Three-Dimensional Structure of Canine Adenovirus Serotype 2 Capsid

Schoehn, Guy; Bakkouri, M. El; Fabry, C.M.S.; Billet, Oliver; Estrozi, Leandro F.; Le, Long P.; Curiel, David T.; Kajava, Andrey V.; Ruigrok, Rob W.H.; Kremer, Eric J.

doi:10.1128/jvi.02393-07

Cited by 62 publications

(70 citation statements)

References 58 publications

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“…5) in the OAdV penton base residues 106 to 131 but is four residues shorter, was aligned with each vertex in the overall pentagonal-shaped density of OAdV (Fig. 7B), as was also done recently in a study of CAdV-2 (27). It is immediately apparent that five ␣-helices around the central axis of the AdV penton base complex (blue arrow) do not fit into any corresponding density in OAdV (Fig.…”

Section: Vol 82 2008supporting

confidence: 61%

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Cryoelectron Microscopy Map of Atadenovirus Reveals Cross-Genus Structural Differences from Human Adenovirus

Pantelic

Lockett

Rothnagel

et al. 2008

J Virol

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A three-dimensional (3D) cryoelectron microscopy reconstruction of the prototype Atadenovirus (OAdV [an ovine adenovirus isolate]) showing information at a 10.6-Å resolution (0.5 Fourier shell correlation) was derived by single-particle analysis. This is the first 3D structure solved for any adenovirus that is not a Mastadenovirus, allowing cross-genus comparisons between structures and the assignment of genus-specific capsid proteins. Viable OAdV mutants that lacked the genus-specific LH3 and p32k proteins in purified virions were also generated. Negatively stained 3D reconstructions of these mutants were used to identify the location of protein LH3 and infer that of p32k within the capsid. The key finding was that LH3 is a critical protein that holds the outer capsid of the virus together. In its absence, the outer viral capsid is unstable. LH3 is located in the same position among the hexon subunits as its protein IX equivalent from mastadenoviruses but sits on top of the hexon trimers, forming prominent "knobs" on the virion surface that visually distinguish OAdV from other known AdVs. Electron density was also assigned to hexon and penton subunits and to proteins IIIa and VIII. There was good correspondence between OAdV density and human AdV hexon structures, which also validated the significant differences that were observed between the penton base protein structures.The Adenoviridae comprise a large family of non-enveloped, icosahedral, generally nonpathogenic viruses with a doublestranded DNA genome (ϳ26.1 to 45 kb) (9). Because of their wide host range, viruses such as human adenovirus type 5 (AdV5) have been developed as gene delivery vectors and applied in the clinic, especially for cancer, monogenic disorders, and vaccination (http://www.wiley.co.uk/genmed /clinical/). While some of these trials have reached phases II and III, there is still considerable room for improvement in vector technology. In particular, there is strong interest in developing vectors that are targeted to specific tissues for improved therapeutic effect (6, 11), and for this purpose, structural information on the virus particle and its constituent molecules is paramount.The family Adenoviridae comprises four genera (Mastadenovirus, Aviadenovirus, Siadenovirus, and Atadenovirus). Of these, the best studied are the mastadenoviruses, which include viruses from numerous mammalian species and all known human AdVs. In contrast, members of the avi-, si-, and atadenoviruses have been isolated from birds, although the latter also occur widely in mammalian and reptilian species (9, 38). The prototype Atadenovirus, an ovine isolate (serotype 7; OAdV), is the only member for which any significant biological studies have been undertaken. OAdV uses a different (unknown) receptor from AdV5 and infects, but does not replicate in, human cells (15). It has also been developed as a gene delivery vector (2) and has regulatory approval to enter a phase I clinical trial for prostate cancer in 2008.Representative genomes from all four genera have now...

