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).     

Drosophila: a model for understanding obesity and diabetic complications

The molecular mechanisms underlying development of obesity and diabetic complications are not well understood. Drosophila has become a popular model organism for studying a variety of human diseases. We discuss here emerging Drosophila models of obesity and diabetic complications.     

Immune response to hepatitis B virus proteins: relevance of the murine model.

The experimental murine models offer unique opportunities to study the genetic, cellular, and molecular basis for variable immune responsiveness to HBV-encoded antigens. The mouse has provided a means of studying the immunogenicity of the pre-S regions of HBsAg and has elucidated the independent H-2-linked genes regulating antibody production to pre-S and S region determinants. The ability to circumvent genetic nonresponsiveness has implications for the design of future HBV vaccines. Mice also afforded the opportunity to examine the ability of HBcAg to activate B cells directly, and to prime Th cells capable of eliciting antibody to envelope proteins. These observations have potential clinical relevance, implications for vaccine design, and may explain the ability of HBcAg vaccination to protect against HBV infection. The murine system ha also facilitated the mapping of T-cell and B-cell recognition sites within HBV proteins, which at least conceptually enhances the prospects for development of a synthetic HBV vaccine. Antibody-binding sites appear to be similar in humans and mice; the extent to which their T-cell repertoires overlap is not known. However, the chemical and structural constraints imposed on T-cell antigenicity, and the fact that MHC-encoded class II molecules and the cellular mechanisms mediating T-cell recognition are conserved across species, make such overlaps appear likely. Furthermore, the ability to generate transgenic mice that express the various HBV proteins has yielded important insights into possible immunopathogenic mechanism and immunologic tolerance mechanisms that may predispose to chronic infection.     

Crystal structures of the trimeric human immunodeficiency virus type 1 matrix protein: implications for membrane association and assembly.

The human immunodeficiency virus type 1 (HIV-1) matrix protein forms a structural shell associated with the inner viral membrane and performs other essential functions throughout the viral life cycle. The crystal structure of the HIV-1 matrix protein, determined at 2.3 angstrom resolution, reveals that individual matrix molecules are composed of five major helices capped by a three-stranded mixed beta-sheet. Unexpectedly, the protein assembles into a trimer in three different crystal lattices, burying 1880 angstrom2 of accessible surface area at the trimer interfaces. Trimerization appears to create a large, bipartite membrane binding surface in which exposed basic residues could cooperate with the N-terminal myristoyl groups to anchor the protein on the acidic inner membrane of the virus.     

Cancer anorexia: a model for the understanding and treatment of secondary anorexia

Under normal conditions, food intake is controlled in the hypothalamus by: (i) transducing metabolic/sensorial inputs arising from the periphery into neuronal response; (ii) integrating the information originating from different tissues; and (iii) triggering the appropriate feeding responses. Thus, the anorexia associated with a number of chronic diseases, including cancer, may result from an abnormal input of information to the hypothalamus, or in its defective transduction and integration, or in the induction of exaggerated and inappropriate feeding responses. Currently available data suggest that the pathogenesis of secondary anorexia is multifactorial, and involves most of the neuronal signalling pathways modulating energy intake, including hormones (e.g. leptin), neuropeptides (e.g. NPY), cytokines (e.g. IL-1, IL-6, TNF) and neurotransmitters (e.g. serotonin and dopamine). However, it is unlikely that they represent separate and distinct pathogenic mechanisms, rather it appears that close interrelationships may exist among them. In line with this reasoning, consistent experimental and human data suggest that the hypothalamic serotonergic neurotransmission may represent a major target on which different anorexia-related factors converge. Thus, interfering pharmacologically with hypothalamic serotonin synthesis and activity may represent an effective therapeutic strategy in anorectic patients, as suggested by recent preliminary clinical data.     

