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• W2080139517 abstract "Avian hepatitis B virus infection is initiated by the specific interaction of the extracellular preS part of the large viral envelope protein with carboxypeptidase D (gp180), the primary cellular receptor. To functionally and biochemically characterize this interaction, we purified a soluble form of duck carboxypeptidase D from a baculovirus expression system, confirmed its receptor function, and investigated the contribution of different preS sequence elements to receptor binding by surface plasmon resonance analysis. We found that preS binds duck carboxypeptidase D with a 1:1 stoichiometry, thereby inducing conformational changes but not oligomerization. The association constant of the complex was determined to be 2.2 × 107m−1 at 37 °C, pH 7.4, with an association rate of 4.0 × 104m−1 s−1 and a dissociation rate of 1.9 × 10−3 s−1, substantiating high affinity interaction of avihepadnaviruses with their receptor carboxypeptidase D. The separately expressed receptor-binding domain, comprising about 50% of preS as defined by mutational analysis, exhibits similar constants. The domain consists of an essential element, probably responsible for the initial receptor contact and a part that contributes to complex stabilization in a conformation sensitive manner. Together with previous results from cell biological studies these data provide new insights into the initial step of hepadnaviral infection. Avian hepatitis B virus infection is initiated by the specific interaction of the extracellular preS part of the large viral envelope protein with carboxypeptidase D (gp180), the primary cellular receptor. To functionally and biochemically characterize this interaction, we purified a soluble form of duck carboxypeptidase D from a baculovirus expression system, confirmed its receptor function, and investigated the contribution of different preS sequence elements to receptor binding by surface plasmon resonance analysis. We found that preS binds duck carboxypeptidase D with a 1:1 stoichiometry, thereby inducing conformational changes but not oligomerization. The association constant of the complex was determined to be 2.2 × 107m−1 at 37 °C, pH 7.4, with an association rate of 4.0 × 104m−1 s−1 and a dissociation rate of 1.9 × 10−3 s−1, substantiating high affinity interaction of avihepadnaviruses with their receptor carboxypeptidase D. The separately expressed receptor-binding domain, comprising about 50% of preS as defined by mutational analysis, exhibits similar constants. The domain consists of an essential element, probably responsible for the initial receptor contact and a part that contributes to complex stabilization in a conformation sensitive manner. Together with previous results from cell biological studies these data provide new insights into the initial step of hepadnaviral infection. hepatitis B virus duck hepatitis B virus carboxypeptidase D duck carboxypeptidase D soluble duck carboxypeptidase D duck hepatitis B virus preS polypeptide Spodoptera frugiperda 4amino-benzoyl-arginine 1-ethyl-3-(3-dimethylaminopropyl)carbo-diimide hydrochloride Autographa californica nuclear polyhedrosis virus Hepatitis B viruses are a group of small enveloped hepatotropic partially double-stranded DNA viruses that cause acute and chronic infections in humans, mammals, and birds (1Mason W.S. Seeger C. Current Top. Microbiol. Immunol. Springer-Verlag KG, Berlin1991: 168Google Scholar). In case of the human hepatitis B virus (HBV)1chronic infections dramatically increases the risk for the development of primary hepatocellular carcinomas, and HBV therefore represents a major health problem to the world population (2Hildt E. Hofschneider P.H. Urban S. Semin. Virol. 1996; 7: 333-347Crossref Scopus (27) Google Scholar). While many details of hepadnaviral genome replication are understood in considerable detail (reviewed in Refs. 3Nassal M. Schaller H. Trends Microbiol. 1993; 1: 221-228Abstract Full Text PDF PubMed Scopus (147) Google Scholar and 4Nassal M. Schaller H. J. Viral Hepatitis. 1996; 3: 217-226Crossref PubMed Scopus (117) Google Scholar), our knowledge of the early events in HBV infection, namely the identity of the cellular receptor for HBV, is poor, and reflects the lack of a suitable infection system (5DeMeyer S. Gong J.Z. Suwandhi W. van Pelt J. Soumillion A. Yap S.H. J. Viral Hepatitis. 