Tài liệu Báo cáo khoa học: Structural and biochemical characterization of a human adenovirus 2/12

Structural and biochemical characterization of a human

adenovirus 2/12 penton base chimera

Chloe Zubieta1,*, Laurent Blanchoin2 and Stephen Cusack1

1 European Molecular Biology Laboratory, Grenoble Outstation, France

2 Laboratoire de Physiologie Cellulaire Vegetale, Commissariat a l’Energie Atomique, Centre National de la Recherche Scientifique,

Institut National de la Recherche Agronomique, Universite Joseph Fourier, Unite Mixte de Recherche 5168, Grenoble, France

Adenoviruses are nonenveloped double-stranded DNA

viruses found in mammalian and non-mammalian

vertebrates. Human adenoviruses are divided into six

subgroups (A–F) based on genetic organization, hema￾gluttination patterns, immuno-crossreactivity, and nuc￾leotide content. Over 50 serotypes have been identified

in humans; these cause generally mild respiratory,

enteric and ocular disease. However, in immunocom￾promised, very young, or elderly individuals, adenoviral

infections can lead to serious illness or death [1,2].

Apart from their role as a common human pathogen,

adenoviruses are one of the most studied vectors for

gene delivery due to extensive knowledge of their

biology and the ability to manipulate the adenoviral

genome [3–5].

The adenoviral T ¼ 25 icosahedral capsid consists

of three major polypeptides: the trimeric hexon, which

forms the facets of the particle, the pentameric penton

base (pb), which forms the vertices, and the trimeric

fiber protein, which extends from the penton base at

the vertex positions. Additionally, cementing proteins

such as IIIa, VI, VIII and IX help stabilize the capsid,

reinforcing the penton–hexon and hexon–hexon inter￾actions. The capsid contains 720 copies of the hexon

Keywords

adenovirus; crystal structure; fiber;

fluorescence anisotropy; penton

Correspondence

C. Zubieta, SLAC ⁄ SSRL, 2575 Sandhill

Road, Menlo Park, CA 94025, USA

Fax: +1 650 926 3292

Tel: +1 650 926 2992

E-mail: [email protected]

*Present address

Stanford Synchrotron Radiation Facility,

Menlo Park, CA, USA

(Received 6 June 2006, revised 11 July

2006, accepted 27 July 2006)

doi:10.1111/j.1742-4658.2006.05430.x

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, trigger￾ing virus internalization. In order to investigate the structural and func￾tional role of this region, we replaced the hypervariable loop of serotype 2

with the corresponding, but much shorter, loop of serotype 12 and com￾pared it to the wild type. Here, we report the 3.6 A˚ 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 lm range. Interestingly, competi￾tion assays using labeled and unlabeled minifiber demonstrated virtually

irreversible binding to the penton base, which we ascribe to a conforma￾tional change, on the basis of comparisons of all available penton base

structures.

Abbreviations

hAd, human adenovirus; MPD, 2-methyl-2,4-pentane diol; NCS, noncrystallographic symmetry averaging; pb, penton base; TMR,

tetramethylrhodamine.

4336 FEBS Journal 273 (2006) 4336–4345 ª 2006 The Authors Journal compilation ª 2006 FEBS