INTRODUCTION

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Adenovirus (Ad) is a group of nonenveloped double-stranded DNA viruses associated with a range of respiratory, ocular, and gastrointestinal infections (15). There is a tendency for virus types within the same subgroups to be associated with similar host tissue tropism and pathogenicity (15, 28). Entry of human Ad into human cells is a stepwise process (10). The primary event in this sequence is attachment that involves an interaction between the Ad fiber protein and its high-affinity cellular receptor (4, 12, 26). Internalization of virus particles as well as cell membrane permeabilization is subsequently mediated through a specific interaction of the viral penton base protein with cell surface integrins (1, 2, 5, 17, 18, 31-33).

The coxsackievirus and Ad receptor (CAR) is a 46-kDa transmembrane protein that was initially identified as a cellular receptor for coxsackie B viruses, Ad type 2 (Ad2), and Ad5 (3, 27). In addition to subgroup C Ad fibers, CAR was also shown to bind to subgroup A, D, E, and F Ad fibers but not to subgroup B Ad fibers like serotype 3 or to the short fiber of subgroup F Ad (24). The CAR sequence predicts a structure related to the immunoglobulin (Ig) superfamily with the extracellular domain consisting of two Ig-related regions, the amino-terminal IgV and IgC2 domains (3). The IgV domain appears sufficient for virus attachment and infection (8), while the transmembrane and intracellular regions appear dispensable for these functions (30). Expression and cellular localization of CAR correlate with Ad5 infection and is therefore an important determinant of Ad tropism (3, 5, 20, 22, 27, 29). The major histocompatibility complex class I 2 domain was also proposed as a high-affinity receptor for Ad2 and Ad5 fibers (14). Its role, however, remains uncertain since the major histocompatibility complex class I allele HLA-A*0201 permanently expressed in hamster cells failed to bind to Ad5 fiber knob with high affinity and there was no evidence of cooperativity between the two proteins (6).

The Ad5 fiber is a homotrimer with each subunit consisting of three domains: the amino-terminal tail that associates with the penton base protein (7, 21); the shaft, which consists of a motif of approximately 15 residues that is repeated 22 times (11); and the knob, which interacts with the cellular receptor. The structure of Ad5 fiber knob monomer is an eight-stranded antiparallel  sandwich composed of two  sheets, with loops and turns connecting the  strands (13, 34).

We recently showed that  strands E and F in Ad5 fiber knob may contain binding residues for CAR (16, 25) and that deletion of two consecutive amino acids in the DG loop of the protein abolished binding to CAR through disruption of local interactions (16). In the present study, we demonstrate that Ser408 and Pro409 in the AB loop, Tyr477 in the DG loop, and Leu485 in  strand F are in direct contact with CAR and that they constitute at least part of the binding site which is located at the periphery of the protein.

