Spacers increase the accessibility of peptide ligands linked to the carboxyl terminus of adenovirus minor capsid protein IX

Abstract

The efficiency and specificity of gene transfer with human adenovirus (hAd)-derived gene transfer vectors would be improved if the native viral tropism could be modified. Here, we demonstrate that the minor capsid protein IX (pIX), which is present in 240 copies in the Ad capsid, can be exploited as an anchor for heterologous polypeptides. Protein IX-deleted hAd5 vectors were propagated in hAd5 helper cells expressing pIX variants, with heterologous carboxyl-terminal extensions of up to 113 amino acids in length. The extensions evaluated consist of alpha-helical spacers up to 75 A in length and to which peptide ligands were fused. The pIX variants were efficiently incorporated into the capsids of Ad particles. On intact particles, the MYC-tagged-pIX molecules were readily accessible to anti-MYC antibodies, as demonstrated by electron microscopic analyses of immunogold-labeled virus particles. The labeling efficiency improved with increasing spacer length, suggesting that the spacers lift and expose the ligand at the capsid surface. Furthermore, we found that the addition of an integrin-binding RGD motif to the pIX markedly stimulated the transduction of coxsackievirus group B and hAd receptor-deficient endothelioma cells, demonstrating the utility of pIX modification in gene transfer. Our data demonstrate that the minor capsid protein IX can be used as an anchor for the addition of polypeptide ligands to Ad particles.

FIG. 1.

(A) Schematic representation of the pIX variants used in the present study. The RGD4c motif and MYC peptide, which are used as ligands, are fused directly with pIX or linked via a spacer. The values 30, 45, and 75 are the predicted lengths of the α-helical spacers in angstroms. (B) Schematic representation of the location of pIX between the hexon capsomers. Electron microscopic image reconstruction suggests that pIX resides between the hexon capsomers, hidden ca. 65 Å below the hexon tops. Introducing spacers between pIX and the ligand may improve the accessibility of ligands fused to the C terminus of pIX.

FIG. 2.

Subcellular localization of pIX variants. The subcellular localization of the various pIX proteins was tested by immunofluorescence microscopy. 911 cells grown on coverslips were transfected with the various pIX.RGD expression plasmids. At 2 days postinfection, the localization of pIX was visualized with anti-pIX and FITC-labeled goat anti-rabbit antibodies. The nuclei were stained with propidium iodide. (A) wt.pIX; (B) pIX.RGD; (C) pIX.flag.RGD (D); pIX.flag.30.RGD; (E) pIX.flag.45.RGD; (F) pIX.flag.75.RGD. All pIX variants are localized in nuclear aggregates, as is wt.pIX.

FIG. 3.

Western blot pIX incorporation assays. (A) To test the specificity of the incorporation assay, wt.pIX and pIX-ΔN expression plasmids were transfected into 911 cells. At 18 h posttransfection, the cells were infected with hAd5dl313. The infected cells were lysed 48 h postinfection. Protein extracts were prepared from aliquots of the lysates, whereas the remainder was used for virus isolation. Protein extracts of the cell lysates and of the purified viruses were analyzed by Western blotting with an anti-pIX serum. Although both pIX variants were expressed in 911 cells, only wt.pIX was incorporated into the virion. (B and C) To test the incorporation of the pIX variants, 911 cells were transfected with the various pIX expression plasmids. After overnight incubation, the cells were infected with hAd5dl313 or hAd5CMV-GFPΔpIX viruses. Protein extracts were prepared from the cell lysate and from the purified viruses and analyzed by Western blotting. The blots were probed with anti-pIX, anti-actin, or anti-hexon as described in the text. All pIX MYC constructs were expressed in equivalent amounts by the 911 cells (B), and all were incorporated in equivalent amounts into hAd5dl313 virions (C). (D and E) An identical analysis was performed for the various pIX.RGD constructs. All pIX variants were expressed in 911 cells (D) and incorporated efficiently into hAd5CMV-GFPΔpIX virions (E). As a negative control (neg), 911 cells were mock transfected and infected with hAd5dl313 and hAd5CMV-GFPΔpIX. The extra bands marked with an asterisk are proteolytic degradation products.

FIG. 4.

Immunoelectron microscopic analysis of ligand accessibility. To test the accessibility of the MYC epitope on hAd5dl313, viruses that were loaded with the various pIX variants were bound on copper grids with a carbon-coated Formvar film. The MYC epitope was detected with anti-MYC antibody, followed by rabbit anti-mouse immunoglobulin and gold-labeled Prot.A. Visualization was done by using a Philips EM 410-LS transmission electron microscope. The viruses were loaded with wt.pIX (A), pIX.MYC (B), pIX.flag.RGD.MYC (C), pIX.flag.30.MYC (D), pIX.flag.45.MYC (E), and pIX.flag.75.MYC (F), respectively.

FIG. 5.

Quantification of the number of gold particles bound to the viruses. The mean number of gold particles bound to the viruses (y axis) is plotted against the length (in amino acids) of the C-terminal extension (x axis). The labeling efficiency of each variant is greater than the pIX.MYC construct (34 amino acids) (P < 0.0001 [unpaired Student t test]).

FIG. 6.

Infection assay. To test whether the spacers increase the infection efficiency, hAd5CMV-GFPΔpIX viruses were propagated in 911 cells expressing the pIX.RGD variants. The capacity of the resulting viruses to transduce cells independent of CAR was tested by exposing CAR-negative Eoma cells to the CsCl purified viruses at 1,000 particles/cell. At 48 h postinfection, the cells were evaluated for GFP expression. (A to F) hAd5CMV-GFPΔpIX with wt.pIX (A), pIX.RGD (B), pIX.flag.RGD (C), pIX.flag.30.RGD (D), pIX.flag.45.RGD (E), or pIX.flag.75.RGD (F). To show that hAd5CMV-GFPΔpIX with wt.pIX (G) and hAd5CMV-GFPΔpIX bearing pIX.flag.75.RGD (H) have equivalent infectious particle numbers, HeLa cells were infected with the corresponding viruses.