Roelvink, D. 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 Alizarin purpose of Ad tropism modification. Human adenovirus (Ad) includes at least 47 viral serotypes grouped into six distinct subgroups (A to F) and represents a large family of nonenveloped viruses containing a linear double-stranded DNA genome of approximately 36 kb (26). Ad is composed of multiple copies of 11 structural proteins, 7 of which (II, hexon; III, penton base; IIIa; IV, fiber; VI; VIII; and IX) form Alizarin the icosahedral capsid, while the other 4 (V, VII, , and tp) are packaged with the DNA genome in the viral particle (VP) core (28). Studies of the mechanism of Ad infection have revealed that penton base and fiber proteins are responsible for recognition of cellular receptors and therefore determine viral tropism. Ad infection is initiated by the binding of the globular knob domain of the fiber with the primary cellular receptor (19, 27). A fiber receptor for Ad of subgroups A, C, D, E, and F has been identified as the coxsackievirus group B and Ad receptor, called CAR (3, 24, 29). The major histocompatibility complex-1 2 subunit was reported as the cellular receptor for subgroup C Ad (15), in addition to CAR and a sialic acid-containing glycoprotein as a receptor moiety for Ad37 of subgroup D (2). Following binding to the fiber receptor, RGD motifs within penton base interact with v integrins and facilitate virus internalization via receptor-mediated endocytosis (13, 21, 32). Ad is widely used as a vector for both in vitro and in vivo gene delivery due to its ability to infect a variety of cell types (37). However, patterns of viral receptor expression vary between different tissues (10), predicating their susceptibilities to Ad infection. The increased knowledge of the Ad capsid structure combined with an understanding of the biology of virus interaction with cellular receptors has facilitated the development of targeted Ad vectors (6, 7). Genetic engineering of Ad capsid proteins to incorporate targeting ligands has been Alizarin employed to generate Ad vectors with novel viral tropism that can overcome the limited infectivity associated with deficiency of viral receptors (18, 31). Several heterologous peptide ligands have been successfully engineered into the HI loop (8, 23, 36) and C terminus of fiber (33, 35), the L1 loop of hexon (30), and the RGD loop of penton base (34), resulting in markedly increased efficiency of Ad infection in a variety CAR-deficient cell types. However, the structural properties of the surface-exposed loops of capsid proteins make them suitable only for incorporation of constrained heterologous sequences, and addition of ligands to the C terminus of fiber apparently has size limitations (35). The present study evaluates the utility of Ad capsid protein IX (pIX) (4, 5) for the purpose of viral tropism modification via genetic incorporation of heterologous peptides. pIX is a minor component of Ad capsid that stabilizes hexon-hexon interactions (11) and is also essential for viral DNA packaging (12). Recent studies have demonstrated that the C terminus of pIX is exposed on the outer surface of the viral capsid (1, 25), suggesting that it could be used as a novel locale for incorporation of targeting ligands. To assess the feasibility of incorporating heterologous sequences into pIX, we engineered Ad vectors encoding recombinant pIX containing either eight consecutive lysines or a polylysine sequence following a C-terminal Flag octapeptide. Here, we demonstrate that modified pIX is incorporated into mature Ad virions and displays Flag-containing carboxy-terminal extensions iNOS antibody which are accessible for binding. Incorporation of polylysine, a heparan sulfate binding motif, resulted in significant augmentation of knob-independent Ad infection of CAR-deficient cell types, illustrating compatibility of pIX ectodoman ligand additions with Ad tropism modification strategies. Our results suggest that the pIX ectodoman may be used for targeting ligand incorporation as.