	MATERIALS AND METHODS

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Site-directed mutagenesis of Ad5 fiber knob. Ad5 fiber knob and 15 residues of the terminal repeating unit of the shaft (nucleotides 32197 to 32783) were cloned into pKK233-3, an isopropyl--D-thiogalactopyranoside-inducible procaryotic expression vector (Pharmacia), to generate plasmid pF5knob, as previously described (16, 25). Plasmid pF5knob was used as the basis for the mutagenesis of Ad5 fiber knob. Single amino acid substitutions were introduced in pF5knob using a QuikChange site-directed mutagenesis kit (Stratagene). All mutations and the integrity of the remaining sequence were confirmed by DNA sequencing. The following pairs of complementary oligonucleotide primers were used to define each mutation in Ad5 fiber: Ala406Lys, 5'-ttgtggaccacaccaaaaccatctcctaactgt and 5'-acagttaggagatggttttggtgtggtccacaa; Ser408Glu, 5'-accacaccagctccagaacctaactgtagacta and 5'-tagtctacagttaggttctggagctggtgtggt; Pro409Lys, 5'-acaccagctccatctaagaactgtagactaaat and 5'-atttagtctacagttcttagatggagctggtgt; Arg412Asp, 5'-ccatctcctaactgtgacctaaatgcagagaaa and 5'-tttctctgcatttaggtcacagttaggagatgg; Asn414Lys, 5'-cctaactggagactaaaggcagagaaagatgct and 5'-agcatctttctctgcctttagtctccagttagg; His456Glu, 5'-acagttcaaagtgctgagcttattataagattt and 5'-aaatcttataataagctcagcactttgaactgt; Ile458Ala, 5'-caaagtgctcatcttgcgataagatttgacgaa and 5'-ttcgtcaaatcttatcgcaagatgagcactttg; Ile458Glu, 5'-caaagtgctcatcttgagataagatttgacgaa and 5'-ttcgtcaaatcttatctcaagatgagcactttg; Asn470Glu, 5'-ggagtgctactaaacgaatccttcctggaccca and 5'-tgggtccaggaaggattcgtttagtagcactcc; Phe472Ala, 5'-ctactaaacaattccgcactggacccagaatat and 5'-atattctgggtccagtgcggaaatgtttagtag; Phe472Lys, 5'-ctactaaacaattcaagactggacccagaatat and 5'-atattctgggtccagcttggaaatgtttagtag; Asp474Arg, 5'-aacaattccttcctgcggccagaatattggaac and 5'-gttccaatattctggccgcaggaaggaattgtt; Tyr477Ala, 5'-ttcctggacccagaagcttggaactttagaaat and 5'-atttctaaagttccaagcttctgggtccaggaa; Arg481Glu, 5'-gaatattggaactttgagaatggagatcttact and 5'-agtaagatctccattctcaaagttccaatattc; Asn482Glu, 5'-attggaactttagagagggagatcttactgaa and 5'-ttcagtaagatctccctctctaaagttccaata; Gly483Glu, 5'-tggaactttagaaatgaagatcttactgaaggc and 5'-gccttcagtaagatcttcatttctaaagttcca; Asp484Ala, 5'-aactttagaaatggagctcttactgaaggcaca and 5'-tgtgccttcagtaagagctccatttctaaagtt; Leu485Lys, 5'-tttagaaatggagataaaactgaaggcacagcc and 5'-ggctgtgccttcagttttatctccatttctaaa; Lys506Ala, 5'-ctatcagcatatccagcatctcacggtaaaact and 5'-agttttaccgtgagatgctggatatgctgatag; Ser507Ala, 5'-tcagcatatccaaaagctcacggtaaaactgcc and 5'-ggcagttttaccgtgagcttttggatatgctga; and Lys510Glu+Lys506Ala, 5'-ctatcagcatatccagcatctcacggtgaaactgccaaaagtaac and 5'-gttacttttggcagtttcaccgtgagatgctgctggatatgctgatag.

Expression and purification recombinant proteins. Expression and purification of wild-type and mutated Ad5 fiber knobs in Escherichia coli were performed as previously described (13, 16, 25, 34).

Surface plasmon resonance (SPR) analysis of interactions between wild-type and mutant Ad5 fibers and soluble CAR. (i) Preparation of SPR sensor surface. Purified recombinant sCAR, IgV, and IgC2 domains were coupled to the CM5 sensor chip, using the amine coupling reaction according to the manufacturer's instructions. Briefly, the CM5 sensor chip was activated using a solution of 50 mM N-hydroxysuccinimide and 200 mM N-ethyl-N'-(dimethylaminopropyl)carbonide to activate the esters on the chip surface. sCAR, IgV, and IgC2 were then injected over the activated surface until a sufficient quantity had bound to the activated esters on the chip surface. A wide range of resonance units (RU) were tested, with immobilization densities of 600 to 800 RU used in all final experiments. Residual esters were inactivated by an injection of 1 M ethanolamine hydrochloride (pH 8.5). Purified soluble Ad5 fiber knob was also immobilized on the chip, using the same approach as described above. Biacore AB supplied the BIAcore system, CM5 sensor chip, and amine coupling kit.

(ii) SPR analysis of wild-type and mutant Ad5 fibers. SPR analysis of immobilized CAR proteins with recombinant wild-type and mutant Ad5 fiber knobs was performed as follows. All interactions were carried out at 25°C, using HBS (10 mM HEPES [pH 7.4], 150 mM NaCl, 3 mM EDTA, 0.005% P-20 surfactant) as the continuous flow buffer at different flow rates (5, 10, 20, and 40 µl/min). A wide range of concentrations (10 nM to 5 µM) of each protein was used as analyte, with the analyte diluted in HBS and injected for a 120-s association phase followed by HBS for approximately 60 s to monitor dissociation of the bound analyte. The chip was then regenerated with two 2-µl pulses of 25 mM HCl to remove analyte bound to the chip surface. Sample injections onto a sensor chip with immobilized bovine serum albumin as an irrelevant protein assessed nonspecific binding of the analyte; injection onto an uncoated sensor chip assessed nonspecific binding to the sensor chip. Nonspecific binding (<1%) was subtracted from total binding for each case.

