The Coiled-Coil Domain of the Adenovirus Type 5 Protein IX Is Dispensable for Capsid Incorporation and Thermostability

Vellinga, Jort; Wollenberg, Diana J.M. van den; Heijdt, Stephanie Van der; Rabelink, Martijn J. W. E.; Hoeben, Rob C.

doi:10.1128/jvi.79.5.3206-3210.2005

Cited by 46 publications

(45 citation statements)

References 21 publications

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“…Second, IX has been shown to be associated with purified GONs (1,32). Third, this spatial arrangement agrees with the known stoichiometry of IX and its apparent ability to form multimers (23,34). Lastly, the mass calculated from the difference imaging of the IX trimers was in close agreement with the actual molecular mass of protein IX: 16.6 to 18.9 kDa by scanning transmission electron microscopy versus 14.3 kDa predicted by sequence analysis (12).…”

supporting

confidence: 65%

“…Therefore, the volume argument for IIIa assignment also supports the assignment of four protein IX molecules to the same location. Although protein IX has been described as a trimer, the only direct evidence of multimerization has been coimmunoprecipitation data with IX-epitope fusions (23,34), as the protein consistently runs as a monomer on nondenaturing polyacrylamide gels (data not shown). Deletion of the coiled-coil multimerization domain has no effect on the encapsidation of IX or its virion stabilization activity (34).…”

mentioning

confidence: 99%

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Cryoelectron Microscopy of Protein IX-Modified Adenoviruses Suggests a New Position for the C Terminus of Protein IX

Marsh

Campos

Baker³

et al. 2006

J Virol

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Recombinant human adenovirus is a useful gene delivery vector for clinical gene therapy. Minor capsid protein IX of adenovirus has been of recent interest since multiple studies have shown that modifications can be made to its C terminus to alter viral tropism or add molecular tags and/or reporter proteins. We examined the structure of an engineered adenovirus displaying the enhanced green fluorescent protein (EGFP) fused to the C terminus of protein IX. Cryoelectron microscopy and reconstruction localized the C-terminal EGFP fusion between the H2 hexon and the H4 hexon, positioned between adjacent facets, directly above the density previously assigned as protein IIIa. The original assignment of IIIa was based largely on indirect evidence, and the data presented herein support the reassignment of the IIIa density as protein IX.The adenoviral capsid is a complex assembly of seven polypeptides, organized into an ϳ900-Å-diameter icosahedral shell. Twelve trimers of hexon, the major capsid component, are arranged onto each of 20 interlocking triangular facets, with penton capsomeres and their protruding fibers occupying each of the 12 vertex positions (24). The icosahedral lattice of hexons is stabilized by a number of so-called "minor" capsid proteins, including IIIa, VI, VIII, and IX. With the exception of protein IX, these minor components provide vital functions and are absolutely necessary for the adenovirus (Ad) life cycle (35). The presence of protein IX is not required for the encapsidation of Ad genomes of subgenomic size (Ͻ36 kb), but virions with capsids lacking IX (⌬IX) are more thermolabile than wild-type virions (5, 6). Previous work (13) suggests that deletion of protein IX prevents the encapsidation of genomes of wild-type or larger sizes (36 to 38 kb). A more recent study indicates that the absence of IX does not prevent encapsidation but rather that the resulting ⌬IX virions are defective in infectivity assays (27).Central to each facet are the groups-of-nine hexons (GONs) that are observed after various methods of capsid disruption (6, 10). The crystal structure of the hexon trimer (25) reveals a pseudohexagonal base with three towers extending upwards, creating a triangular top approximately 113 Å tall. The maximum radius of the hexon trimer is 50 Å. The triangular top is rotated roughly 10 degrees counterclockwise with respect to the hexagonal base. The hexon trimers present within each facet are closely packed along their hexagonal bases, creating a continuous protein shell that is approximately 33 to 44 Å thick. The three towers of each hexon trimer extend roughly 69 Å above the capsid floor, thereby creating cavities between the towers of different hexon trimers. Due to the different relative angles between the bases and towers of individual hexon trimers, each GON contains four large and three small cavities between the nine different hexon trimers.Structurally, protein IX is thought to function as capsid cement, and trimers of IX are thought to reside at the bottom of the four large cavitie...