Structural basis for scaffolding-mediated assembly and maturation of a dsDNA virus

Formation of many dsDNA viruses begins with the assembly of a procapsid, containing scaffolding proteins and a multisubunit portal but lacking DNA, which matures into an infectious virion. This process, conserved among dsDNA viruses such as herpes viruses and bacteriophages, is key to forming infectious virions. Bacteriophage P22 has served as a model system for this study in the past several decades. However, how capsid assembly is initiated, where and how scaffolding proteins bind to coat proteins in the procapsid, and the conformational changes upon capsid maturation still remain elusive. Here, we report Cα backbone models for the P22 procapsid and infectious virion derived from electron cryomicroscopy density maps determined at 3.8- and 4.0-Å resolution, respectively, and the first procapsid structure at subnanometer resolution without imposing symmetry. The procapsid structures show the scaffolding protein interacting electrostatically with the N terminus (N arm) of the coat protein through its C-terminal helix-loop-helix motif, as well as unexpected interactions between 10 scaffolding proteins and the 12-fold portal located at a unique vertex. These suggest a critical role for the scaffolding proteins both in initiating the capsid assembly at the portal vertex and propagating its growth on a T = 7 icosahedral lattice. Comparison of the procapsid and the virion backbone models reveals coordinated and complex conformational changes. These structural observations allow us to propose a more detailed molecular mechanism for the scaffolding-mediated capsid assembly initiation including portal incorporation, release of scaffolding proteins upon DNA packaging, and maturation into infectious virions.     

Matchmaking and species marriage: a game-theory model of community assembly

Theories developed to explain the processes that govern the assembly and composition of natural plant communities can be divided into two broad categories. Niche-based theories propose that coevolutionary changes among species lead to character divergence (displacement), which allows for coexistence by partitioning resources among species. In contrast, ecological-drift theories propose that species diversity results from a balance of migration, speciation, and extinction, with little microevolutionary change. We use a game-theory model to reconcile drift and niche perspectives by developing a theory of species "marriage." Initially, ecological drift may determine which species encounter each other in a competitive arena. Once species come into contact, historical niche development as a result of prior coevolutionary molding of competitive ability determines which species may coexist. The model shows that only species that display the well-known tradeoff between seed size and competitive ability as a result of past competitive histories comply with the requisite for mutual evolutionary stability. Mutual evolutionary stability of competitive ability tends to make reproductive outputs more similar between species, increasing the chances of ecological equilibrium, i.e., the coexistence of species competing for a single resource. Moreover, mutual evolutionary stability guarantees that such an ecological equilibrium will be stable. The species-marriage model predicts that two or more plant species will coexist indefinitely (i.e., "marry") when their difference in seed size, their densities, and the resource availability obey a specific quantitative relation. For example, when resource availability is high, married species should be characterized by a greater asymmetry in seed size than when resource availability is low. Thus, in the species-marriage model, competition can shape the detailed properties of communities without violating the postulates of ecological-drift theory.     

Axonal transport of herpes simplex virions to epidermal cells: evidence for a specialized mode of virus transport and assembly.

To examine the transmission of herpes simplex virus (HSV) from axon to epidermal cell, an in vitro model was constructed consisting of human fetal dorsal root ganglia cultured in the central chamber of a dual-chamber tissue culture system separated from autologous skin explants in an exterior chamber by concentric steel cylinders adhering to the substratum through silicon grease and agarose. Axons grew through the agarose viral diffusion barrier and terminated on epidermal cells in the exterior chamber. After inoculation of HSV onto dorsal root ganglia, anterograde axonal transport of glycoprotein and nucleocapsid antigen was observed by confocal microscopy to appear in exterior chamber axons within 12 h and in epidermal cells within 16 h, moving at 2-3 mm/h. Although both enveloped and unenveloped nucleocapsids were observed in the neuronal soma by transmission electron microscopy, only nucleocapsids were observed in the axons, closely associated with microtubules. Nodule formation at the surface of HSV-infected axons, becoming more dense at the axon terminus on epidermal cells, and patches of axolemmal HSV glycoprotein D expression were observed by scanning (immuno)electron microscopy, probably representing virus emerging from the axolemma. These findings strongly suggest a specialized mode of viral transport, assembly, and egress in sensory neurons: microtubule-associated intermediate-fast anterograde axonal transport of unenveloped nucleocapsids with separate transport of glycoproteins to the distal regions of the axon and assembly prior to virus emergence at the axon terminus.     