(iii) Kinetic analysis of SPR data. The association (k_a) and dissociation (k_d) rate constants for a monophasic model of binding were obtained using the BIAevaluation analysis package (version 2.1). The ability of the models to describe the experimental data was determined by examination of the residual plots, which were calculated by subtracting the experimental data points from the fitted curve. Residual was small and randomly distributed around zero (range between 0.5 to 0.5). Nonspecific binding (<1%) was subtracted from specific binding prior to kinetic analysis.

Cell binding competition experiments. The efficiency and affinity of wild-type and mutant recombinant Ad5 mutant fibers to functional receptors on CHO-CAR cells (3) was determined from cell binding competition assays between Ad5Luc3 (Ad5Luc3, obtained from P. Boulanger, is a replication-competent virus which contains the luciferase gene under the control of the simian virus 40 early promoter inserted in the E3 region of the Ad5 genome) and recombinant Ad5 fiber knobs, using the level of luciferase gene expression as the endpoint assay.

Phenotypic analysis of recombinant mutated fibers. Circular dichroism (CD) measurements were performed on Jobin-Yvon (Longjumeau, France) CD6 spectrophotometer using cylindrical quartz cells of 0.2-cm path lengths. The spectrophotometer was calibrated for wavelength and ellipticity using d-10-camphorsulfonic acid. Samples were measured in the concentration range 0.9 to 1.4 mg/ml in 50 mM sodium perchlorate (pH 6.5) at constant temperature in thermostated cell holder. The CD data were analyzed for percentage  helix and  sheet as previously described (23).

Cells and viruses. CHO cells and CHO-CAR cells (3) were maintained in alpha medium (Gibco) supplemented with 10% fetal calf serum. Ad5Luc3 was plaque purified twice and propagated in 293 cells in accordance with established methods (9).

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Selection of Ad5 fiber knob mutations. Ad5 fiber knob mutants deleted of two consecutive amino acids between residues 485 and 493 in the DG loop of the protein abolished high-affinity binding to CAR (16). These five deletion mutant fibers accumulated as stable trimers, but their CD spectra indicated that although there was no disruption of the overall folding and secondary structure of the protein, local conformational changes had occurred (16). This implied the involvement of residues in neighboring loop regions. One candidate region was the segment containing the  strands E and F and the turn between them (residues 479 to 486) because substitution of this sequence with the corresponding sequence in Ad3 resulted in reduced receptor binding (16).

View larger version (7884K): [in this window] [in a new window]   FIG. 1.   (a) Stereo image showing the course of the polypeptide chain (Ca atoms only) within a subunit of the fiber knob protein, and the residues implicated in CAR binding in this study. In yellow are residues which, when mutated, profoundly affect the affinity for CAR (Ser408 and Pro409 in the AB loop, Tyr477 in the DG loop, and Leu485 in strand F) and implicated as contact residues. In orange are residues that may be only peripherally or indirectly involved in CAR binding (Ala406 and Arg412 in the AB loop, Arg481 in strand E [this report], and Tyr491 in the DG loop [16]). In green are locations of residues (Ca atoms only) which, when mutated, have no effect on CAR binding (Asn414, His456, Ile458, Asn470, Phe472, Asp474, Asn482, Gly483, Asp484, Lys506, Ser507, His508, and Lys510 [this report]; Thr492, Asn493, and Val495 [16]; and Cys428, Thr451, Lys526, Gly538, and Glu566 [data not shown]). The four-stranded  sheet known as the R sheet (see text) can be seen edge-on at the top right. (Coordinates were taken from the crystal structure [13, 34], and the images were produced by INSIGHTII [Molecular Simulations Inc., Cambridge, United Kingdom]). (b) Space-filling image (showing all nonhydrogen atoms) of the fiber knob subunit in the same orientation as in panel a, together with the two other subunits that form the trimer. The three subunits are distinguished by color (purple, magenta, and maroon), but the residues investigated by mutagenesis are colored in the same way as in panel a, in each subunit. Only the CAR binding site in the purple subunit is visible in this orientation, in which the trimer is viewed at 90° to the trimer axis with the shaft connection at the bottom. (c) The trimeric fiber knob protein as shown in panel b, rotated 90° and viewed along the threefold axis, from the side connected to the shaft. All three CAR-binding sites are visible in this orientation.

