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 Å 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.

Adenovirus targeting to HLA-A1/MAGE-A1-positive tumor cells by fusing a single-chain T-cell receptor with minor capsid protein IX

“…During purification, particle-associated pIX molecules were separated from the nonassociated Adenovirus targeting to cancer-testis antigens J de Vrij et al pIX molecules, as nonassociated variants do not copurify with the virus particles in the CsCl gradient. 17,33 A schematic representation of the pIX_TCR A1M1 fusion protein, with its exposed single-chain TCR A1M1 positioned above the hexon capsomers, is depicted in Figure 3a.…”

“…These approaches have the potential to incorporate an increased number of complex ligands per virion. To date, the capsid proteins hexon, 10,11 penton base, 12 minor capsid protein IX (pIX) [13][14][15][16][17][18] and protein pIIIa 19 have been explored as platforms for the incorporation of heterologous peptides (reviewed by Vellinga et al 20 ).…”

“…17,18 Protein IX is a small (14.3 kDa) protein of unknown structure that acts as capsid cement, stabilizing the interactions between hexon trimers on each facet of the virion. 21 Twelve molecules of pIX are associated with each facet of the icosahedron, with an overall stoichiometry of 240 pIX monomers per virion.…”

“…To ensure enhanced protrusion of the scTCR at the virus surface, a 75-Angstrom a-helical spacer was included between pIX and the TCR. 17 We produced virus particles that were efficiently loaded with the pIX_TCR A1M1 fusion protein. The transduction efficiency of the HLA-A1/ MAGE-A1-positive melanoma cell lines MZ2-MEL3.0 and MZ2-MEL43 was strongly increased upon incorporation of the fusion protein.…”

Transductional targeting of adenovirus vectors for gene therapy

“…In a similar approach, Vellinga et al 130 fused an integrin-binding RGD peptide to pIX, and used a-helical spacers (up to 7.5 nm in length and 113 amino acids) to extend the RGD motif away from the virion surface. Increased gene transfer to CAR-deficient endothelioma cells was observed with increased spacer length, giving support to the notion that the pIX C-terminus may reside in a cavity formed by surrounding hexons.…”