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

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 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 significant information on the structure and location of protein VI, which serves key roles in the virus life cycle. 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. However, based on our X-ray crystallographic data and considering the principles of quasi-equivalence (16), we earlier suggested that although the IX molecules form triskelion structures, it is rather unlikely that the C termini of IX would form 4-HLXB structures (12). Instead, we proposed that this 4-HLXB is most likely derived from a subdomain of IIIa (12).Open in a separate windowFig. 1.Structure and organization of human adenovirus. (A) A schematic illustration of the organization of capsid and core proteins in human adenovirus. The locations of various proteins are represented by different-colored symbols and the corresponding names are shown (Right). The indicated locations of the core proteins are approximate. Shown in blue-colored lettering are the proteins whose structures have been identified in this study. (B) Overall organization of hexon and penton base subunits exhibiting pseudo-T = 25 icosahedral symmetry. Structurally unique hexons (1–4) are color-coded in light blue, pink, green, and khaki, respectively. Penton vertices are shown in magenta. Outer cement proteins IIIa and IX are shown in purple and blue, respectively. Fiber molecules associated with the penton base are disordered. The outline of the triangular icosahedral facet is shown as a gray triangle, whereas the border of the GON hexons is indicated by yellow-colored rope. (C) An exterior view of the triangular icosahedral facet that comprises 12 hexons along with penton base vertices shown in magenta. Color representations are the same as in B. (D) An interior view of the facet in C, with three minor proteins, V (green), VI (red), and VIII (orange). It is noteworthy that a copy of V, VI, and VIII forms a ternary complex beneath the vertices, whereas VIII (orange) molecules are arranged as staples along the border (yellow-colored rope) of the GON hexons.Here we report a revised interpretation, a paradigm shift, of the structures and locations of all of the cement proteins based on the refined crystal structure of Ad5F35 (HAdV5 vector encoding the type 35 fiber) that includes detailed models for the ordered regions of all four cement proteins (IIIa, VI, VIII, and IX). Additionally, we identified a segment of core protein V, which associates closely with protein VI. The 4-HLXB structure on the capsid exterior is a subdomain of IIIa (amino acids 101–355) that mediates interactions between group-of-nine (GON) (17) hexons (Fig. 1 C and D). The backbone path of each IX molecule is reversed from what was assigned by the cryo-EM studies (11, 14). Proteins V, VI, and VIII form a ternary complex that stabilizes the adjacent peripentonal hexons (PPHs) underneath each of the 12 vertex regions (Fig. 1D). This complex was incorrectly assigned to protein IIIa in cryo-EM studies (11, 14). Following virion maturation, the cleaved propeptide(s) of VI (pVIn; amino acids 1–33) is observed in the inner cavities of the PPHs, in agreement with recent interpretations from hydrogen–deuterium exchange mass spectrometry studies (18).