The first extracellular domain of CAR is responsible for binding to Ad5 fiber knob. It was recently shown that IgV domain of CAR formed complexes with Ad2 fiber knob in vitro and was sufficient to inhibit Ad2 infection of HeLa cells (8). The kinetics of binding for this interaction and for the interaction between the IgC2 domain IgC2 and CAR were, however, not assessed. Recombinant CAR proteins expressed in bacteria are produced as mainly insoluble and aggregated proteins (8); we therefore expressed the two extracellular domains of CAR together (sCAR) and independently (IgV and IgC2) in insect cells. The biological activity of purified recombinant sCAR, IgV, and IgC2 proteins was assessed first in cell binding competition experiments. We found that sCAR and IgV domains inhibited Ad5 infection of CHO-CAR cells with similar efficiencies and that the IgC2 domain had no effect on Ad5 infection of these cells (data not shown).

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View larger version (20K): [in this window] [in a new window]   FIG. 2.   SPR sensograms for wild-type Ad5 fiber knob interacting with sCAR (A) and IgV domain alone (B). Ad5 fiber knob was injected over the sensor surface at multiple concentrations in the range of 10 nM to 5 µM. A 90- to 120-s association phase was followed by a 60-s dissociation phase with HBS buffer flowing over the sensor surface at 10, 20, 30, and 40 µl/min. Representative sensograms of Ad5 fiber knob as analyte at concentrations of 200 nM, 400 nM, and 5 µM are shown.

Leu485 in  strand F is critical for Ad5 fiber knob binding to CAR. We mutated all residues with surface-exposed side chains within short  strands E and F and the turn between them (residues 479 to 486) in Ad5 fiber knob and assessed their effect on fiber knob binding to CAR.

View larger version (31K): [in this window] [in a new window]   FIG. 3.   SPR analysis of interactions between sCAR and wild-type and mutant Ad5 fiber knobs. sCAR was immobilized on the CM5 chip. The filled bars represent the maximal association response at equilibrium in RU, and the open bars represent the response at a fixed time point (60 s) into the dissociation phase. This permits comparison to be made between wild-type and mutant fiber knob proteins in relation to their association and dissociation kinetics.

View larger version (15K): [in this window] [in a new window]   FIG. 4.   SPR sensograms of wild-type Ad5 fiber knob (a) and of mutant Ad5 fiber knobs Ala406Lys (b), Arg481Glu (c), and Arg412Asp (d) binding to immobilized sCAR. The various Ad5 fiber ligands were injected over the sensor surface at three different concentrations (10, 50, and 200 nM). A 2-min association phase was followed by a 1-min dissociation phase with HBS buffer flowing over the sensor surface at 10, 20, 30, and 40 µl/min. Representative sensograms with wild-type and mutant Ad5 fiber knob proteins at a concentration of 200 nM used as analyte are shown.

View larger version (33K): [in this window] [in a new window]   FIG. 5.   CHO-CAR cell binding competition experiments between wild-type or mutant Ad5 fiber knobs and Ad5Luc3. Cells were infected with Ad5Luc3 (MOI of 10) in the presence of large excess of recombinant full-length Ad5 fiber proteins (100 µg/ml). Ad5Luc3 was preincubated with recombinant proteins at room temperature, and the mixture was added to CHO-CAR cells precooled on ice. After incubation for 1 h at 0°C, unabsorbed virus was rinsed off; the cell monolayers were covered with prewarmed medium, transferred to 37°C, further incubated at that temperature for 18 h, and then processed for luciferase assay. Luciferase activity, expressed in relative light units, was assayed in cell lysates using luciferase substrate solution. Results were expressed as percentage reduction in luciferase activity compared to the control cells (i.e., no recombinant fiber = 0%). The data presented are means and standard errors of the means (n = 3) of three representative experiments.

