The Structural Basis for the Integrity of Adenovirus Ad3 Dodecahedron

Abstract

During the viral life cycle adenoviruses produce excess capsid proteins. Human adenovirus serotype 3 (Ad3) synthesizes predominantly an excess of free pentons, the complexes of pentameric penton base and trimeric fiber proteins, which are responsible for virus penetration. In infected cells Ad3 pentons spontaneously assemble into dodecahedral virus-like nano-particles containing twelve pentons. They also form in insect cells during expression in the baculovirus system. Similarly, in the absence of fiber protein dodecahedric particles built of 12 penton base pentamers can be produced. Both kinds of dodecahedra show remarkable efficiency of intracellular penetration and can be engineered to deliver several millions of foreign cargo molecules to a single target cell. For this reason, they are of great interest as a delivery vector. In order to successfully manipulate this potential vector for drug and/or gene delivery, an understanding of the molecular basis of vector assembly and integrity is critical. Crystallographic data in conjunction with site-directed mutagenesis and biochemical analysis provide a model for the molecular determinants of dodecamer particle assembly and the requirements for stability. The 3.8 Å crystal structure of Ad3 penton base dodecamer (Dd) shows that the dodecahedric structure is stabilized by strand-swapping between neighboring penton base molecules. Such N-terminal strand-swapping does not occur for Dd of Ad2, a serotype which does not form Dd under physiological conditions. This unique stabilization of the Ad3 dodecamer is controlled by residues 59–61 located at the site of strand switching, the residues involved in putative salt bridges between pentamers and by the disordered N-terminus (residues 1–47), as confirmed by site directed mutagenesis and biochemical analysis of mutant and wild type protein. We also provide evidence that the distal N-terminal residues are externally exposed and available for attaching cargo.

Penton-Dodecahedral Particles Trigger Opening of Intercellular Junctions and Facilitate Viral Spread during Adenovirus Serotype 3 Infection of Epithelial Cells

“…Importantly, PtDd signals were detectable for Ad14 but not for Ad5. The latter is in agreement with earlier studies demonstrating that Ad2 and Ad5 are unable to form stable pentondodecahedra [9].…”

“…Furthermore, penton base mutants 100-D→R or 425-R→E drastically reduced the yield and stability of penton base dodecamers [9]. We therefore attempted to generate Ad3 penton base mutants that contained the SELS→SDVA mutation, the 100-D→R mutation, and the 425-R→E mutation individually or in combination ( Figures 1A–C ).…”

“…It is thought that Ad2 pentons can form salt bridges but the penton N-termini do not rearrange as is the case for Ad3. Ad2 penton dodecamerization is only possible under artificial conditions, such as in the presence of dioxane and ammonium sulphate [9]. This raises the question why Ad3 pentons form dodecamers.…”

“…Western blot analysis did not indicate differences in post-translational modification of PtDd produced from baculovirus vectors in insect cells and PtDd produced from Ad3 in infected HeLa cells ( Figure S1 ). The crystal structure of recombinant penton base dodecahedra has recently been delineated at 3.8 Å resolution, which allowed for the elucidation of the mechanisms of Ad3 PtDd formation [9]. PtDd self-assembly is initiated through relative weak salt bridges involving residues D100 and R425.…”

Development of Adenovirus Capsid Proteins for Targeted Therapeutic Delivery

“…The Ad3 penton base noncovalently self-assembles into structurally stable dodecahedral particles, which comprises 12 pentamers (or 60 penton base monomers) when expressed as a recombinant protein in insect cells [48,49]. These particles, called Dds, can mimic viral cell entry by binding to integrins as well as HS-GAG [50,51], undergoing receptor-mediated endocytosis, and trafficking toward the nuclear periphery where Dds accumulate inside the cell.…”