Characterization of an adenovirus vector containing a heterologous peptide epitope in the HI loop of the fiber knob

Abstract

The utility of the present generation of recombinant adenovirus vectors for gene therapy applications could potentially be improved by designing targeted vectors capable of gene delivery to selected cell types in vivo. In order to achieve such targeting, we are investigating the possibilities of incorporation of ligands in the adenovirus fiber protein, which mediates primary binding of adenovirus to its cell surface receptor. Based on the proposed structure of the cell-binding domain of the fiber, we hypothesized that the HI loop of the fiber knob can be utilized as a convenient locale for incorporation of heterologous ligands. In this study, we utilized recombinant fiber proteins expressed in baculovirus-infected insect cells to demonstrate that the incorporation of the FLAG octapeptide into the HI loop does not ablate fiber trimerization and does not disturb formation of the cell-binding site localized in the knob. We then generated a recombinant adenovirus containing this modified fiber and showed that the short peptide sequence engineered in the knob is compatible with the biological functions of the fiber. In addition, by using a ligand-specific antibody, we have shown that the peptide incorporated into the knob remains available for binding in the context of mature virions containing modified fibers. These findings suggest that heterologous ligands can be incorporated into the HI loop of the fiber knob and that this locale possesses properties consistent with its employment in adenovirus retargeting strategies.

FIG. 1

3D model of the Ad5 fiber knob. The trimer forms a propeller-like structure when it is viewed along the threefold-symmetry axis from above. The HI loop, exposed outside the knob, connects the β-strands H and I, which are involved in the formation of the cell-binding site. (Reproduced from reference by permission.)

FIG. 2

Modifications of the HI loop of the fiber knob. PCR-based mutagenesis was employed to delete a portion of the fiber gene encoding the hypervariable region of the HI loop. A unique EcoRV restriction site was incorporated in place of the deletion to allow the cloning of segments of DNA coding for heterologous protein sequences. In the fiber-FLAG protein, deleted amino acids of the HI loop were restored, and FLAG octapeptide was incorporated between threonine-546 and proline-547. The site of deletion is indicated by a filled triangle.

FIG. 3

Analysis of recombinant fiber proteins by polyacrylamide gel electrophoresis. Fiber proteins expressed in insect cells were analyzed by gel electrophoresis to confirm their trimeric configurations. To dissociate trimers to monomers, the proteins were denatured by boiling them in the sample buffer prior to loading them on a 7.5% polyacrylamide gel. The bands were visualized by Coomassie blue staining. (A) Six-histidine-tagged fiber proteins purified on an Ni-NTA–Sepharose column. Lane 1, wild-type fiber, boiled; lane 2, wild-type fiber, unboiled; lane 3, fiber-FLAG, boiled; lane 4, fiber-FLAG, unboiled; lane M, broad-range protein standards. (B) Fiber-FLAG protein purified by immunoprecipitation with anti-FLAG M2-affinity gel. Lane 1, unboiled protein; lane 2, boiled protein; lane M, broad-range protein standards. The numbers on the left indicate molecular masses of marker proteins in kilodaltons.

FIG. 4

Inhibition of adenovirus infectivity by recombinant fiber proteins. HeLa cells were preincubated with either the wild-type (wt) fiber or fiber-FLAG at the indicated concentrations for 10 min at room temperature. AdCMVLuc was then added at a multiplicity of infection of 10, and incubation was continued for another 30 min at room temperature. The unbound virus was aspirated, complete medium was added, and the cells were transferred to 37°C. After 30 h, the cells were lysed and luciferase activity was determined. Luciferase activities are given as percentages of the activity in the absence of blocking fiber protein. Each point represents the mean of four determinations obtained in one experiment.

FIG. 5

Generation of Ad5F_HIFLAG. The master plasmid, pTG3602, was modified to incorporate a unique SwaI restriction site in the fiber gene, thereby creating plasmid pVK50, suitable for fiber modifications. The genome of Ad5F_HIFLAG was generated by homologous DNA recombination in E. coli between the DNA fragment containing the fiber-FLAG gene and plasmid pVK50 linearized by SwaI digestion. To rescue the virus, the resulting plasmid, pVK300, which contains the complete adenovirus genome with a modified fiber gene, was cleaved with PacI and was then used to transfect 293 cells.

FIG. 6

Adenovirus binding assay. Aliquots of A549 cells containing 10⁵ cells per sample were incubated for 1 h at 4°C with serial dilutions of either wild-type (wt) Ad5 fiber or fiber-FLAG (see Materials and Methods). Virions of Ad5CMVLacZ (A) and Ad5F_HIFLAG (B) labeled with ¹²⁵I were added to samples, and incubation was continued for an additional hour. The cells were washed with 4 ml of PBS containing 0.1% BSA and pelleted by low-speed centrifugation. Radioactivities of samples were determined with a gamma counter. Each point represents the mean of two determinations obtained in one experiment.

FIG. 7

Accessibility of the FLAG peptide in the context of intact Ad5F_HIFLAG virions. Virions of Ad5F_HIFLAG purified on a CsCl gradient were dialyzed, immunoprecipitated with anti-FLAG M2-affinity gel as described in Materials and Methods, and eluted from the gel with free FLAG peptide. Recombinant adenovirus vector Ad5CMVLuc containing unmodified fiber was used as a negative control for binding. Aliquots of all the fractions collected throughout the purification procedure were treated with DNase I to digest traces of the cellular DNA and then treated with SDS, EDTA, and proteinase K to release adenovirus DNA from the virions. The samples obtained were analyzed on a 0.8% agarose gel, and DNA was detected by ethidium bromide staining. Lanes 1 through 3, AdCMVLuc in the supernatant containing unbound material, buffer wash, and FLAG-eluate, respectively; lanes 4 through 6, Ad5F_HIFLAG in the supernatant, buffer wash, and FLAG-eluate, respectively; lanes M, DNA molecular weight standards (the bands corresponding to marker fragments ranging from 3 to 12 kb are seen on the gel).