1997; 4: 145-153Crossref PubMed Scopus (84) Google Scholar). However, promising results have been achieved in the duck hepatitis B virus (DHBV) model system where systematic infection studies using primary duck hepatocytes can reproducibly be performed (6Tuttleman J.S. Pugh J.C. Summers J.W. J. Virol. 1986; 58: 17-25Crossref PubMed Google Scholar). In this system infections can be suppressed by the simultaneous application of nucleocapsid-free, non-infectious subviral particles which are produced in an about 1000-fold excess over virus during infection (7Klingmüller U. Schaller H. J. Virol. 1993; 67: 7414-7422Crossref PubMed Google Scholar). Both subviral particles and virions contain the same two envelope proteins, namely the large viral surface protein (L-protein) and the small viral surface protein (S-protein). Both proteins are transcribed from a common open reading frame and share the hydrophobic S moiety which is responsible for membrane anchoring. The L-protein additionally contains the 161-amino acid amino-terminal hydrophilic preS sequence which, on its own, inhibits the DHBV infection of primary duck hepatocytes. A subsequent deletion analysis showed that an 85-amino acid preS element constitutes the receptor-binding domain within preS (8Urban S. Breiner K.M. Fehler F. Klingmüller U. Schaller H. J. Virol. 1998; 72: 8089-8097Crossref PubMed Google Scholar). In an attempt to identify receptor candidates for DHBV, Kuroki et al. (9Kuroki K. Cheung R. Marion P.L. Ganem D. J. Virol. 1994; 68: 2091-2096Crossref PubMed Google Scholar) and independently Tong et al. (10Tong S. Li J. Wands J.R. J. Virol. 1995; 69: 7106-7112Crossref PubMed Google Scholar) identified a glycoprotein of 170–180 kDa (gp180/p170) which binds DHBV particles and Escherichia coli-derived GST-preS polypeptides. Sequence comparisons of gp180/p170 cDNA with known sequences suggested that it represents the prototype of a new family of membrane bound carboxypeptidases (10Tong S. Li J. Wands J.R. J. Virol. 1995; 69: 7106-7112Crossref PubMed Google Scholar, 11Kuroki K. Eng F. Ishikawa T. Turck C. Harada F. Ganem D. J. Biol. Chem. 1995; 270: 15022-15028Crossref PubMed Scopus (119) Google Scholar). Independently, several mammalian homologues of gp180/p170 were discovered in bovine, rat, mouse, and humans, and have been classified as metallocarboxypeptidase D (CPD) (12Song L. Fricker L.D. J. Biol. Chem. 1995; 270: 25007-25013Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 13Xin X. Varlamov O. Day R. Dong W.J. Bridgett M.M. Leiter E.H. Fricker L.D. DNA Cell Biol. 1997; 16: 897-909Crossref PubMed Scopus (75) Google Scholar, 14McGwire G.B. Tan F. Michel B. Rehli M. Skidgel R. Life Sci. 1997; 60: 715-724Crossref PubMed Scopus (20) Google Scholar, 15Tan F. Rehli M. Krause S.W. Skidgel R.A. Biochem. J. 1997; 327: 81-87Crossref PubMed Scopus (61) Google Scholar). The primary sequence analyses indicate that CPDs consist of three luminal/extracellular carboxypeptidase B-like domains (called A, B, and C), a hydrophobic transmembrane anchor and a highly conserved cytoplasmic tail. While in the A and B domains all essential amino acids for the enzymatic activity of CPDs investigated so far are conserved, the C domains lost most of them, despite their high overall sequence homology. It has therefore been hypothesized that this domain of CPD is catalytically inactive and serves a different, as yet unknown function (15Tan F. Rehli M. Krause S.W. Skidgel R.A. Biochem. J. 1997; 327: 81-87Crossref PubMed Scopus (61) Google Scholar). This assumption was recently confirmed for duck CPD, whose A and B domains displayed CPD activity, while the C-domain contains the DHBV preS-binding site (16Eng F.J. Novikova E.G. Kuroki K. Ganem D. Fricker L.D. J. Biol. Chem. 1998; 273: 8382-8388Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). CPDs from rat and duck have been shown to be residenttrans-Golgi network membrane proteins which cycle between the trans-Golgi network and the plasma membrane (17Varlamov O. Fricker L.D. J. Cell Sci. 1998; 111: 877-885Crossref PubMed Google Scholar, 18Breiner K.M. Urban S. Schaller H. J. Virol. 