin (OVA) (43 kDa), fitting the expected monomeric molecular mass of 53 kDa. The monomer mass of hCD81LEL-MBP was also confirmed by sedimentation equilibrium analysis. Molecular masses of 51,498 (47,737–55,338) at 8000 rpm and 57,376 (53,123–61,590) at 10,000 rpm were obtained for the CD81LEL-MBP, indicating a monomer. We then injected the monomeric hCD81LEL-MBP to the sensorchips. Binding of hCD81LEL-MBP to HCV E1E2 (Fig. 4A) or HCV E2 (Fig. 4B) was reduced compared to hCD81LEL-GST, nevertheless, the calculated association rate constants using the (1:1) Langmuir fitting model (Table 2) were similar to those calculated for hCD81LEL-GST (Table 1). However, the calculated dissociation rate constants differed from the ones obtained with the hCD81LEL-GST by about 10-fold (Table 1). In contrast to the differences in the dissociation rates of E1E2 and E2 from hCD81LEL-GST, the dissociation rates of E1E2 and E2 from hCD81LEL-MBP were similar. In this experiment, we also analyzed the kinetics of E2661 binding to hCD81LEL-MBP (data not shown). The kinetic constants for E2661 were similar to E2 (Table 2).

H. Nakajima et al. / Biochemical and Biophysical Research Communications 328 (2005) 1091–1100

Fig. 3. Analysis of monomeric and multimeric forms of the hCD81LEL fusion proteins. Size exclusion chromatography profile of hCD81LEL-GST (A) and hCD81LEL-MBP (B). Proteins were ˚ column as described in chromatographed on a Macrosphere 100 A Materials and methods. Elution points of blue dextran at the void volume (a), BSA (69 kDa) (b), and OVA (43 kDa) (c) are indicated by arrows. The theoretical molecular masses of hCD81LEL-GST and hCD81LEL-MBP monomers are 36 and 53 kDa, respectively. Gray lines indicate reactivity of fractions with anti-CD81. No peaks corresponding to the hCD81LEL-GST monomer (36 kDa) or the hCD81LEL-MBP dimer (106 kDa) were identified.

Fitting models for the interaction of the HCV envelope proteins with hCD81LEL fitting of interaction models To evaluate the form of the HCV envelope proteins that were immobilized by the H53 mAb on the sensorchip, we performed immunoprecipitation, followed by SDS–PAGE. The H53-immunoprecipitated E2 and E1E2 proteins contain both lower molecular weight monomeric E2 and misfolded disulfide-linked aggregated complexes, as seen upon electrophoresis under non-reducing conditions (Fig. 5, lanes 2 and 3). Upon exposure to reducing conditions the high molecular

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Fig. 4. Kinetics of interaction between hCD81LEL-MBP and HCV envelope proteins. Increasing concentrations of hCD81LEL-MBP at 62.5, 125, 250, 500, 1000, and 2000 nM were injected into E1E2captured (302 RU) (A) or E2-captured (732 RU) (B) sensorchip at a flow rate of 10 ll/min.

Table 2 Kinetic constant of interaction between CD81LEL-MBP and HCV envelope protein obtained by (1:1) Langmuir fitting model Analyte

Ligand

kon (1/Ms)

koff (1/s)

KD (M)

hCD81LEL-MBP

E1E2 E2 E2661

1.05 · 104 1.20 · 104 1.95 · 104

1.60 · 10
3 1.10 · 10
3 1.64 · 10
3

1.52 · 10
7 0.92 · 10
7 0.84 · 10
7

weight aggregates collapse into the monomeric E2 band (Fig. 5, lanes 4 and 5). Thus, although H53 interacts with a conformational epitope on the native HCV envelope proteins [32], this mAb can react with some disulfide-linked proteins. We proceeded to use calculation formulas to interrogate sensorgrams as to their fit with various interaction models [41–43]. As we expected, global fitting of interaction between the HCV envelope protein and hCD81LEL-GST eliminated the pseudo-first ordered kinetics ‘‘(1:1) Langmuir binding’’ model, the ‘‘bivalent analyte’’ model, and the ‘‘two state reaction (conformation change)’’ model, because the Ch2 (x2) of those

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the ‘‘two state reaction (conformation change)’’ model for the interactions with E1E2 were 3.12, 3.2, and 3.8, respectively, and Ch2 between E2 and hCD81LELMBP were 27.4, 23.5, and 35.5, respectively. The kinetic constants calculated by the ‘‘heterogeneous ligand’’ model are summarized in Table 3. There are two kinds of ligands on the sensor in this interaction model. The interaction between hCD81LEL and one ligand measured as kon1 and koff1 rate constants has stronger affinity than that of the second ligand (kon2 and koff2), it has faster association rate constants and slower dissociation constants. The kon1 are similar between hCD81LEL-GST and hCD81LEL-MBP, but the koff1 between hCD81LEL-GST and HCV envelope protein is approximately one order lower than that of hCD81LEL-MBP. Simulation of curves representing the binding components of the ‘‘heterogeneous ligand’’ model