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Identification of further candidate contact residues in the CAR-binding site of Ad5 fiber knob. Following the identification of Leu485 as a critical residue for the interaction of Ad5 fiber knob with CAR, we obtained additional mutants in order to characterize the CAR-binding site. Four residues, Ala406, Ser408, and Pro409 in the AB loop and Tyr477 in the DG loop, have exposed side chains in the same region as Leu485 (Fig. 1a and b). We found that Ser408, Pro409, and Tyr477 failed to bind to sCAR at all concentrations used (Fig. 3) and also failed to compete efficiently with Ad5Luc3 for CHO-CAR cell receptors (Fig. 5). In addition, their IC₅₀s were strikingly higher than that of Ad5 fiber knob (for Tyr477Ala, 100 µg/10⁵ cells; for Ser408Glu, >100 µg/10⁵ cells; for Ser408Glu, >100 µg/10⁵ cells). These results demonstrate that Ser408, Pro409, and Tyr477 are critical for Ad5 fiber knob binding to CAR. Two other mutants, Ala406Lys and Arg412Asp, competed as efficiently as the wild-type protein for CHO-CAR cell receptors (Fig. 5), but their IC₅₀s (0.5 and 2.5 µg/10⁵ cells) were 5- and 25-fold higher, respectively, than the IC₅₀ of Ad5 fiber knob. Ala406Lys and Arg412Asp both exhibited significantly lower levels of binding to sCAR, principally due to a higher dissociation rate in the case of Arg412Asp at least (Table 1 and Fig. 4). These finding suggest that Ala406 and Arg412 are likely to be peripheral to the binding site (Fig. 1b).

Residues in  strand D and the DG loop (residues Asn470 to Asn474 and Lys506 to Lys510) do not interact with CAR. Our previous findings indicated that deletions in the DG loop dramatically reduced Ad5 fiber knob binding to CAR due to local conformational changes (16). One possibility was that these conformational changes had disrupted the interaction between neighboring residues in the R sheet of the protein and CAR. We therefore mutated residues Asn414 in the AB loop, His456 and Ile458 in  strand D, and Asn470, Phe472, and Asn474 in the DG loop. All of these residues have exposed and accessible side chains at the edge of the R sheet and in the region between this sheet and those residues already identified as contact residues (Fig. 1a).

Analysis of wild-type mutant Ad5 fiber knob proteins by CD spectroscopy. The consequence each mutation on the folding and secondary structure of the Ad5 fiber knob was analyzed by CD spectroscopy and by the ability to form stable trimers.

View larger version (24K): [in this window] [in a new window]   FIG. 6.   Comparisons of the CD spectrum of wild-type Ad5 fiber knob with those of mutant fiber knob proteins. All samples were measured in the concentration range of 0.9 to 1.4 mg/ml in 50 mM sodium perchlorate (pH 6.5) in a 0.2-cm path length cell at constant temperature.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

In this study, we set out to characterize the CAR-binding site in Ad5 fiber knob and to identify specific residues that may be in direct contact with CAR. This work built on our previous studies, which showed that the segment containing  strands E and F and the turn between them (residues 479 to 486) in Ad5 fiber knob might contain residues that are critical for receptor recognition (16).

Before discussing individual mutations, the interaction between wild-type Ad5 fiber knob and CAR needs to be addressed. We found that the dissociation constant determined by SPR for wild-type Ad5 fiber knob binding to sCAR was similar to that derived using conventional binding assays on CAR-transfected hamster cells (6) and KB and HeLa cells (19) and almost identical to the dissociation constant for Ad5 fiber knob binding to IgV alone. Since the IgC2 domain failed to bind to Ad5 fiber knob at all concentrations used, it is clear that the IgV domain of CAR is both necessary and sufficient for binding. It is also clear that there is no requirement for additional membrane components to confer high-affinity binding to Ad5 fiber knob. These observations support and extend those of Freimuth et al. (8).