1998; 72: 8098-8104Crossref PubMed Google Scholar). Their localization, enzymatic activity, and the broad tissue distribution support the notion that CPDs are involved in processing of a variety of polypeptides that traverse the secretory pathway of various tissues (19Song L. Fricker L.D. J. Biol. Chem. 1996; 271: 28884-28889Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). However, evolutionary conservation of an enzymatically inactive C-domain, recycling from the cell surface to thetrans-Golgi network, and the uptake of DHBV particles strongly implies a yet unidentified function of CPDs, possibly involving binding of a natural ligand. We have recently characterized the receptor-binding domain within the preS region of the DHBV L-protein and demonstrated that duck carboxypeptidase D is the primary receptor for avian hepatitis B viruses (8Urban S. Breiner K.M. Fehler F. Klingmüller U. Schaller H. J. Virol. 1998; 72: 8089-8097Crossref PubMed Google Scholar, 18Breiner K.M. Urban S. Schaller H. J. Virol. 1998; 72: 8098-8104Crossref PubMed Google Scholar). In the present study we have extended these results by the detailed biochemical analysis of the DHBV preS-duck carboxypeptidase D interaction. Using a set of preS deletion mutants, we investigated binding and dissociation rates of the preS·receptor complex by real time surface plasmon resonance spectroscopy. We defined a receptor-binding domain, comprising about one-half of preS, which consists of a short receptor attachment site and a conformation dependent stabilizing element. Binding of dCPD to this domain is strong and, considering the presence of multiple binding sites in viral particles, implies that DHBV binding to hepatocytes is probably irreversible. Together with findings from earlier studies our results allow a model to be proposed for the early steps in hepadnaviral infection with several implication regarding the mechanisms of hepadnaviral infection. The baculovirus transfer vector pVL-sdCPD was constructed by ligating the NcoI/XhoI fragment of plasmid pBKRSV-gp180 (kindly provided by K. Kuroki, Kanasawa, Japan) into a modified version of plasmid pVL1393 (Fig. 1 A). This vector contains an additional NcoI site as a part of the start codon and a polylinker at the 3′ end which introduces an artificial stop codon. The NcoI/XhoI fragment of pBKRSV-gp180 encodes the signal sequence of duck CPD and the three carboxypeptidase-like domains but lacks the carboxyl-terminal transmembrane anchor and the cytosolic tail. Recombinant baculoviruses were obtained by co-lipofection ofSpodoptera frugiperda (Sf9) cells with a mixture of 100 ng of linearized Baculo-Gold DNA (Pharmingen) and 5 μg of the baculovirus transfer vector pVL-sdCPD using the manufacturer's protocol for lipofection with DOTAP (Boehringer-Mannheim). Two hours after lipofection, the medium was changed and recombinant viruses were collected after 5 days. The virus was purified according to the protocol of O′Reilly et al. (20O'Reilly D. Miller L.K. Luckow V.A. Baculovirus Expression Vectors: A Laboratory Manual. W. H. Freeman and Co., New York1992Google Scholar), amplified inSf9 cells by two additional rounds of infection and used for infection of High Five cells as described below. Protein samples for SDS-PAGE were dissolved in sample buffer (200 mm Tris/Cl, pH 6.8, 6% SDS, 20% glycerol, 10% dithiothreitol, 0.1 mg/ml bromphenol blue, 0.1 mg/ml Orange G), boiled for 5 min, and subjected to electrophoresis in 7.5 or 13% polyacrylamide-SDS gels (21Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar). After electrophoresis, proteins were either silver-stained according to the method described by Heukeshoven and Dernick (22Heukeshoven J. Dernick R. Electrophoresis. 1988; 9: 28-32Crossref PubMed Scopus (643) Google Scholar) or transferred to a nitrocellulose filter for immunological analysis. As a primary antibody we used 1:5000 dilutions of a polyclonal rabbit antiserum raised against a recombinant polypeptide of the COOH-terminal third of dCPD (kindly provided by K. Breiner). As a secondary antibody we used a horseradish peroxidase-conjugated goat anti-mouse antibody (Dianova). Detection was done by enhanced chemoluminescence (ECL, Amersham) according to the manufacturer's instructions. 