Fig. 5. HCV envelope proteins immobilized by H53. Lysates of cells transfected with E2 (lanes 2 and 4) or E1E2 (lanes 3 and 5) were immunoprecipitated by H53. Immunoprecipitates were electrophoresed under non-reducing (left panel) or reducing (right panel) condition and blotted with an anti-HCV E2 mAb (3/11), which recognizes linear epitope in HCV E2. Lane 1, H53 immobilized beads alone show crossreactivity with the immunoglobulin. M, molecular weight.

interaction models between E1E2 and CD81LEL-GST were 12.7, 10.5, and 10.1, and between E2 and CD81LEL-GST were 18.5, 9.18, and 16.2, respectively. The best fit was obtained with the ‘‘heterogeneous ligand’’ model. Global fitting of the data using this model yielded the Ch2 value of 1.38 (E1E2) and 1.34 (E2), indicating that the fitting procedure by the ‘‘heterogeneous ligand’’ model best described the kinetic data. Next, we examined the sensorgrams obtained by the interaction between monomeric hCD81LEL-MBP and the HCV envelope proteins using the same models. Again, the best fit was obtained with the ‘‘heterogeneous ligand’’ model. Ch2 values for the ‘‘heterogeneous ligand’’ model for this monomeric form hCD81LEL-MBP and E1E2 or E2 were 2.43 and 9.33, respectively. The Ch2 of the ‘‘(1:1) Langmuir binding’’ model, the ‘‘bivalent analyte’’ model, and

The interactions between the HCV envelope proteins with either the monomeric hCD81LEL-MBP or with the dimeric and multimeric hCD81LEL-GST fitted the same model of interaction. It is therefore likely that there are two kinds of ligands on the sensor, which is the case for both the E1E2 or E2 proteins. These interactions can be simulated by curves representing the two components, as acquired from fitting with the E1E2 or E2 sensorgrams (Fig. 6). The acquired total signal is shown as the bold solid line (c). The interaction with one ligand is shown as a thin solid line (a) and the interaction with the second ligand is shown as the dotted line (b). At the start of injection during the association phase, the thin solid lines ‘‘a’’ are steeper representing faster association kinetics than ‘‘b’’. This interaction is also slow to dissociate, maintaining the binding mass for several minutes. In contrast, the interactions with the second ligand, represented by the dotted lines, have slower association and steeper dissociation slopes. Taken together, component ‘‘a’’ (solid lines) defines stronger interactions than ‘‘b’’ (dotted lines). Comparison of these simulation curves demonstrates that the strong interactions ‘‘a’’ are similar for both the hCD81LEL-GST and hCD81LEL-MBP, whereas the weak interactions have steeper dissociation phases for the hCD81LEL-MBP fusion protein. In addition, comparison of the weaker interaction ‘‘b’’ obtained with the hCD81LEL-GST fusion protein shows a faster

Table 3 Kinetic constant of interaction between CD81LEL fusion proteins and HCV envelope protein obtained by ‘‘heterogeneous ligands’’ model Analyte

Ligand

kon1 (1/Ms)

koff1 (1/s)

kon2 (1/Ms)

koff2 (1/s)

hCD81LEL-GST

E1E2 E2

3.17 · 104 1.88 · 104

1.33 · 10
7 3.34 · 10
7

4.43 · 103 1.51 · 103

3.27 · 10
4 7.96 · 10
5

hCD81LEL-MBP

E1E2 E2

4.62 · 104 3.73 · 104

3.76 · 10
5 7.89 · 10
6

5.09 · 103 1.94 · 103

1.58 · 10
3 3.22 · 10
3

H. Nakajima et al. / Biochemical and Biophysical Research Communications 328 (2005) 1091–1100

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Fig. 6. Simulation of curves representing binding components of the ‘‘heterogeneous ligand’’ model. Those sensorgrams correspond to the result obtained by the injection of 1000 nM hCD81LEL fusion protein. The acquired sensorgrams are shown as the bold solid line (c). The globally fitted curves from the ‘‘heterogeneous ligand’’ model are shown by the narrow solid lines (a), dotted lines (b), and represent the two ongoing interactions between hCD81LEL-GST and E1E2 (A) or E2 (C), and between hCD81LEL-MBP and E1E2 (B) or E2 (D).

dissociation from E2 than from E1E2 (compare A and C, Fig. 6).

