Engineering of adenovirus vectors containing heterologous peptide sequences in the C terminus of capsid protein IX

Abstract

The utility of the present generation of adenovirus (Ad) vectors for gene therapy applications could be improved by restricting native viral tropism to selected cell types. In order to achieve modification of Ad tropism, we proposed to exploit a minor component of viral capsid, protein IX (pIX), for genetic incorporation of targeting ligands. Based on the proposed structure of pIX, we hypothesized that its C terminus could be used as a site for incorporation of heterologous peptide sequences. We engineered recombinant Ad vectors containing modified pIX carrying a carboxy-terminal Flag epitope along with a heparan sulfate binding motif consisting of either eight consecutive lysines or a polylysine sequence. Using an anti-Flag antibody, we have shown that modified pIXs are incorporated into virions and display Flag-containing C-terminal sequences on the capsid surface. In addition, both lysine octapeptide and polylysine ligands were accessible for binding to heparin-coated beads. In contrast to virus bearing lysine octapeptide, Ad vector displaying a polylysine was capable of recognizing cellular heparan sulfate receptors. We have demonstrated that incorporation of a polylysine motif into the pIX ectodomain results in a significant augmentation of Ad fiber knob-independent infection of CAR-deficient cell types. Our data suggest that the pIX ectodomain can serve as an alternative to the fiber knob, penton base, and hexon proteins for incorporation of targeting ligands for the purpose of Ad tropism modification.

FIG. 1.

Schema of Ad pIX modifications. To generate the AdLucIXflag vector, the Flag octapeptide (DYKDDDDK) coding sequence was introduced into the DraI site of the wild-type pIX gene. Introduction of the NheI site following the Flag coding sequence in AdLucIXpK caused a pIX stop codon deletion and translation through the poly(A) signal, resulting in incorporation of a poly(Lys) tail into the carboxy terminus of pIX. Cloning of the DNA sequence encoding eight consecutive lysines resulted in restoration of the stop codon and incorporation of a Lys₈ peptide into the C terminus of pIX. Modified DNA and protein sequences of pIX are designated by italics. Translated amino acid sequences are presented in capital letters. Restriction sites are underlined, and the poly(A) signal is indicated in boldface. Incorporated Flag, poly(Lys), and Lys₈ peptide sequences are underlined.

FIG. 2.

Western blot analysis of pIX-modified Ad vectors. Samples of CsCl-purified AdLuc (pIXwt), AdLucIXflag (pIXflag), AdLucIX8K (pIX8K), and AdLucIXpK (pIXpK) were boiled in Laemmli loading sample buffer and separated by 4 to 20% gradient SDS-PAGE. Electrophoretically resolved viral proteins were transferred to polyvinylidene difluoride membranes, probed with anti-Flag M2 MAb, and detected with secondary alkaline phosphatase-conjugated goat anti-mouse antibodies. The numbers on the left indicate the molecular masses of marker proteins (lane MW) in kilodaltons.

FIG. 3.

Presentation of modified pIX C terminus on Ad capsid. Dilutions of CsCl-purified AdLuc, AdLucIXflag, AdLucIX8K, and AdLucIXpK were immobilized on an ELISA plate and probed with anti-Flag M2 MAb. Anti-Flag MAb bound to the VPs was detected with alkaline phosphatase-conjugated secondary antibody. Each point represents the cumulative mean ± SD of triplicate determinations. Some error bars depicting SDs are smaller than the symbols.

FIG. 4.

Accessibility of pIX-incorporated ligands for binding. (A) Binding of pIX-modified Ad to heparin-coated beads. Suspensions of heparin-coated ceramic beads were incubated with 10¹⁰ VP (2 × 10⁵ to 8 × 10⁵ cpm) of ³H-labeled AdLuc (pIX wt), AdLucIXflag (pIXflag), AdLucIX8K (pIX8K), and AdLucIXpK (pIXpK). The beads were washed by centrifugation to remove unbound virions, and then bound radioactivity was calculated as a percentage of input radioactivity for each Ad sample. Each bar represents the cumulative mean ± SD of triplicate determinations. (B) Binding of pIX-modified Ad to AU-565 cells. Aliquots of AU-565 cells (10⁶) were preincubated separately with Ad5 knob protein, heparin, or PBS (Virus alone). Samples of radiolabeled AdLuc (pIX wt), AdLucIXflag (pIXflag), AdLucIX8K (pIX8K), and AdLucIXpK (pIXpK) containing 10¹⁰ VP (2 × 10⁵ to 8 × 10⁵ cpm) were added to the cells and incubated for 1 h at 4°C. Bound radioactivity was determined after washing the cell samples by centrifugation, and the VP/cell ratio was calculated for each Ad vector. Each bar represents the cumulative mean ± SD of triplicate determinations.

FIG. 5.

Infection properties of pIX-modified Ad vectors. (A) Monolayers of GI-101A, AU-565, and HUVEC CAR-deficient cell lines and CAR-positive 293 cells were infected with pIX-modified AdLucIXflag, AdLucIX8K, AdLucIXpK, or control AdLuc vector in the presence or absence of Ad5 knob protein. The levels of luciferase activity were determined in lysates of infected cells 20 h postinfection. The results are presented as the percentages of luciferase activity detected in the cells infected in the presence of Ad5 knob protein calculated with respect to luciferase activity determined in the cells infected in the absence of knob (100%). Each bar represents the cumulative mean ± SD of triplicate determinations. (B) Cell monolayers were infected with AdLucIXflag, AdLucIX8K, AdLucIXpK, or control AdLuc vectors in the presence or absence of free heparin. The luciferase from cell lysate activities was analyzed 20 h postinfection. The results are presented as the percentages of luciferase activity detected in the cells infected in the presence of free heparin calculated with respect to luciferase activity determined in the absence of heparin (100%). Each bar represents the cumulative mean ± SD of triplicate determinations.

FIG. 6.

Thermostabilities of pIX-modified Ad vectors. Aliquots of AdLucIXflag, AdLucIX8K, AdLucIXpK, or AdLuc vector were incubated at 45°C for different time intervals and then used to infect 293 cells. The results are presented as the percentages of luciferase activity detected in the cells infected with a heat-treated viral sample with respect to luciferase activity determined in the cells infected with untreated virus (100%). Each bar represents the cumulative mean ± SD of triplicate determinations. Some error bars depicting SDs are smaller than the symbols.