4-Amino-benzoyl-arginine (PABA) was synthesized by a combination of the methods described by Hitchcock and Smith (23Hitchcock M. Smith J.N. Biochem. J. 1964; 93: 392-400Crossref PubMed Scopus (7) Google Scholar) and Plummer and Hurwitz (24Plummer Jr., T.H. Hurwitz M.Y. J. Biol. Chem. 1978; 253: 3907-3912Abstract Full Text PDF PubMed Google Scholar). The product was recrystallized and its structure verified by 3H,13C NMR, and mass spectrometry. Coupling of PABA to activated CH-Sepharose 4B (Pharmacia) was performed according to the suppliers standard protocol. 5.4 × 107 High Five insect cells (three T175 flasks), grown in 100 ml of Express Five serum-free medium (Life Technologies, Inc.) were infected with 9 ml of culture supernatant of AcNPV-sdCPD infected Sf9 cells (multiplicity of infection = 100). Cells were incubated at 27 °C for 72 h to allow protein expression. The culture supernatant was centrifuged (5000 × g, 15 min) to remove cells, passed through a 0.22-μm nitrocellulose filter, adjusted to pH 5.5 with acetic acid and applied to a 4-amino-benzoyl-arginine-Sepharose column (15-ml bed volume, flow rate of 0.5 ml/min) equilibrated with 20 mm NaAc, 1m NaCl, pH 5.5. Due to the absence of the transmembrane domain, detergent was not required during purification. All purification steps were performed at 4 °C on a FPLC system (Pharmacia). After extensive washing (15 bed volumes), the buffer was changed to 10 mm NaAc, pH 5.5 (5 bed volumes), and sdCPD was eluted with 50 mml-arginine in 20 mm Tris/Cl, pH 8.0. Protein containing fractions were dialyzed against 25 mm NaPi, pH 7.0, and concentrated to a final volume of 2–4 ml using a Centriplus 30 concentrator (Amicon). Concentration was determined by measuring the extinction at 280 nm as described by Gill and von Hippel (25Gill S.C. von Hippel P.H. Anal. Biochem. 1989; 182: 319-326Crossref PubMed Scopus (5073) Google Scholar) based on the molar extinction coefficient of 159,400 calculated from the primary sequence using the program Protean (Lasergene). Primary duck hepatocytes were prepared and cultivated as described previously (26Rigg R.J. Schaller H. J. Virol. 1992; 66: 2829-2836Crossref PubMed Google Scholar). For infection, competition assays 8 × 105 cells were cultivated for 3–8 days in 12-well plates, and infected with 4 × 107 DNA-containing DHBV particles (determined by DNA dot blot) in the absence or presence of increasing concentrations of sdCPD. To this aim DHBV-containing duck serum was mixed with appropriate stock solutions of sdCPD, completed to a final volume of 500 μl with maintenance medium, and applied to a 12-well plate of primary duck hepatocytes. After infection for 14 h at 37 °C, cells were washed twice with phosphate-buffered saline and cultured for 6 additional days. Intracellular viral DNA was prepared, using the QIAamp blood kit (Qiagen) and subjected to DNA dot-blot analysis as described by Rigg and Schaller (26Rigg R.J. Schaller H. J. Virol. 1992; 66: 2829-2836Crossref PubMed Google Scholar). All assays were quantified using a Molecular Dynamics PhosphoImager. Isolation of monomeric sdCPD and determination of apparent molecular weights of proteins and protein complexes were achieved by exclusion chromatography on a calibrated Superdex 200 column (1.6 × 60 cm; Pharmacia), connected to a FPLC system (Pharmacia) and equilibrated in 5% sucrose, 150 mmNaCl, 25 mm NaPi, pH 7.0. All chromatographic steps were performed at 4 °C with a flow rate of 2.2 ml/min. Sample volumes were 0.5 ml for analytical and up to 2 ml for preparative purposes. Eluted proteins were collected in fractions of 2.2 ml, subjected to SDS-PAGE, and analyzed by silver staining or immunoblotting. The column was calibrated with thyroglobulin (670 kDa), fraction 12; γ-globulin (158 kDa), fraction 16; ovalbumin (44 kDa), fraction 20; myoglobin (17 kDa), fraction 23 and vitamin B-12 (1.3 kDa), fraction 28. PreS polypeptides were prepared as described previously (8Urban S. Breiner K.M. Fehler F. Klingmüller U. Schaller H. J. Virol. 