Discussion The interaction between the HCV envelope glycoproteins and CD81 has been documented at the cell surface [8] and confirmed by numerous assays including protein precipitation [38], immunosorbent [39], and immunoblot [33], and by quenching of the intrinsic tryptophan fluorescence [27]. The SPR system used in the current study allows very sensitive real time monitoring of association and dissociation of components interacting in a continuous flow. As a result, this method not only determines the dissociation constant (KD: affinity) but also the association rate constant (kon) and the dissociation rate constant (koff). An additional advantage of this method is that it can determine the association and dissociation between the immobilized ‘‘ligand’’ proteins of an undefined concentration with a defined concentration of its ‘‘analyte’’ protein.

Here we investigated the interaction between the HCV envelope proteins (E1E2 or E2 and E2661), that were captured by an antibody to the sensorchip and a defined concentration of the injected soluble CD81LEL fusion proteins. The specificity of this interaction with the human CD81LEL but not with the African green monkey CD81LEL is demonstrated in Fig. 1 and Table 1. The human and monkey CD81LEL differ only in four amino acid residues [33]. By increasing the concentrations of agmCD81LEL to micromolar order (data not shown), we could detect some binding because of the high sensitivity and the ability of the SPR system to detect binding in real time. However, the bound agmCD81LEL protein immediately dissociates from the HCV envelope proteins. The affinity (KD) of interaction between the agmCD81LEL interaction with E1E2 or E2 was about 100-fold lower than that measured with hCD81LEL (the association rate constants were reduced about 10-fold and the dissociation rate constants increased about 10-fold: KD = koff/kon). The affinities of agmCD81LEL to HCV envelope protein were too low to detect using the other procedure [7,33,44,45].

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Functionally, such low kinetic constants may not suffice for the HCV envelope proteins to bind cells that express agmCD81. Additional binding kinetics analysis determined that the association rate constants between hCD81LEL and the various HCV envelope proteins were similar (Tables 1–3). However, the dissociation of hCD81LEL-GST from E1E2 was four times longer than from E2 (Fig. 2 and Table 1). These data fit with recent studies demonstrating the dependence on the heterodimeric E1E2 proteins for cell entry that has been recently demonstrated by the use of HCV pseudotyped particles [13–16]. Cell entry was observed only when both envelope proteins, E1 and E2, were assembled in the viral particles [13,14]. Moreover, the binding of heterodimeric E1E2 to cell surface expressed CD81 induced downstream signaling events, as we previously demonstrated [29]. It is therefore likely that the inclusion of HCV E1, which also improves the stability of membrane insertion of E2 [47], plays a conformational role in viral–cell interactions. We assume that while E2 binds to target cells, E1 may act to stabilize the envelope protein complex and to facilitate membrane interactions and cell entry. The lower dissociation rate constant obtained with the HCV E1E2 proteins, compared with the HCV E2 protein, may prolong its chance to interact with cells. These data are in accord with several studies that demonstrated the biological importance of slow dissociation rates. For example, the protease inhibitor of HIV, saquinavir, binds WT and mutant protease at similar association rate constants, however, the dissociation rate constant of the mutant protease is higher, consequently it is less sensitive to the drug [46]. Another example is the interaction of a soluble form of the 2B4 TCR with its ligand, a moth cytochrome c (MCC) peptide bound to the mouse MHC class II molecule Ek (MCC-Ek). MCC-Ek and an altered ligand, T102-Ek, bound the TCR with similar association rate constants, but only MCC-Ek displayed longer dissociation leading to increased specificity of interaction [28]. By analogy, the lower dissociation rate constant obtained with the HCV E1E2 protein, compared with the HCV E2 protein, may prolong its chance to interact with cells. The extremely low dissociation constant which we measured occurred only between the CD81LEL-GST, but not the CD81LEL-MBP, and HCV envelope proteins (Table 3, KD = koff1/kon1). We propose that these measurements reflect the conformation of the interacting native molecules, as discussed below. This slower dissociation from the HCV E1E2 protein, observed with dimeric and multimeric hCD81LELGST, may explain how this fusion protein could block the interaction of HCV envelope proteins with cellular CD81, as demonstrated in multiple studies [13,14,36,37]. It was not seen with the monomeric hCD81LEL-MBP form (Figs. 2 and 5, Tables 1–3).

