Localization of the N-terminus of minor coat protein IIIa in the adenovirus capsid |
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Authors: | San Martín Carmen Glasgow Joel N Borovjagin Anton Beatty Matthew S Kashentseva Elena A Curiel David T Marabini Roberto Dmitriev Igor P |
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Institution: | 1 Department of Macromolecular Structure, Centro Nacional de Biotecnología (CNB-CSIC), Darwin 3, 28049 Madrid, Spain 2 Division of Human Gene Therapy, Departments of Medicine, Obstetrics and Gynecology, Pathology and Surgery, University of Alabama at Birmingham, Birmingham, AL 35294, USA 3 Division of Cardiovascular Disease, University of Alabama at Birmingham, Birmingham, AL 35294, USA 4 The Gene Therapy Center, University of Alabama at Birmingham, Birmingham, AL 35294, USA 5 Escuela Politécnica Superior, Universidad Autónoma de Madrid, Francisco Tomás y Valiente 11, 28049 Madrid, Spain |
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Abstract: | Minor coat protein IIIa is conserved in all adenoviruses (Ads) and is required for correct viral assembly, but its precise function in capsid organization is unknown. The latest Ad capsid model proposes that IIIa is located underneath the vertex region. To obtain experimental evidence on the location of IIIa and to further define its role, we engineered the IIIa gene to encode heterologous N-terminal peptide extensions. Recombinant Ad variants with IIIa encoding six-histidine (6His) tag, 6His, and FLAG peptides, or with 6His linked to FLAG with a (Gly4Ser)3 linker were rescued and analyzed for virus yield, capsid incorporation of heterologous peptides, and capsid stability. Longer extensions could not be rescued. Western blot analysis confirmed that the modified IIIa proteins were expressed in infected cells and incorporated into virions. In the Ad encoding the 6His-linker-FLAG-IIIa gene, the 6His tag was present in light particles, but not in mature virions. Immunoelectron microscopy of this virus showed that the FLAG epitope is not accessible to antibodies on the viral particles. Three-dimensional electron microscopy and difference mapping located the IIIa N-terminal extension beneath the vertex complex, wedged at the interface between the penton base and peripentonal hexons, therefore supporting the latest proposed model. The position of the IIIa N-terminus and its low tolerance for modification provide new clues for understanding the role of this minor coat protein in Ad capsid assembly and disassembly. |
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Keywords: | Ad adenovirus 6His six-histidine Ad5 human adenovirus type 5 3DEM three-dimensional electron microscopy eGFP enhanced green fluorescent protein ORF open reading frame mAb monoclonal antibodies immuno-EM immunoelectron microscopy 3D three-dimensional cryoEM cryoelectron microscopy CTF contrast transfer function SPR single particle reconstruction TBS Tris-buffered saline BSA bovine serum albumin |
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