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Section: Vol 82 2008supporting

confidence: 61%

“…7C) are also apparent, but the role and identity of the OAdV sequences involved remain to be determined. These appear to have no counterpart in the AdV or CAdV complexes (26,27). In addition, there is a groove between the OAdV monomers at the base of the complex which is occupied by AdV sequences in the composite image (Fig.…”

Section: Vol 82 2008mentioning

confidence: 99%

Cryoelectron Microscopy Map of Atadenovirus Reveals Cross-Genus Structural Differences from Human Adenovirus

Pantelic

Lockett

Rothnagel

et al. 2008

J Virol

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“…Significantly, the revised polypeptide direction allows the C termini of all IX molecules to be surface-exposed, in agreement with previous biochemical and immunoelectron microscopy experiments (20,21,23). This change in chain direction also allows the organization of IX molecules in HAdVs to be consistent with the arrangement of trimeric C-terminal spikes (helical bundles) seen in nonhuman adenoviruses (24,25), even though the corresponding trimeric spikes are disordered in human serotypes.…”

Section: Resultsmentioning

confidence: 58%

Structures and organization of adenovirus cement proteins provide insights into the role of capsid maturation in virus entry and infection

Reddy¹,

Nemerow

2014

Proc. Natl. Acad. Sci. U.S.A.

118

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Adenovirus cement proteins play crucial roles in virion assembly, disassembly, cell entry, and infection. Based on a refined crystal structure of the adenovirus virion at 3.8-Å resolution, we have determined the structures of all of the cement proteins (IIIa, VI, VIII, and IX) and their organization in two distinct layers. We have significantly revised the recent cryoelectron microscopy models for proteins IIIa and IX and show that both are located on the capsid exterior. Together, the cement proteins exclusively stabilize the hexon shell, thus rendering penton vertices the weakest links of the adenovirus capsid. We describe, for the first time to our knowledge, the structure of protein VI, a key membrane-lytic molecule, and unveil its associations with VIII and core protein V, which together glue peripentonal hexons beneath the vertex region and connect them to the rest of the capsid on the interior. Following virion maturation, the cleaved N-terminal propeptide of VI is observed, reaching deep into the peripentonal hexon cavity, detached from the membrane-lytic domain, so that the latter can be released. Our results thus provide the molecular basis for the requirement of maturation cleavage of protein VI. This process is essential for untethering and release of the membrane-lytic region, which is known to mediate endosome rupture and delivery of partially disassembled virions into the host cell cytoplasm.human adenovirus | cement protein scaffold | structure-function relationships H uman adenoviruses (HAdVs) are large (∼150 nm in diameter, 150-MDa) nonenveloped double-stranded DNA (dsDNA) viruses that cause respiratory, ocular, and enteric diseases (1). Although these diseases are self-limiting in immunocompetent individuals, they cause significant morbidity in AIDS, cancer, and organ transplant patients with compromised immune systems (2-4). Because of their broad cell tropism and ease of genome manipulation, replication-deficient or conditionally replicating HAdVs are also being evaluated in the clinic as potential vaccine and gene therapy vectors (5).The capsid shell of an adenovirus (Ad) comprises multiple copies of three major capsid proteins (MCPs; hexon, penton base, and fiber) and four minor/cement proteins (IIIa, VI, VIII, and IX) that are organized with pseudo-T = 25 icosahedral symmetry ( Fig. 1 A and B). In addition, six other proteins (V, VII, μ, IVa2, terminal protein, and adenovirus protease) are encapsidated along with the 36-kb dsDNA genome inside the capsid (Fig. 1A). The crystal structures of all three MCPs are known, and so is their organization in the capsid from prior X-ray crystallography (6-8) and cryoelectron microscopy (cryo-EM) analyses (9, 10). Recently, high-resolution structures of recombinant HAdV5 vectors have been determined using cryo-EM (11) and X-ray methods (12) that revealed the structures and organization of some of the cement proteins. Both studies agree closely on the organization of the MCPs and confirm the earlier cryo-EM observations (9, 10, 13), but neither provided...