Our data suggest that this monomeric form may not mimic cell surface expressed CD81 (see below). A previous study that compared the binding of monomeric and dimeric hCD81LEL-MBP to E2661 in solid phase also showed that the dimeric forms were 10-fold more effective than the monomeric ones [39]. The study that previously determined the affinity of hCD81LEL to truncated E2 also used a trimeric form of hCD81LEL. The KD determined in that study, 1.8 nM, is lower than our measurements (Tables 1 and 2). The difference could be possibly explained by the use of the trimeric hCD81LEL or by the different method applied [27]. It is of interest that hCD81LEL crystallizes as homodimers in two types of crystals [25,26,48]. A molecular model of E2 also shows this protein as a head-to-tail homodimer, possibly forming a heterodimeric association with E1 [49]. CD81 and other tetraspanins tend to associate on the plasma membrane in the ‘‘tetraspanin web’’ [19–21]. Tetraspanin homodimers, which form in the Golgi, were suggested to be the core unit for the assembly of the tetraspanin web [50]. It is therefore tempting to speculate that CD81LEL-GST, which forms dimers (Fig. 3), is in the preferable conformation for avid binding to the HCV envelope proteins. Indeed, CD81LEL-GST, which was used in numerous studies [13,14,36,37], is an effective blocker of binding of HCV envelope proteins to cell surface expressed CD81. It is therefore likely that clustering of CD81 on the cell surface stabilizes the complex of CD81 and the HCV envelope proteins thereby increasing the chance for viral entry. The current study tested several interaction models, one of which, the ‘‘heterogeneous ligand’’ model, bestfitted the experimental results. The ‘‘heterogeneous ligand’’ model simulates binding of one kind of analyte to two kinds of ligands, and it also calculates the contribution of each ligand to the total signal. This model distinguished two kinds of contributing ligands, the first forming strong interactions (Fig. 6 ‘‘a’’), while the second formed weak ones having slower association and faster dissociation (Fig. 6 ‘‘b’’). The monomer, hCD81LEL-MBP, has faster dissociation rate constants in both strong interaction and weak interaction (Figs. 6B and D, Table 3). The difference between the hCD81LEL-GST and the monomeric hCD81LELMBP is most evident in their weaker interactions, having gentler association and steeper dissociation slopes (Fig. 6). Nevertheless, these weak interactions between CD81LEL and HCV envelope protein have 10
6 > order of dissociation constant (KD = koff2/kon2) (Table 3). It is therefore tempting to speculate that forming dimers or multimers of CD81 on the cell surface could strengthen such weak interactions. A concern for a fit to the ‘‘heterogeneous ligand’’ model is that it could be due to improper ligand immobilization [43]. However, we immobilized the HCV envelope proteins via a monoclonal antibody that does not inhibit the interac-

H. Nakajima et al. / Biochemical and Biophysical Research Communications 328 (2005) 1091–1100

tion between CD81 and HCV E2 protein [35]. Thus, all of the HCV envelope proteins used in this study were anchored via the same epitope onto the sensor. The HCV envelope proteins have been shown to assemble as non-covalent heterodimer and as disulfidelinked aggregate [5]. The heterodimers are thought to represent the native viral conformation while the disulfide-linked aggregates are likely to be dead-end products [6]. We have previously shown that immobilized hCD81LEL-GST bound both heterodimer and disulfide-linked aggregate [38]. Similar results were obtained with hCD81LEL-MBP (data not shown). Thus, even monomeric hCD81LEL binds the non-covalently linked E1E2 heterodimer and the covalent misfolded aggregates of these HCV proteins. A possibility exists that the two interactions modeled by the ‘‘heterogeneous ligand’’ model represent interactions between hCD81LEL and these two forms of the viral envelope proteins. Our experimental system cannot determine which component, namely the native or the disulfide-linked aggregates, contributed to the strong and weak interactions measured. Nevertheless, the presence of E1 affects the kinetics of interaction of CD81LEL with both the properly folded and the misfolded E2 proteins (Table 3). In conclusion, two forms or conformations of the HCV envelope proteins were distinctly separated by their affinities of interaction with the hCD81LEL fusion proteins, as measured by SPR. The form of the hCD81LEL fusion proteins also mattered, the multimeric form was more difficult to dissociate from both conformations of the HCV envelope proteins. The multimeric hCD81LEL was also more difficult to dissociate from the E1E2 complex than from E2. CD81 is thought to form multimeric complexes in the ‘‘tetraspanin web’’ [19–21], a feature that may increase the avidity of interaction between HCV and its cellular receptor.

Acknowledgments This work was supported in part by National Institute of Health Grants CA34233 and OverseaÕs Research Fellowship from the Japan Society for the promotion of Science. We thank Ms. Jenifer Theresa Schulte (Department of Chemical Engineering, Stanford University) for the advice on the HPLC experiment.

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