Structural and functional characterisation of animal adenovirus capsid proteins

Abstract

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 24-10-2014Adenoviruses are disease-causing agents, but are also explored as a model system to decipher mechanisms of molecular biology and used as vectors for gene and cancer therapy trials. Vectors wholly or partially based on animal adenoviruses may be interesting in this respect, both for avoiding pre-existing immunity in humans and the possibility of having novel cell-targeting properties. The capsid of the icosahedral, double-stranded DNA-containing adenovirus maintains the integrity of virus particle, but also has proteins involved in virus entry. The fibre is one such protein and is responsible for primary virus-host interaction. Hence, the homo-trimeric fibre protein is an important factor in adenoviral biology and to successfully exploit these viruses for medical applications. In this thesis work, X-ray crystallographic structures of four animal adenovirus fibre heads have been determined, three of which belong to recently established genera of Adenoviridae, namely Siadenovirus and Atadenovirus. Despite being members of distinct genera and targeting various organisms, the structures revealed overall conserved topology of the central beta-sandwich in their head domains. However, other features of these fibre heads, such as their size, shape and the length of connecting loops show more diversity, which may, in turn, have an impact on their tropism. Structures determined from two atadenoviruses have established their fibre heads as the smallest among all known adenovirus fibre head structures. Both the snake adenovirus 1 fibre head and the lizard adenovirus 2 short fibre heads are compact, with relatively short loops connecting their beta-strands. The presence of a prominent alpha-helix in their structures is also interesting. Turkey adenovirus-3, which exists in avirulent and virulent forms, has a fibre head more similar to reovirus fibre heads than to those of other adenoviruses. The presence of a betahairpin insertion is unique to the structure. Moreover, the striking similarity of turkey adenovirus-3 fibre head with reovirus attachment proteins has strengthened the theory of an evolutionary link between adenoviruses and reoviruses. Sialic acid-containing carbohydrates were identified as in vitro ligands by NMR spectroscopy and calorimetry and co-crystal structures were successfully determined. The structure of ligand-bound turkey adenovirus 3 fibre head revealed the sialic acid binding site located on the side of the trimer. Murine adenovirus 2, like most mammalian adenoviruses and all known human adenoviruses, belongs to the Mastadenovirus genus and could potentially be developed into a model system to study adenovirus pathogenicity. I have determined the high-resolution crystal XVI structure of the fibre head domain of murine adenovirus 2, along with the two directly preceding shaft domain repeats, which shows an overall resemblance to other adenovirus fibre heads of Mastadenovirus genus. Structural comparison with other fibre head structures, however, clearly shows variations that may have functional implications. Nacetylglucosamine, which is common component of glycosaminoglycans such as heparan sulfate and has a wide tissue distribution, has been identified as a ligand for murine adenovirus 2 fibre head in this thesis. In summary, I present new structural and functional findings on distinct adenovirus fibre heads that will be useful in elucidating their tropism and biology. The new structures may also be useful in the design and development of animal adenovirus-based applications