Mutations in the DG loop of adenovirus type 5 fiber knob protein abolish high-affinity binding to its cellular receptor CAR

Abstract

The amino acid residues in adenovirus type 5 (Ad5) fiber that interact with its cellular receptor, the coxsackie B virus and Ad receptor (CAR), have not been defined. To investigate this, multiple mutations were constructed in the region between residues 479 and 497 in Ad5 fiber (beta-strands E and F and the adjacent region of the DG loop). The effects of these mutations on binding to CAR were determined by use of cell-binding competition experiments, surface plasmon resonance, and direct binding studies. The mutation effects on the overall folding and secondary structure of the protein were assessed by circular dichroism (CD) spectroscopy. Deletions of two consecutive amino acids between residues 485 and 493 abolished high-affinity binding to CAR; the CD spectra indicated that although there was no disruption of the overall folding and secondary structure of the protein, local conformational changes did occur. Moreover, single site mutations in this region of residues with exposed, surface-accessible side chains, such as Thr492, Asn493, and Val495, had no effect on receptor binding, which demonstrates that these residues are not in contact with CAR themselves. This implies the involvement of residues in neighboring loop regions. Replacement of the segment containing the two very short beta-strands E and F and the turn between them (residues 479 to 486) with the corresponding sequence from Ad3 (betaEFAd3-->5 mutation) resulted in the loss of receptor binding. The identical CD spectra for betaEFAd3-->5 and wild-type proteins suggest that these substitutions caused no conformational rearrangement and that the loss of binding may thus be due to the substitution of one or more critical contact residues. These findings have implications for our understanding of the interaction of Ad5 fiber with CAR and for the construction of targeted recombinant Ad5 vectors for gene therapy purposes.

FIG. 1

(A) Schematic representation of the trimeric structure of the fiber knob protein, viewed along the threefold axis from the side connected to the fiber shaft. Sheets are indicated by arrows, and the segment in solid black in each protein subunit corresponds to the part of the DG loop (residues 479 to 497) that was mutagenized in these experiments. This region is shown in detail in Fig. 1B. The coordinates were obtained from the crystal structure of Ad5 fiber knob (40). (B) Representation of the conformation adopted in the native structure by the peptide sequence Asn479 to Phe497, showing the short β-strands E and F, and the side chains of residues discussed in the text. Thr492, Asn493, and Val495 residues are all exposed, in contrast to Tyr491, whose bulky aromatic side chain is almost totally buried. For example, the surface accessibility of the Tyr491 side chain was calculated to be 10.2% of the maximum possible compared to the surface accessibility of the adjacent Thr492, which was calculated to be 99.7% of the maximum possible (18a).

FIG. 2

Reduction in luciferase activity in CHO-CAR cells in the presence of maximal amounts of wild-type and mutant Ad5 fiber knobs. Cells were infected at constant MOIs of Ad5Luc3 (MOI = 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, and the cell monolayers were covered with prewarmed medium, transferred to 37°C, and further incubated at that temperature for 18 h; they were then processed for the luciferase assay. The luciferase activity, expressed in RLUs, was assayed in cell lysates by using luciferase substrate solution. Results were expressed as percentages of the control cells (i.e., no recombinant fiber = 100%). The data presented are means and standard errors of the means (n = 3) of three representative experiments.

FIG. 3

Inhibition of luciferase activity in CHO-CAR cells infected with Ad5Luc3 (MOI = 10) in the presence of increasing concentrations (0 to 50 μg/ml) of wild-type Ad5 fiber knob and of mutant fibers Tyr491Gly, Thr492Val, and βEFAd3→5. Luciferase activity was assayed as described in Fig. 2. The IC₅₀ for wild-type and mutant fibers was obtained from individual curves by calculating the amount of each protein required to achieve 50% of maximal inhibition of luciferase activity.

FIG. 4

SPR analysis of the biomolecular interactions between the soluble extracellular domains of CAR (sCAR) and wild-type and mutant fiber knobs. sCAR was immobilized on the CM5 chip. The signal correlates with the degree of surface binding of the fiber knob protein to the immobilized sCAR and was expressed in RU. Sensograms were used to obtain a quantitative indication of biomolecular interactions in RUs.

FIG. 5

Comparison of the CD spectrum of wild-type Ad5 fiber knob with those of mutant fibers (a) Thr492Val (a), Tyr491Gly (b), βEFAd3→5 (c), and dl491-492 (d). 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 a constant temperature.