1998; 72: 8089-8097Crossref PubMed Google Scholar) and covalently immobilized on covalink immunoplates (Nunc) via COOH groups by NHS/EDC activation chemistry. Each well consisted of 15–30 μg of preS polypeptide in 100 μl of 25 mm NaPi, pH 6.3, and were mixed with 50 μl ofN-hydroxysulfosuccinimide (3.48 mg/ml in aqua bidest.) and 50 μl of EDC (3.07 mg/ml in aqua bidest.). Coupling was allowed to proceed for 30 min at room temperature with gentle agitation. To remove uncoupled polypeptide, plates were washed 3 times with 300 μl of water and 3 times with 300 μl of phosphate-buffered saline, 0.2% Tween 20. After blocking with 200 μl of 2% BSA in phosphate-buffered saline for 30 min, CPD containing protein samples were added in either standard binding buffer (1% Triton X-100, 50 mm Tris/Cl, 150 mm NaCl, pH 7.4) for solubilized membrane fractions or detergent-free binding buffer (50 mm Tris/Cl, 150 mm NaCl) for recombinant soluble dCPD. Binding assays at different pH values were performed in sodium acetate buffer, pH 3.5–5.5, sodium phosphate buffer, pH 6.0–8.0, and Tris buffer, pH 8.5–10.0. Binding was allowed to occur for at least 4 h at 4 °C. Unbound proteins were removed and the plate was washed 3 times with binding buffer at 4 °C. Bound proteins were eluted with 50 μl of SDS sample buffer at 80 °C and analyzed by PAGE and Western blotting. Surface plasmon resonance analysis (BIAcore-Upgrade, BIAcore-System) of DHBV preS protein binding to sdCPD was done at 37 °C in 1 × HBS buffer (10 mm HEPES, pH 7.4, 150 mm NaCl, 0.005% Surfactant P-20 (Pharmacia)) at flow rates of 5–30 μl/min according to standard protocols provided by the manufacturer. DHBV preS proteins were coupled to a CM5 sensor chip via standard NHS/EDC activation chemistry (BIAcore amine coupling kit) in amounts that yielded 2,200–3,500 response units. Monomeric and oligomeric sdCPD at the indicated concentrations were injected for 5 min followed by 3 min elution with HBS buffer. The sensor chip was cleaned of sdCPD bound to immobilized DpreS by injection of 30 μl of regeneration solution (20 mm HCl). Binding constants of the sdCPD-DpreS interaction were calculated with the BIAevaluation program version 2.1 (Pharmacia). The baselines for the curves shown in Fig. 4 were adjusted to zero before calculation. Calculation of K d was done by fitting the data to the equation R =R 0e −K d(t−t 0). To facilitate biochemical studies on the interaction of duck hepatitis B virus (DHBV) with duck carboxypeptidase D (dCPD), we constructed recombinant baculoviruses that encode the three extracellular carboxypeptidase-like domains of dCPD and the amino-terminal export signal but lack the COOH-terminal transmembrane anchor and the highly conserved cytoplasmic tail (Fig.1 A). Recombinant baculoviruses were amplified in S. frugiperda 9 (Sf9) cells and used for infection of the High Five insect cell line (see “Experimental Procedures”). Omission of the transmembrane anchor and the cytoplasmic part resulted, as intended, in the secretion of a soluble dCPD variant of 170 kDa (sdCPD), as shown by Western blot analysis of culture supernatants using an antiserum against gp180 (Fig.1 B). Treatment with endoglycosidases H and F led to a decrease of the molecular weight indicating that sdCPD had been modified by both complex and high mannose-type glycosylation (Fig.1 B). Purification of sdCPD from the culture supernatant of infected High Five cells was achieved by affinity chromatography on a PABA-Sepharose column (Fig. 1 C) as described previously (12Song L. Fricker L.D. J. Biol. Chem. 1995; 270: 25007-25013Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). Silver staining of proteins from pooled fractions eluting from the PABA column revealed a single band at about 170 kDa with only minor contaminants. Western blot analysis using an anti-dCPD specific antibody verified the identity of the 170 kDa as dCPD (Fig. 1 D) as does analysis by Edman degradation. The NH2-terminal sequence AHIKKAEAA … indicated cleavage of the leader sequence at position 25 in insect cells, and was found to be identical to previously published data for endogenous duck CPD (11Kuroki K. Eng F. Ishikawa T. Turck C. Harada F. Ganem D. J. Biol. Chem. 1995; 270: 15022-15028Crossref PubMed Scopus (119) Google Scholar) and bovine CPD (19Song L. Fricker L.D. J. Biol. Chem. 1996; 271: 28884-28889Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). In addition, detection of a second sequence KAEAA … , lacking 4 amino acids, suggests that processing can also occur after lysine 28 in insect cells. The comparison of the yields of recombinant sdCPD obtained from Sf9 cells with the yields obtained from High Five cells revealed an about 8-fold higher expression rate in the High Five cell line (25 μg of sdCPD per 107 Sf9 cells in contrast to 200 μg/107 High Five cells). Further analysis of affinity purified sdCPD on a calibrated Superdex 200 gel filtration column (Fig.2 A) resulted in two forms with different apparent molecular weights, which were, however, indistinguishable in SDS-PAGE (Fig. 2 B) and Western blot analysis (data not shown). About 35% of sdCPD eluted in the void volume and represent oligomers with molecular masses of approximately 3,600 kDa as determined by electron microscopy (data not shown). 