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supporting

confidence: 65%

mentioning

confidence: 99%

Cryoelectron Microscopy of Protein IX-Modified Adenoviruses Suggests a New Position for the C Terminus of Protein IX

Marsh

Campos

Baker³

et al. 2006

J Virol

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“…Protein IX is a relatively small capsid protein of 14.4 kDa, but it has gained prominence as a platform for ligand addition for the purposes of vector retargeting and fluorescence labeling (30). Recent studies have shown that only the conserved N-terminal domain of protein IX (amino acids [aa] 1 to 39) is necessary for stabilization of the Ad capsid (34,48). A cryoEM study at 9-Å resolution from our laboratory indicated that the locations identified by STEM for protein IX are likely to correspond to only the N-terminal viral interaction domains (37).…”

mentioning

confidence: 99%

Visualization of α-Helices in a 6-Ångstrom Resolution Cryoelectron Microscopy Structure of Adenovirus Allows Refinement of Capsid Protein Assignments

Saban

Silvestry

Nemerow

et al. 2006

J Virol

112

203

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The structure of adenovirus was determined to a resolution of 6 Å by cryoelectron microscopy (cryoEM) single-particle image reconstruction. Docking of the hexon and penton base crystal structures into the cryoEM density established that ␣-helices of 10 or more residues are resolved as rods. A difference map was calculated by subtracting a pseudoatomic capsid from the cryoEM reconstruction. The resulting density was analyzed in terms of observed ␣-helices and secondary structure predictions for the additional capsid proteins that currently lack atomic resolution structures (proteins IIIa, VI, VIII, and IX). Protein IIIa, which is predicted to be highly ␣-helical, is assigned to a cluster of helices observed below the penton base on the inner capsid surface. Protein VI is present in ϳ1.5 copies per hexon trimer and is predicted to have two long ␣-helices, one of which appears to lie inside the hexon cavity. Protein VIII is cleaved by the adenovirus protease into two fragments of 7.6 and 12.1 kDa, and the larger fragment is predicted to have one long ␣-helix, in agreement with the observed density for protein VIII on the inner capsid surface. Protein IX is predicted to have one long ␣-helix, which also has a strongly indicated propensity for coiled-coil formation. A region of density near the facet edge is now resolved as a four-helix bundle and is assigned to four copies of the C-terminal ␣-helix from protein IX.Adenovirus (Ad) is a common etiologic agent of respiratory, gastrointestinal, and ocular infections. Ad vectors also have substantial potential for diverse gene therapy approaches to treat cancer and other chronic diseases, as well as promise for vaccine delivery (33). The lack of an atomic resolution structure of an intact Ad virion has hindered further development of practical applications, as well as limited our understanding of Ad cell entry. The mature Ad virion has an icosahedral protein capsid composed of three major capsid proteins (hexon, penton base, and fiber) and four additional capsid proteins (proteins IIIa, VI, VIII, and IX). The capsid is ϳ926 Å in diameter, not including the fibers that vary in length from 120 to 315 Å depending on the serotype. The double-stranded DNA Ad genome is packaged inside of the capsid, together with four additional proteins (V, VII, mu, and terminal protein) and multiple copies of the Ad protease. Cryoelectron microscopy (cryoEM) structures of Ad and Ad vectors (12, 37), Ad/receptor (7), and Ad/antibody (46) complexes, combined with crystal structures for hexon (36), penton base (53), and fiber (45), have provided a wealth of structural information on Ad, but significant gaps remain in our knowledge of the virus capsid structure. In particular, the structure and locations of

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“…During purification, particle-associated pIX molecules were separated from the nonassociated Adenovirus targeting to cancer-testis antigens J de Vrij et al pIX molecules, as nonassociated variants do not copurify with the virus particles in the CsCl gradient. 17,33 A schematic representation of the pIX_TCR A1M1 fusion protein, with its exposed single-chain TCR A1M1 positioned above the hexon capsomers, is depicted in Figure 3a.…”

Section: Efficient Incorporation Of Pix_tcr A1m1 In the Virus Capsidmentioning

confidence: 99%

Adenovirus targeting to HLA-A1/MAGE-A1-positive tumor cells by fusing a single-chain T-cell receptor with minor capsid protein IX

et al. 2008

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The Coiled-Coil Domain of the Adenovirus Type 5 Protein IX Is Dispensable for Capsid Incorporation and Thermostability

Cited by 46 publications

References 21 publications

Cryoelectron Microscopy of Protein IX-Modified Adenoviruses Suggests a New Position for the C Terminus of Protein IX

Cryoelectron Microscopy of Protein IX-Modified Adenoviruses Suggests a New Position for the C Terminus of Protein IX

Visualization of α-Helices in a 6-Ångstrom Resolution Cryoelectron Microscopy Structure of Adenovirus Allows Refinement of Capsid Protein Assignments

Adenovirus targeting to HLA-A1/MAGE-A1-positive tumor cells by fusing a single-chain T-cell receptor with minor capsid protein IX

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