Modeling Viral Capsid Assembly.

Decision letter: Many-molecule encapsulation by an icosahedral shell

An automobile body assembly is a complex system consisting of hundreds of compliant sheet metal parts. A number of locating schemes are used throughout the assembly and inspection processes. This paper presents a methodology to represent the assembly and inspection processes of an automobile body for tolerance analysis. The proposed representation methodology consistently describes dimensional variations with respect to various locating schemes that change through the assembly process.

Timing of the synthesis of empty shells and minor nucleoproteins in relation to turnip yellow mosaic virus synthesis in Brassica protoplasts

Prematurity and Perinatal Antibiotics: A Tale of Two Factors Influencing Development of the Neonatal Gut Microbiota

In order to effectively represent assembly process information based on three dimensional (3D) models and rapidly extract the necessary information for assembly process planning, analysis and simulation, this paper aims to research the multiple view representation of assembly process information based on 3D models. Assembly process information based on 3D models is summarized and considered as the single source of the multiple view representation. The definition and organization of assembly process view is defined to implement assembly process representation based on view. Assembly process views based on assembly process planning work flow are established. View mapping approach is investigated to rapidly extract the necessary information for assembly process planning, analysis and simulation. The use of the multiple view assembly process representation is illustrated by building detailed assembly process view and assembly process simulation view and by extracting relevant information for the assembly process simulation view from detailed assembly process view.

This paper uses combinatorics and group theory to answer questions about the assembly of icosahedral viral shells. Although the geometric structure of the capsid (shell) is fairly well understood in terms of its constituent subunits, the assembly process is not. For the purpose of this paper, the capsid is modeled by a polyhedron whose facets represent the monomers. The assembly process is modeled by a rooted tree, the leaves representing the facets of the polyhedron, the root representing the assembled polyhedron, and the internal vertices representing intermediate stages of assembly (subsets of facets). Besides its virological motivation, the enumeration of orbits of trees under the action of a finite group is of independent mathematical interest. If G is a finite group acting on a finite set X, then there is a natural induced action of G on the set T(x) of trees whose leaves are bijectively labeled by the elements of X. If G acts simply on X, then |X|:=|X(n)|=n·|G|, where n is the number of G-orbits in X. The basic combinatorial results in this paper are (1) a formula for the number of orbits of each size in the action of G on T(x)(n), for every n, and (2) a simple algorithm to find the stabilizer of a tree τ ∈T(x) in G that runs in linear time and does not need memory in addition to its input tree. These results help to clarify the effect of symmetry on the probability and number of assembly pathways for icosahedral viral capsids, and more generally for any finite, symmetric macromolecular assembly.

Okra mosaic virus empty protein shells in nuclei

During the life cycle of a virus, viral proteins and other components self-assemble to form an ordered protein shell called a capsid. This assembly process is subject to multiple competing constraints, including the need to form a thermostable shell while avoiding kinetic traps. It has been proposed that viral assembly satisfies these constraints through allosteric regulation, including the interconversion of capsid proteins among conformations with different propensities for assembly. In this article, we use computational and theoretical modeling to explore how such allostery affects the assembly of icosahedral shells. We simulate assembly under a wide range of protein concentrations, protein binding affinities, and two different mechanisms of allosteric control. We find that above a threshold strength of allosteric control, assembly becomes robust over a broad range of subunit binding affinities and concentrations, allowing the formation of highly thermostable capsids. Our results suggest that allostery can significantly shift the range of protein binding affinities that lead to successful assembly and thus should be taken into account in models that are used to estimate interaction parameters from experimental data.

Both β-galactosidase (GAL) and β-glucuronidase (GUS) are tetrameric enzymes used widely as reporter proteins. However, little is known about the folding and assembly of these enzymes. Although the refolding kinetics of GAL from a denatured enzyme have been reported, it is not known how the kinetics differ when coupled with a protein translation reaction. Elucidating the assembly kinetics of GAL and GUS when coupled with protein translation will illustrate the differences between these two reporter proteins and also the assembly process under conditions more relevant to those in vivo. In this study, we used an in vitro translation/transcription system to synthesize GAL and GUS, measured the time development of the activity and oligomerization state of these enzymes, and determined the rate constants of the monomer to tetramer assembly process. We found that at similar concentrations, GAL assembles into tetramers faster than GUS. The rate constant of monomer to dimer assembly of GAL was 50-fold faster when coupled with protein translation than that of refolding from the denatured state. Furthermore, GAL synthesis was found to lack the rate-limiting step in the assembly process, whereas GUS has two rate-limiting steps: monomer to dimer assembly and dimer to tetramer assembly. The consequence of these differences when used as reporter proteins is discussed.