65% of sdCPD eluted at 248 kDa and represents monomeric sdCPD eluting at a slightly higher molecular mass than expected. Since proteins were initially purified by affinity chromatography on PABA-Sepharose it can be assumed that both forms have carboxypeptidase activity but differ considerably in their binding properties to DHBV preS, as shown below. To investigate whether recombinant sdCPD exhibits receptor function comparable to the cell surface bound molecule on hepatocytes, we performed infection inhibition assays essentially as described previously for DHBV preS (DpreS) polypeptides (8Urban S. Breiner K.M. Fehler F. Klingmüller U. Schaller H. J. Virol. 1998; 72: 8089-8097Crossref PubMed Google Scholar). Primary duck hepatocytes were infected with DHBV in the presence of increasing amounts of monomeric or oligomeric sdCPD, and viral markers (intracellular viral DNA or secreted DHBV e-antigen) were quantified 6 days post-infection. As shown in Fig.3 A, both forms of sdCPD efficiently inhibit DHBV infection. About 7 molecules of monomeric sdCPD per viral particle were sufficient for 50% inhibition of infection (18Breiner K.M. Urban S. Schaller H. J. Virol. 1998; 72: 8098-8104Crossref PubMed Google Scholar). In comparison, oligomeric sdCPD, despite its inability to bind recombinant DHBV preS, as shown below, competed DHBV infection also remarkably well; with about 15 molecules needed for 50% inhibition. In these calculations we assumed that an approximately 1,000-fold excess of non-infective subviral particles is present in the infective serum. Taking into account that one particle consists of approximately 20 L-protein molecules with 50% having an inverse topology and therefore their receptor-binding domain is located inside the viral particle (27Swameye I. Schaller H. J. Virol. 1997; 71: 9434-9441Crossref PubMed Google Scholar), we conclude that every single sdCPD molecule is able to bind viral particles. Ratios of 100 sdCPD molecules/particle almost completely block DHBV infection, indicating that viral particles bound to sdCPD are unable to infect hepatocytes. Insect cell-derived soluble duck carboxypeptidase D therefore constitutes a suitable tool for virus-receptor interaction studies at the molecular level. Competitive inhibition of infection was also performed with theE. coli-derived preS polypeptides used in the binding studies described below. As shown in Fig. 3 B, full-length DpreS and the DHBV preS fragment DpreS30–115, representing the receptor-binding domain, inhibit DHBV infection equally well in a concentration-dependent manner. Under the conditions used, the IC50 was determined to be 0.4–0.8 μm, corresponding to approximately 3000 DpreS molecules per viral particle. To confirm that the biological activity of sdCPD in infection competition experiments correlates with its physical binding properties to DHBV preS polypeptides, we performed binding assays with the solid phase bound preS polypeptides depicted in Fig.4 A and compared the results with those obtained for authentic dCPD from duck liver lysates. As shown in Fig. 4 B, authentic dCPD binds DpreS and the DpreS fragment consisting of amino acids 30–115 (DpreS30–115). The deletion mutant DpreSΔ85–96, inactive in DHBV infection competition (8Urban S. Breiner K.M. Fehler F. Klingmüller U. Schaller H. J. Virol. 