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

Abstract

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

Adenovirus serotype 5 hexon is critical for virus infection of hepatocytes in vivo

“…The Ad5-FX reconstruction compared with a previous reconstruction of the Ad5 capsid (18) revealed that the point of contact between FX and the Ad5 hexon is within the central depression of each hexon trimer (Fig. 2C).…”

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

“…Of note, however, the two studies differ significantly in their assignments of the cement proteins IIIa and IX. Recent cryo-EM studies reported that only protein IX molecules form "triskelion" as well as "four-helix bundle" (4-HLXB) structures and mediate the network of interactions between hexon subunits on the capsid exterior (11,14,15). They also suggested that the densities ascribed to α-helices beneath the vertex region belong to protein IIIa.…”

Molecular Rearrangements Involved in the Capsid Shell Maturation of Bacteriophage T7

“…The contact sites for the accessory proteins are usually exposed after the rearrangement of the shell protein monomers during maturation, thus avoiding premature interactions leading to abortive assemblies. This mechanism applies to HSV-1 (21) and adenovirus (22), as well as to the bacteriophages, lambda (23), T4 (24), and ⑀15 (25). A second mechanism involves the generation of new interactions between neighboring subunits by using additional domains inserted within the primitive protein sequence.…”