Membrane emulsification for the production of suspensions of uniform microcapsules with tunable mechanical properties

In recent years, assembly system has been developing towards multi-state, complexity, large-scale and intelligent trend, and complex assembly tasks and assembly processes as well as dynamic and changeable assembly environment have reduced the mission reliability of assembly system, resulting in low quality and low reliability of assembly products. In addition, with the large-scale and complex structure of assembly product and the deterioration trend of service environment is becoming more and more obvious, people have higher and higher requirements for its reliability and quality. Therefore, this paper proposes a mission reliability modeling method of assembly process considering workpiece quality deviation. Firstly, the connotation of assembly process mission reliability is proposed on the basis of clarifying assembly system components and operation principle. Secondly, from the perspective of system theory, the conceptual model of PQR chain, which is embodied as the chain conceptual model of assembly machine performance state (P), assembly process quality (Q) and product reliability (R), is innovatively proposed to analyze the relationship among assembly machine performance state, assembly process quality and assembly product reliability. Thirdly, the assembly process operation quality data is highly integrated and applied to the PQR chain, and the assembly process mission reliability model considering the workpiece quality deviation is established. Finally, the feasibility of the proposed method is verified by a double nozzle flapper valve assembling example.

The genetic code expansion strategy, the recently emerged pyrrolysine (Pyl)-based system in particular, has become a generally applicable method for site-specific incorporation of unnatural amino acids (UAAs) into a protein of interest in bacteria, yeast, mammalian cells, and even in animals. However, this technique has yet to be applied to intact and live viruses, which is largely due to the fragile nature as well as the complicated assembly process of many human viruses. To address this challenge, we here coupled the genetic-code expansion strategy with an engineered virus assembly process in human hepatocytes to site-specifically introduce unnatural chemical groups onto virus surface proteins by using hepatitis D virus (HDV) as a model system. HDV has infectedmore than 15 million people worldwide, and currently there are no drugs clinically available against this virus. HDV is a satellite virus of human hepatitis B virus (HBV), which has infected two billion people and among them about 240 million are currently chronically infected. Both HBV and HDV share the same envelope proteins for infection of hepatocytes. Study of HBV and HDV infection has long been hampered by the lack of efficient and easily accessible in vitro infection system. Recently, a bile acid transporter predominantly expressed in liver, sodium taurocholate cotransporting polypeptide (NTCP) was identified as a functional receptor for HDV and HBV. The NTCP complemented human hepatoma cell line HepG2 provided a feasible in vitro infection system for studying HBV and HDV infection. However, the lack of methods to selectively label, monitor, and/or manipulate an intact virus under living conditions still restricts investigations into molecular details of the infection. Many problems are due to the distinct topological features of the critical viral proteins, as well as complex virus assembly processes. For example, HDV has developed a tightly regulated assembly process to produce infectious viral particles in human hepatocytes: the HDV RNAs were first encapsulated with delta antigens and then packaged with three HBV envelope proteins, namely large (L), middle (M), and small (S) proteins, to produce the intact viral particle before being secreted to the extracellular space (Supporting Information, Figure S1). The resulting HDV, with a diameter of 36 nm, is one of the smallest animal viruses known to date. It is therefore exceedingly difficult to chemically label this tiny virus with delicate structures under living conditions. Furthermore, the virus surface envelope proteins contain many chemically active amino acids (for example, cysteine and lysine) that are essential for virus entry in host cells. Conjugation or modification of these natural residues will severely compromise viral infectivity. A noninvasive strategy for manipulation of living viral particles without impairment of their viability and infectivity is thus highly desired. Bioorthogonal reactions have revolutionized our ability to label and manipulate various biomolecules and even whole cells and organisms under living conditions. As a critical step for applying such chemistry for virus labeling, several approaches have been reported for installation of bioorthogonal handles, typically in the form of UAAs into proteins from sub-viral-like particle (SVP) or intact virus. For instance, site-specific or residue-specific incorporation of UAAs bearing an azide or an alkyne moiety into SVP has been demonstrated in bacterial cells by several laboratories. These methods allow the conjugation of SVPs with various fluorescent dyes or therapeutic reagents for biomedical or biomaterial applications. However, SVPs are non-infectious and not suitable for investigating virus infection mechanisms. Indeed, SVPs produced from prokaryotic cells lack posttranslational modifications, particularly on their surface envelope proteins, and therefore differ from the native SVPs. Although attempts have been made to extend some of these methods for virus production in mammalian cells, such strategies typically require the metabolic replacement of a specific type of amino acid permissive only to simple groups (for example, azide and ketone) from the entire virus proteome, which may disrupt the virion assembly process or permute the vulnerable virion structure, resulting in compromised viral infectivity. Taken together, a general approach for precise labeling and manipulation of intact [*] S.-X. Lin, M.-Y. Yang, Prof. Dr. P. R. Chen Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, Department of Chemical Biology, College of Chemistry and Molecular Engineering, Synthetic and Functional Biomolecules Center and Peking-Tsinghua Center for Life Sciences Peking University, Beijing 100871 (China) E-mail: pengchen@pku.edu.cn H. Yan Graduate program in School of Life Sciences Peking University, Beijing (China) H. Yan, L. Li, B. Peng, S. Chen, Prof. Dr. W.-H. Li National Institute of Biological Sciences, Beijing (China) E-mail: liwenhui@nibs.ac.cn [] These authors contributed equally to this work.

