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

Abstract

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...

The Amphipathic Helix of Adenovirus Capsid Protein VI Contributes to Penton Release and Postentry Sorting

“…6A to D, green), making it a possible location of VI, as VI had not been assigned in this atomic model (34). This location in the difference map is also close to the location where Reddy and Nemerow (36) assigned parts of proteins VI (called IIIa in reference 34) (Fig. 6H, blue) and V (Fig.…”

“…Next, we fitted both the previously published Ad5-wt atomic structure (34) (Fig. 6A to D) and the newly assigned structure of the adenovirus capsid cement protein from a recent X-ray crystallography study (36) (Fig. 6E to H) into the wt map and overlaid it with the difference map.…”

“…Most tentative allocations based on high-resolution structures place protein VI at the inner capsid surface under the penton/vertex region, with the N termini of protein VI contacting peripentonal hexons (5,33), or in the internal cavity of the hexon trimer (11,34,35). Recently, a large part of the structure of protein VI was assigned using a fullparticle crystallography approach (36). However, the assigned location of protein VI is in disagreement with a cryo-EM-based high-resolution model that assigns the same density to protein IIIa (34).…”

Structures of Adenovirus Incomplete Particles Clarify Capsid Architecture and Show Maturation Changes of Packaging Protein L1 52/55k

“…HAdV-C5 is the largest structure solved by either of the two techniques. However, even after this tour de force, the location of some of the minor coat proteins is still a subject of debate (11)(12)(13). One issue is the location of polypeptide IIIa.…”

Nuclear Import of Adenovirus DNA Involves Direct Interaction of Hexon with an N-Terminal Domain of the Nucleoporin Nup214

“…On the outer surface of the capsid at each of the 12 vertices, fiber proteins are anchored to the penton base. A number of minor capsid proteins on the outer and inner surfaces of the virus particle help to stabilize the capsid (4). The DNA is directly associated with the core proteins, including protein X, the "terminal protein," which is covalently linked to the 5= DNA termini, protein VII, and protein V, which connects the core to the outer capsid.…”