ABSTRACT
The virulent form of turkey adenovirus 3 (TAdV-3), also known as turkey hemorrhagic enteritis virus (THEV), is an economically important poultry pathogen, while the avirulent form is used as a vaccine. TAdV-3 belongs to the genus Siadenovirus. The carboxy-terminal region of its fibre does not have significant sequence similarity to any other adenovirus fibre heads of known structure. Two amino acid sequence differences between virulent and avirulent TAdV-3 map on the fibre head: where virulent TAdV-3 contains Ile354 and Thr376, avirulent TAdV-3 contains Met354 and Met376. We determined the crystal structures of the trimeric virulent and avirulent TAdV-3 fibre head domains at 2.2 Å resolution. Each monomer contains a beta-sandwich, which, surprisingly, resembles reovirus fibre head more than other adenovirus fibres, although the ABCJ-GHID topology is conserved in all. A beta-hairpin insertion in the C-strand of each trimer subunit embraces its neighbouring monomer. The avirulent and virulent TAdV-3 fibre heads are identical apart from the exact orientation of the beta-hairpin insertion. In vitro, sialyllactose was identified as a ligand by glycan microarray analysis, nuclear magnetic resonance spectroscopy, and crystallography. Its dissociation constant was measured to be in the mM range by isothermal titration calorimetry. The ligand binds to the side of the fibre head, involving amino acids Glu392, Thr419, Val420, Lys421, Asn422, and Gly423 binding to the sialic acid group. It binds slightly more strongly to the avirulent form. We propose that, in vivo, the TAdV-3 fibre may bind a sialic acid-containing cell surface component.
Subject(s)
Lactose/analogs & derivatives , Protein Structure, Tertiary , Siadenovirus/metabolism , Sialic Acids/chemistry , Viral Proteins/chemistry , Amino Acid Sequence , Binding Sites/genetics , Calorimetry/methods , Carbohydrate Conformation , Carbohydrate Sequence , Crystallography, X-Ray , Lactose/chemistry , Lactose/metabolism , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Sequence Data , Mutation , Polysaccharides/chemistry , Polysaccharides/metabolism , Protein Binding , Siadenovirus/genetics , Siadenovirus/pathogenicity , Sialic Acids/metabolism , Thermodynamics , Viral Proteins/genetics , Viral Proteins/metabolism , Virulence/geneticsABSTRACT
Turkey adenovirus 3 (TAdV-3) causes high mortality and significant economic losses to the turkey industry. However, little is known about the molecular determinants required for viral replication and pathogenesis. Moreover, TAdV-3 does not grow well in cell culture, thus detailed structural studies of the infectious particle is particularly challenging. To develop a better understanding of virus-host interactions, we performed a comprehensive proteomic analysis of proteinase K treated purified TAdV-3 virions isolated from spleens of infected turkeys, by utilizing one-dimensional liquid chromatography mass spectrometry. Our analysis resulted in the identification of 13 viral proteins associated with TAdV-3 virions including a novel uncharacterized TaV3gp04 protein. Further, we detected 18 host proteins in purified virions, many of which are involved in cell-to cell spread, cytoskeleton dynamics and virus replication. Notably, seven of these host proteins have not yet been reported to be present in any other purified virus. In addition, five of these proteins are known antiviral host restriction factors. The availability of reagents allowed us to identify two cellular proteins (collagen alpha-1 (VI) chain and haemoglobin) in the purified TAdV-3 preparations. These results represent the first comprehensive proteomic profile of TAdV-3 and may provide information for illustrating TAdV-3 replication and pathogenesis.
Subject(s)
Adenoviridae Infections/veterinary , Poultry Diseases/genetics , Proteome/genetics , Siadenovirus/genetics , Turkeys , Viral Proteins/genetics , Adenoviridae Infections/genetics , Adenoviridae Infections/virology , Animals , Blotting, Western/veterinary , Chromatography, Liquid/veterinary , Poultry Diseases/virology , Proteome/metabolism , Proteomics , Siadenovirus/metabolism , Tandem Mass Spectrometry/veterinary , Viral Proteins/metabolism , Viral Structural Proteins/genetics , Viral Structural Proteins/metabolismABSTRACT
Turkey adenovirus 3 belongs to the genus Siadenovirus. Its predicted fibre protein consists of an N-terminal virus-attachment domain, a central shaft domain and a head domain at the C-terminus. The head domain has little sequence identity to known adenovirus fibre head structures. Crystals of the fibre head domain consisting of amino acids 304-454 with an N-terminal purification tag were produced. Crystals of native and selenomethionine-derivatized protein belonged to space group I23 (unit-cell parameter 99â Å). They diffracted synchrotron radiation to 2.0 and 2.14â Å resolution, respectively, and are expected to contain one monomer in the asymmetric unit.