1998; 72: 8089-8097Crossref PubMed Google Scholar), was used as a control and showed no binding. Likewise, recombinant sdCPD eluted from the PABA column binds DpreS but not DpreSΔ85–96 (Fig.4 C). However, even at a large excess of DpreS we did not observe complete binding of sdCPD (Fig. 4 C, left frame, left lane) and therefore assumed that a fraction of sdCPD might be inactive in DpreS binding. This interpretation was confirmed in a quantitative BIAcore analysis showing that oligomeric sdCPD does not bind DpreS (see below). In Fig. 5 the pH dependence of the DpreS-sdCPD interaction is shown. Binding was observed between pH 5.5 and 10.0 with an optimum at pH 6.5. Binding still occurred at pH 5.0. However, further protonation abolished binding completely. To determine association and dissociation rates of the DHBV preS·sdCPD complex and accordingly deduce the affinity of DHBV to its cellular receptor we followed complex formation and dissociation by real time surface plasmon resonance analysis (BIAcore). DHBV preS polypeptides were covalently immobilized to CM5 sensor chips (see “Experimental Procedures”) and three different concentrations (0.06, 0.15, and 0.29 μm) of monomeric sdCPD were injected onto the surface at 37 °C (Fig. 6 A). The kinetics of binding (100–400 s) and release (400–575 s) of sdCPD were calculated from the slopes of the curves. The association ratek a was determined to 4.0 × 104m−1 s−1, the dissociation ratek d to 1.9 × 10−3s−1, corresponding to a half-life for the complex of about 6.0 min at 37 °C. From these data we calculated the dissociation constant K d to 4.6 × 10−8m. Using the DHBV preS fragment from amino acid 30 to 115 (DpreS30–115), which has been identified by infection competition experiments as the receptor-binding domain (8Urban S. Breiner K.M. Fehler F. Klingmüller U. Schaller H. J. Virol. 1998; 72: 8089-8097Crossref PubMed Google Scholar), we observed similar constants: k a, 7.1 × 104m−1 s−1; k d, 2.7 × 10−3 s−1;t 12, 4.3 min; K d, 3.8 × 10−8m (Fig. 6 B). Consequently full-length DpreS and DpreS30–115 are indistinguishable in both infection competition and dCPD interaction. Consistent with our observation in the qualitative binding assay (Fig.4 B), the deletion mutant DpreSΔ85–96 showed no obvious interaction with sdCPD in the BIAcore experiment (Fig. 6 D). This observation led us to conclude that amino acid residues 85–96 of DpreS contain absolutely essential elements for receptor interaction. Interestingly, anti-preS antibodies which are capable of blocking DHBV infection have been mapped to bind epitopes within this region 2C. Kuhn, personal communication., 3L. Cova, personal communication.indicating their surface exposure. Despite its ability to bind the substrate PABA, most likely via the two enzymatically active A and B domains, oligomeric sdCPD was found to interact only weakly with DpreS (Fig. 6 C). Thus oligomerization restricts DpreS binding. We extended surface plasmon resonance analysis to investigate the contribution of particular sequence elements within DpreS to receptor binding, by testing the same set of deletion mutants (Fig.7 A) that have been characterized in infection competition experiments (8Urban S. Breiner K.M. Fehler F. Klingmüller U. Schaller H. J. Virol. 1998; 72: 8089-8097Crossref PubMed Google Scholar) for sdCPD interaction (Fig. 7, B-D). Deletions outside the receptor-binding domain (mutants DpreSΔ22–30 and DpreSΔ128–139) showed kinetics of association and dissociation similar to full-length DpreS (Fig. 7 B). This indicates that the part of preS that binds the receptor folds independently of the deleted flanking amino acids and constitutes a distinct domain within the viral L-protein. In contrast, DpreSΔ85–96 and deletion mutant DpreSΔ101–109 were completely impaired in sdCPD binding (Fig. 7 D). This region of preS (amino acids 85–109) therefore contains elements which are absolutely required for receptor interaction. An intermediate phenotype was found for deletion mutants DpreSΔ67–70 and DpreSΔ74–84. They were able to interact with sdCPD but displayed drastically diminished binding and dissociation rates (Fig. 7 D). This was confirmed using the terminal deletion mutants depicted in Fig. 7 C. While DpreS30–115 binds sdCPD as strongly as full-length DpreS, mutants with successive deletions beyond amino acid 30 (DpreS38–115, DpreS43–115, and DpreS52–130) were" @default.
• W2080139517 created "2016-06-24" @default.
• W2080139517 creator A5014580542 @default.
• W2080139517 creator A5034130805 @default.
• W2080139517 creator A5090868065 @default.
• W2080139517 date "1999-02-01" @default.
• W2080139517 modified "2023-09-29" @default.
• W2080139517 title "A Soluble Form of the Avian Hepatitis B Virus Receptor" @default.
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