134. Structural Requirements of Adenovirus Targeting Via by Modification of Minor Capsid Protein IX

Abstract

In mastadenoviruses, the hexon-associated protein IX is a 14.3kDa protein that is strongly conserved between the different members of the genus. It associates with nine hexon capsomers to form group-of-nine hexons (GON). These make up the central region of each of the facets of the icosahedron. Protein IX is believed to function as a capsid 'cement'. So far, its structure in not known. Although viruses lacking pIX do not form GONs and are less heat-stable than wild-type viruses, they can be propagated with the same kinetics and yields as the wild-type viruses. Deletion mutants lacking (parts of) the conserved N-terminal domain are not incorporated into the virion, implicating this domain in capsid formation. In contrast, pIX molecules with mutations in other conserved domains (i.e. the central alanine stretch, and the putative leucine zipper domain) are efficiently incorporated into virions. The carboxyl terminal domain contains a putative leucine-zipper domain that may be responsible for trimerization via the formation of a coiled-coil structure. Mutants in this region are efficiently incorporated into the capsid, suggesting that trimerization is not essential for capsid formation. To get more insight into the structure/function relationship we studied the incorporation of heterologous pIX molecules into the HAdV-5 capsid. Our data show that the pIX proteins of HAdV-12 (species A) and HAdV-7 (species B) can be efficiently incorporated into the capsid of pIX-deleted HAdV-5 (species C). In addition, further deletion mutagenesis confirmed that the N-terminus of the HAdV-5 pIX is required and sufficient for incorporation into the capsid. These data further our knowledge on pIX and facilitated the development of pIX as an anchor for the incorporation of heterologous peptides into the capsid. In this way receptor-binding ligands were incorporated in the capsid. Inclusion of alphahelical spacers of up to 7.5 nm between the pIX anchor and the ligand resulted in more efficient binding of the virions to antibodies and cells. These data conform that modification of pIX can be used to incorporate heterologous peptides into the adenoviral capsid, and support the feasibility of this approach to amend the tissue tropism of the adenovirus vectors.