Four mutations, Ser408Glu, Pro409Lys, Tyr477Ala, and Leu485Lys, abolished specific binding to sCAR as assessed by SPR and cell binding competition experiments. These findings indicate that Ser408, Pro409, Tyr477, and Leu485 are critical residues in the Ad5 fiber knob-CAR binding interaction. Two other mutations, Ala406Lys and Arg412Asp, had significant effects on binding kinetics, with dissociation constants 5 and 16 times lower, respectively, than those for their wild-type counterpart. The side chain of Ala406 lies very close to those of Ser408, Pro409, Tyr477, and Leu485, whereas that of Arg412 is further away (Fig. 1a). However, the long side chain of arginine may allow sufficient flexibility of movement to bring it closer to the other exposed side chains (Fig. 1a). All six mutant proteins were analyzed by CD spectroscopy and found to be indistinguishable from the wild-type Ad5 fiber knob. This, and the fact that these proteins accumulated as stable trimers, would indicate that these mutations did not alter the secondary or tertiary structure of the protein. This leads us to propose that Ser408, Pro409, Tyr477, and Leu485 are critical contact residues and together with Ala406 and Arg412, which may be only peripherally involved, constitute at least part of the high-affinity binding site for CAR. Determination of the crystal structure of Ad5 fiber-CAR complex will resolve the precise contribution of these residues in the binding interaction.

The only other residue in  strands E and F that, when mutated, altered the binding kinetics significantly was Arg481. Replacement of this residue with glutamic acid reduced the dissociation constant mainly through an increase in the rate of dissociation. In contrast, adjacent residues Asn482, Glu483, and Asp484 bound to CAR with similar kinetics and overall affinity as wild-type protein. This and the fact that the side chain of Arg481 is partially buried at the subunit interface, with the head group exposed at a distance from the other contact residues (Fig. 1), suggests that Arg481 is unlikely to be a contact residue. However, it may well exert an indirect effect on binding by causing a subtle local structural rearrangement.

Residues Ala406, Ser408, and Pro409 lie close to the interface between adjacent subunits, raising the possibility that the binding site may span two subunits. However, this was not supported by our findings, which showed that Lys506Ala, Ser507Ala, His508Glu, and Lys510Glu+Lys506Ala mutant fiber knobs bound to CAR with almost the same affinity as their wild-type counterpart. These residues lie close to residues Ala406, Ser408, and Pro409 across the subunit interface (Fig. 1b and c). Another adjacent region that might include contact residues for receptor binding lies toward and encompasses the edge of the R sheet. The conformation of this could be affected by  strands E and F and the adjoining part of the DG loop, which, when rearranged, reduced Ad5 fiber binding to CAR. We therefore mutated Asn414 in the AB loop, His456 and Iso458 in  strand D, as well as Asn470, Phe472, and Asp474 in the DG loop, since all of these residues lie within this region and have surface-exposed side chains. All fiber knob proteins with mutations in this region of the structure bound to CAR with the same affinity as the wild-type protein, demonstrating that none of the above residues is involved in the binding interaction and that the boundaries of the binding patch do not extend toward this part of the R sheet.

Our findings are consistent with the known pattern of reactivity of different Ad serotypes with CAR. Two of the proposed contact residues, Ser408 and Pro409, are identically conserved in Ad2 and Ad9 fibers as well as the long fibers of Ad40 and Ad41 that bind to CAR but are not conserved in Ad3 fiber and the short fiber of Ad41 which do not bind to CAR. Residues Tyr477 and Leu485 are either nonconservatively substituted or have no counterpart at all in Ad3 fiber or the short fiber of Ad41. Epitope mapping of two monoclonal antibodies that neutralized Ad5 infection of HeLa cells by blocking the Ad5 fiber-binding site characterized two contiguous regions in Ad5 fiber knob spanning residues 438 to 486 (14). The fact that critical residues Arg477 and Leu485 are within a region spanned by one of the two epitopes (residues 473 to 486) provides a molecular explanation for those observations.

The three binding sites for CAR are located around the periphery of the trimer (Fig. 1c). They are, however, disposed toward the underside of the protein near the shaft domain and not, as had been speculated, on the upper more exposed surface (Fig. 1a and b) (34). The definition of the CAR binding site and the identification of individual contact residues in Ad5 fiber knob have important implications for our understanding of the molecular determinants of Ad tropism, will permit modulation of the Ad-host cell interaction for the development of novel target-specific Ad vectors for human gene therapy, and will allow the development of peptide inhibitors of Ad infection.