VirusesVirion components, including viral genome, CPs forming the coat around the genome, and membranes associated with nucleocapsids (NCs), come together in a process called assembly. The self-assembly process of viruses was first demonstrated by Fraenkel-Conrat and Williams in 1955 [21]. This study showed that disassembled TMV particles could be reconstituted in vitro and the particles maintained their infectivity. Virion assembly occurs basically with two mechanisms: (i) They are coassembled with the genome, or (ii) viral genomes are packaged into an empty preformed procapsid in a process powered by nucleoside triphosphate (NTP) hydrolysis. The assembly process is driven by the laws of thermodynamics, meaning that the subunits forming the particle are energetically in a more favorable environment within the virion than the free subunits [22]. The structure of icosahedral virus particles can be described on the basis of the triangulation number defining the possible icosahedral surface lattice designs, quasi-equivalence describing the nearly equivalent bonding, and self-assembly. As many copies of one protein compose the particle the required information for selfassembly resides in the specific bonding patterns of the individual proteins, although with some viruses the assembly process is regulated by scaffolding proteins that are not present in the final virion [23]. Cementing or glue proteins may exist in final virions to give stability, and some other proteins may be incorporated into virions to serve specific functions like binding of the virus to its receptor on the cell surface. To fully understand the assembly mechanism it is important to know the composition of the virion as well as all intermediates formed during the assembly pathway. The biology of virion assembly as well as the requirements to form stable mature particles set boundaries for the doable genetic and chemical modifications ofthe virions. Therefore it is especially important for ENC applications to solve the surface exposed areas that do not take part in CP interactions needed in the assembly pathway or for the stability of the mature virions. 1.2.1 Particle Formation via CoassemblyThe capsids of helical viruses, both rod-shaped and flexuous filaments, and small icosahedral RNA viruses are coassembled with the genome. Helical viruses are viruses that have their RNA enclosed in a protective shell consisting of identical protein subunits arranged in a helical manner. This basic structure was first proposed by Crick and Watson in 1956 [4]. Their studies led them to conclude that the structure of small viruses consisted of identical units with specific bonding properties arranged to form a close surface around the viral genetic material, the main events being nucleation and elongation in the order mentioned. Virus assembly and stability of virus particles is governed by several molecular interactions. Viruses of the genera Tymovirus and Comovirus, for example, are stabilized mostly by protein-protein interactions and hence can form VLPs in the absence of viral RNA, while viruses of the genera Alfamovirus, Bromovirus, and Cucumovirus are primarily stabilized by RNA-protein interactions and require other virus-encoded proteins for successful packaging [24, 25]. In spite of these two being the major acting forces, other sequence-dependent/independent RNA-protein interactions as well as structure-dependent interactions are also important. One amongst other constraints in coassembly of icosahedral viruses is the dimension of the capsid, which inflicts a restriction on the size of the RNA to be encapsidated. Studies on turnip crinkle virus (TCV) by Qu and Morris exemplify this aspect [26]. The filamentous virus structure is more flexible in this respect, as the virion size can increase with increasing genome size, as shown with potyviruses [27]. Next we attempt to picturize the assembly process of small simple viruses using TMV as a model to enlighten the major steps. 1.2.1.1 Assembly of TMVTMV is a rod-shaped RNA virus, 300 nm in length, 18 nm in diameter, and a central radius of 2 nm [28]. Its virus particle contains about2100 protein subunits enclosing a single RNA molecule. Hence, RNA makes only 5% of the particle, while 95% contains the protein shell. The length of the protein shell seems to be dependent on the length of the viral RNA. Intensive and informative studies on the in vitro assembly process enabling the proper understanding and uncovering of the TMV assembly have been communicated. These studies are used here as a prime example of the simple assembly process involving condensation of genomic single-stranded RNA (ssRNA)(+) and CPs. The major component of the TMV rod is the 17 kDa CP comprising 154 amino acids. Depending on certain conditions, pH being the most important of all, protein monomers constituting the virion can self-assemble to form different aggregates (Fig. 1.3).

Intracellular compartmentalization enhances biological reactions, crucial for cellular function and survival. An example is the carboxysome, a bacterial microcompartment for CO2 fixation. The carboxysome uses a polyhedral protein shell made of hexamers, pentamers, and trimers to encapsulate Rubisco, increasing CO2 levels near Rubisco to enhance carboxylation. Despite their role in the global carbon cycle, the molecular mechanisms behind carboxysome shell assembly remain unclear. Here, we present a structural characterization of α-carboxysome shells generated from recombinant systems, which contain all shell proteins and the scaffolding protein CsoS2. Atomic-resolution cryo-electron microscopy of the shell assemblies, with a maximal size of 54 nm, unveil diverse assembly interfaces between shell proteins, detailed interactions of CsoS2 with shell proteins to drive shell assembly, and the formation of heterohexamers and heteropentamers by different shell protein paralogs, facilitating the assembly of larger empty shells. Our findings provide mechanistic insights into the construction principles of α-carboxysome shells and the role of CsoS2 in governing α-carboxysome assembly and functionality.

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