ABSTRACT
The vesivirus feline calicivirus (FCV) is a positive strand RNA virus encapsidated by an icosahedral T=3 shell formed by the viral VP1 protein. Upon its expression in the insect cell - baculovirus system in the context of vaccine development, two types of virus-like particles (VLPs) were formed, a majority built of 60 subunits (T=1) and a minority probably built of 180 subunits (T=3). The structure of the small particles was determined by x-ray crystallography at 0.8 nm resolution helped by cryo-electron microscopy in order to understand their formation. Cubic crystals belonged to space group P213. Their self-rotation function showed the presence of an octahedral pseudo-symmetry similar to the one described previously by Agerbandje and co-workers for human parvovirus VLPs. The crystal structure could be solved starting from the published VP1 structure in the context of the T=3 viral capsid. In contrast to viral capsids, where the capsomers are interlocked by the exchange of the N-terminal arm (NTA) domain, this domain is disordered in the T=1 capsid of the VLPs. Furthermore it is prone to proteolytic cleavage. The relative orientation of P (protrusion) and S (shell) domains is alerted so as to fit VP1 to the smaller T=1 particle whereas the intermolecular contacts around 2-fold, 3-fold and 5-fold axes are conserved. By consequence the surface of the VLP is very similar compared to the viral capsid and suggests a similar antigenicity. The knowledge of the structure of the VLPs will help to improve their stability, in respect to a use for vaccination.
Subject(s)
Calicivirus, Feline/ultrastructure , Virion/ultrastructure , Amino Acid Sequence , Animals , Calicivirus, Feline/genetics , Capsid Proteins/chemistry , Capsid Proteins/genetics , Cats , Models, Molecular , Molecular Sequence Data , Protein Conformation , Sequence AlignmentABSTRACT
Although it has been known for 50 years that adenoviruses (Ads) interact with erythrocytes ex vivo, the molecular and structural basis for this interaction, which has been serendipitously exploited for diagnostic tests, is unknown. In this study, we characterized the interaction between erythrocytes and unrelated Ad serotypes, human 5 (HAd5) and 37 (HAd37), and canine 2 (CAV-2). While these serotypes agglutinate human erythrocytes, they use different receptors, have different tropisms and/or infect different species. Using molecular, biochemical, structural and transgenic animal-based analyses, we found that the primary erythrocyte interaction domain for HAd37 is its sialic acid binding site, while CAV-2 binding depends on at least three factors: electrostatic interactions, sialic acid binding and, unexpectedly, binding to the coxsackievirus and adenovirus receptor (CAR) on human erythrocytes. We show that the presence of CAR on erythrocytes leads to prolonged in vivo blood half-life and significantly reduced liver infection when a CAR-tropic Ad is injected intravenously. This study provides i) a molecular and structural rationale for Ad-erythrocyte interactions, ii) a basis to improve vector-mediated gene transfer and iii) a mechanism that may explain the biodistribution and pathogenic inconsistencies found between human and animal models.
Subject(s)
Adenoviridae/pathogenicity , Cell Adhesion Molecules/metabolism , Erythrocytes/pathology , N-Acetylneuraminic Acid/metabolism , ATPases Associated with Diverse Cellular Activities , Animals , Binding Sites , Dogs , Erythrocytes/virology , Hemagglutination , Humans , MetalloendopeptidasesABSTRACT
Adenovirus fibers from most serotypes bind the D1 domain of coxsackie and adenovirus receptor (CAR), although the binding residues are not strictly conserved. To understand this further, we determined the crystal structures of canine adenovirus serotype 2 (CAV-2) and the human adenovirus serotype 37 (HAd37) in complex with human CAR D1 at 2.3 and 1.5A resolution, respectively. Structure comparison with the HAd12 fiber head-CAR D1 complex showed that the overall topology of the interaction is conserved but that the interfaces differ in number and identity of interacting residues, shape complementarity, and degree of conformational adaptation. Using surface plasmon resonance, we characterized the binding affinity to CAR D1 of wild type and mutant CAV-2 and HAd37 fiber heads. We found that CAV-2 has the highest affinity but fewest direct interactions, with the reverse being true for HAd37. Moreover, we found that conserved interactions can have a minor contribution, whereas serotype-specific interactions can be essential. These results are discussed in the light of virus evolution and design of adenovirus vectors for gene transfer.
Subject(s)
Adenoviridae/chemistry , Adenoviridae/metabolism , Receptors, Virus/chemistry , Receptors, Virus/metabolism , Adenoviridae/classification , Adenoviridae/genetics , Amino Acid Sequence , Animals , Conserved Sequence , Coxsackie and Adenovirus Receptor-Like Membrane Protein , Crystallography, X-Ray , Dogs , Humans , Models, Molecular , Molecular Sequence Data , Mutation/genetics , Protein Binding , Protein Structure, Quaternary , Protein Structure, Tertiary , Sequence Alignment , Surface Plasmon Resonance , Water/chemistrySubject(s)
Adenoviruses, Canine/genetics , Gene Transfer Techniques , Genetic Vectors , Animals , HumansABSTRACT
Two DNA minor-groove binding polyamides 1 and 2 were designed and synthesized and evaluated for inhibition of FIV-34TF10 replication. Both 1 and 2 decreased the replication of FIV-34TF10 by 75% by acting at the level of the virus but outside of the LTR or env region.
