873. Adenovirus Polypeptide IIIa as a Novel Locale for Incorporation of Heterologous Peptides

Abstract

Listen

Translate article

Top of pageAbstract The utility of the present generation of recombinant adenovirus (Ad) vectors for gene therapy applications could potentially be improved by restricting broad native viral tropism to selected cell types. In order to achieve cell-specific targeting of Ad vectors, we have proposed modification of viral tropism via genetic engineering of the viral coat proteins to include targeting ligands. To this end, major Ad capsid proteins fiber, hexon, penton and the minor capsid protein polypeptide IX (pIX) have been exploited as platforms for the presentation of ligand peptides. While each of these capsid locales may be of utility, additional capsid locales for the presentation of heterologous peptides should be investigated. The adenovirus capsid protein pIIIa is a 570 amino acid protein present in the virion as a 63.5 kDa monomer. Structural studies indicate that sixty copies of pIIIa are present in the Ad5 virion, with two pIIIa monomers positioned along each of the thirty icosahedral edges. Further, cryo-electron microscopy studies indicate that the bulk of pIIIa is localized to the outer surface of the virion. Based on these considerations, we hypothesized that the minor capsid protein pIIIa could be exploited as a novel locale for ligand incorporation. In this study, we engineered recombinant Ad vector genomes encoding pIIIa modified to incorporate a 6-Histidine tag or FLAG octapeptide/6-Histidine at the N-terminus. Both pIIIa Ad variants were rescued, indicating that the presence of these non-native pIIIa proteins does not significantly disturb viral replication and/or assembly. Further, by using the relevant specific antibodies, we have confirmed that FLAG- and 6His-containing pIIIa chimeric capsomers are incorporated into virions, and are accessible for binding. These findings suggest that pIIIa may possess properties consistent with its employment in Ad capsid modification strategies and may be a promising locale for incorporation of targeting moieties.