Separation-individuation conflict as a model for understanding distressed caregivers: psychodynamic and cognitive case studies

Caring for a frail relative can be a stressful undertaking. Caregivers are faced with a series of losses, and old interpersonal wounds are often reopened. Project Assist was designed to study the efficacy of time-limited psychodynamic and cognitive/behavioral therapies for experienced and recent caregivers. It offers psychotherapy that can assist caregivers in separating their own emotions, identity, and well-being from those of the care receiver. This process appears necessary for the individuation and mental health of caregivers.     

《2016年澳大利亚HCV感染管理共识意见》摘译

HCV感染是澳大利亚重要的公共卫生问题,约230 000人会因进展性肝纤维化最终导致肝硬化、肝衰竭甚至肝细胞癌(HCC).在澳大利亚,HCV感染是肝脏疾病需行肝移植的最主要原因.由HCV所致肝脏疾病的负担预计到2030年会增加3倍.然而,HCV感染是可治愈的,且病毒学清除可带来多重临床获益,包括提高生活质量、丧失传染性、肝硬化的逆转,降低肝脏衰竭和HCC的发生,并且降低死亡率.     

Retrostructural analysis of metalloproteins: application to the design of a minimal model for diiron proteins

De novo protein design provides an attractive approach for the construction of models to probe the features required for function of complex metalloproteins. The metal-binding sites of many metalloproteins lie between multiple elements of secondary structure, inviting a retrostructural approach to constructing minimal models of their active sites. The backbone geometries comprising the metal-binding sites of zinc fingers, diiron proteins, and rubredoxins may be described to within approximately 1 A rms deviation by using a simple geometric model with only six adjustable parameters. These geometric models provide excellent starting points for the design of metalloproteins, as illustrated in the construction of Due Ferro 1 (DF1), a minimal model for the Glu-Xxx-Xxx-His class of dinuclear metalloproteins. This protein was synthesized and structurally characterized as the di-Zn(II) complex by x-ray crystallography, by using data that extend to 2.5 A. This four-helix bundle protein is comprised of two noncovalently associated helix-loop-helix motifs. The dinuclear center is formed by two bridging Glu and two chelating Glu side chains, as well as two monodentate His ligands. The primary ligands are mostly buried in the protein interior, and their geometries are stabilized by a network of hydrogen bonds to second-shell ligands. In particular, a Tyr residue forms a hydrogen bond to a chelating Glu ligand, similar to a motif found in the diiron-containing R2 subunit of Escherichia coli ribonucleotide reductase and the ferritins. DF1 also binds cobalt and iron ions and should provide an attractive model for a variety of diiron proteins that use oxygen for processes including iron storage, radical formation, and hydrocarbon oxidation.     

Use of a zinc-finger consensus sequence framework and specificity rules to design specific DNA binding proteins.

We have designed three zinc-finger proteins with different DNA binding specificities. The design strategy combines a consensus zinc-finger framework sequence with previously characterized recognition regions such that the specificity of each protein is predictable. The first protein consists of three identical zinc fingers, each of which was expected to recognize the subsite GCG. This protein binds specifically to the sequence 5'-GCG-GCG-GCG-3' with a dissociation constant of approximately 11 microM. The second protein has three zinc fingers with different predicted preferred subsites. This protein binds to the predicted recognition site 5'-GGG-GCG-GCT-3' with a dissociation constant of 2 nM. Furthermore, selection experiments indicate that this is the optimal binding site. A permuted version of the second protein was also constructed and shown to preferentially recognize the corresponding permuted site 5'-GGG-GCT-GCG-3' over the non-permuted site. These results indicate that earlier observations on the specificity of zinc fingers can be extended to generalized zinc-finger structures and realize the use of zinc fingers for the design of site-specific DNA binding proteins. This consensus-based design system provides a useful model system with which to study details of zinc-finger-DNA specificity.