Structural and biochemical characterization of a human adenovirus 2/12 penton base chimera

The vertex of the adenoviral capsid is formed by the penton, a complex of two proteins, the pentameric penton base and the trimeric fiber protein. The penton contains all necessary components for viral attachment and entry into the host cell. After initial attachment via the head domain of the fiber protein, the penton base interacts with cellular integrins through an Arg-Gly-Asp (RGD) motif located in a hypervariable surface loop, triggering virus internalization. In order to investigate the structural and functional role of this region, we replaced the hypervariable loop of serotype 2 with the corresponding, but much shorter, loop of serotype 12 and compared it to the wild type. Here, we report the 3.6 Å crystal structure of a human adenovirus 2/12 penton base chimera crystallized as a dodecamer. The structure is generally similar to human adenovirus 2 penton base, with the main differences localized to the fiber protein-binding site. Fluorescence anisotropy assays using a trimeric fiber protein mimetic called the minifiber and wild-type human adenovirus 2 and chimeric penton base demonstrate that fiber protein binding is independent of the hypervariable loop, with a Kd for fiber binding estimated in the 1–2 µm range. Interestingly, competition assays using labeled and unlabeled minifiber demonstrated virtually irreversible binding to the penton base, which we ascribe to a conformational change, on the basis of comparisons of all available penton base structures

3PO, a novel nonviral gene delivery system using engineered Ad5 penton proteins

This study describes the development of 3PO, a nonviral, protein-based gene delivery vector which utilizes the highly evolved cell-binding, cell-entry and intracellular transport functions of the adenovirus serotype 5 (Ad5) capsid penton protein. A penton fusion protein containing a polylysine sequence was produced by recombinant methods and tested for gene delivery capability. As the protein itself is known to bind integrins through a conserved consensus motif, the penton inherently possesses the ability to bind and enter cells through receptor-mediated internalization. The ability to lyse the cellular endosome encapsulating internalized receptors is also attributed to the penton. The recombinant protein gains the additional function of DNA binding and transport with the appendage of a polylysine motif. This protein retains the ability to form pentamers and mediates delivery of a reporter gene to cultured cells. Interference by oligopeptides bearing the integrin binding motif suggests that delivery is mediated specifically through integrin receptor binding and internalization. The addition of protamine to penton-DNA complexes allows gene delivery in the presence of serum