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“…A sample of the Ad5LacZ-pIX-SY12 virion was prepared for cryo-EM observation as described previously (10). Two thousand, nine hundred forty-three single Ad particles out of a total of 4,905 coming from 18 micrographs imaged with a JEOL 2010 FEG electron microscope at a magnification of ϫ30,000 were used to generate the 3D structure of the pIXmodified virus (Fig.…”

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confidence: 99%

The C-Terminal Domains of Adenovirus Serotype 5 Protein IX Assemble into an Antiparallel Structure on the Facets of the Capsid

Fabry

Rosa‐Calatrava

Moriscot

et al. 2009

J Virol

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Adenovirus serotype 5 protein IX (pIX) has two domains connected by a flexible linker. Three Nterminal domains form triskelions on the capsid facets that cement hexons together, and the C-terminal domains of four monomers form complexes toward the facet periphery. Here we present a cryoelectron microscopy structure of recombinant adenovirus with a peptide tag added to the C terminus of pIX. The structure, made up by several C termini of pIX, is longer at both ends than the wild-type protein, and Fabs directed against the tag bind to both ends of the oligomer, demonstrating that the pIX C termini associate in an antiparallel manner.Adenoviruses (Ad) are double-stranded DNA viruses which are found in all vertebrate species (6). More than 50 human serotypes provoke mild diseases and induce a robust and longlasting immunity, which can be detrimental to the use of Ad vectors in substitutive gene therapy and anticancer treatments (1). The viral capsid is built up by 7 out of the 12 polypeptides present in the virion. The major structural component is the trimeric hexon, with 240 copies per virion that make up the 20 facets of the icosahedron (11). At each of the 12 vertices is a pentameric penton base and the externally projecting trimeric fiber. Among the four remaining minor proteins, protein IIIa and protein IX (pIX) have been tentatively positioned in the first three-dimensional (3D) electron microscopy (EM) map. It had been assumed that both protein IIIa and pIX were located on the outside of the capsid: IIIa would bridge two capsid facets, whereas pIX would stabilize the group-of-nine hexons as four pIX trimers per group-of-nine (11).More recently, a new location for protein IIIa and pIX was proposed, based on a high-resolution cryo-EM 3D reconstruction of the Ad37 capsid, combined with secondary structure predictions. Protein IIIa is located inside the capsid and connected to the N-terminal domain of the penton base oriented inwards (9). pIX would form a connecting network of four trimers in the middle of each facet (9). The N-terminal domains of three pIX monomers form a triskelion structure that cements three hexons together (Fig. 1A and B). The C-terminal domains would be located near the edge between two facets and would form a tetramer at that position ( Fig. 1A and B). The N-and C-terminal domains would be connected by the central alanine-rich domain. In this model, three out of the four C-terminal domains of pIX would associate together in a parallel structure, whereas the fourth domain would associate to the trimer in an antiparallel manner (Fig. 1A).In order to verify this hypothesis, we fused the dodecapeptide TAYSSYMKGGKF (abbreviated SY12) (5) to the C terminus of pIX (Fig. 1B), and a recombinant Ad5LacZ-pIX-SY12 vector was constructed as described previously (2, 8), amplified, and purified by CsCl gradient ultracentrifugation (3). Ad5LacZ-pIX-SY12 showed the same infectivity efficiency as wild-type (WT) virions, i.e., a ratio of 1:25 to 1:50 in terms of PFU to physical particles, and was as stable...

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Three-Dimensional Structure of Canine Adenovirus Serotype 2 Capsid

Cited by 62 publications

References 58 publications

Cryoelectron Microscopy Map of Atadenovirus Reveals Cross-Genus Structural Differences from Human Adenovirus

Cryoelectron Microscopy Map of Atadenovirus Reveals Cross-Genus Structural Differences from Human Adenovirus

Structures and organization of adenovirus cement proteins provide insights into the role of capsid maturation in virus entry and infection

The C-Terminal Domains of Adenovirus Serotype 5 Protein IX Assemble into an Antiparallel Structure on the Facets of the Capsid

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