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Medusavirus is a giant virus classified into an independent family of Mamonoviridae. Amoebae infected with medusavirus release immature particles in addition to virions. These particles were suggested to exhibit the maturation process of this virus, but the structure of these capsids during maturation remains unknown. Here, we apply a block-based reconstruction method in cryo-electron microscopy (cryo-EM) single particle analysis to these viral capsids, extending the resolution to 7–10 Å. The maps reveal a novel network composed of minor capsid proteins (mCPs) supporting major capsid proteins (MCPs). A predicted molecular model of the MCP fitted into the cryo-EM maps clarified the boundaries between the MCP and the underlining mCPs, as well as between the MCP and the outer spikes, and identified molecular interactions between the MCP and these components. Several structural changes of the mCPs under the fivefold vertices of the immature particles were observed, depending on the presence or absence of the underlying internal membrane. In addition, the lower part of the penton proteins on the fivefold vertices was also missing in mature virions. These dynamic conformational changes of mCPs indicate an important function in the maturation process of medusavirus. IMPORTANCE The structural changes of giant virus capsids during maturation have not thus far been well clarified. Medusavirus is a unique giant virus in which infected amoebae release immature particles in addition to mature virus particles. In this study, we used cryo-electron microscopy to investigate immature and mature medusavirus particles and elucidate the structural changes of the viral capsid during the maturation process. In DNA-empty particles, the conformation of the minor capsid proteins changed dynamically depending on the presence or absence of the underlying internal membranes. In DNA-full particles, the lower part of the penton proteins was lost. This is the first report of structural changes of the viral capsid during the maturation process of giant viruses. [ABSTRACT FROM AUTHOR]

The foot-and-mouth disease virus is a highly contagious and economically devastating virus of cloven-hooved animals, including cattle, buffalo, sheep, and goats, causing reduced animal productivity and posing international trade restrictions. For decades, chemically inactivated vaccines have been serving as the most effective strategy for the management of foot-and-mouth disease. Inactivated vaccines are commercially produced in cell culture systems, which require successful propagation and adaptation of field isolates, demanding a high cost and laborious time. Cell culture adaptation is chiefly indebted to amino acid substitutions in surface-exposed capsid proteins, altering the necessity of RGD-dependent receptors to heparan sulfate macromolecules for virus binding. Several amino acid substations in VP1, VP2, and VP3 capsid proteins of FMDV, both at structural and functional levels, have been characterized previously. This literature review combines frequently reported amino acid substitutions in virus capsid proteins, their critical roles in virus adaptation, and functional characterization of the substitutions. Furthermore, this data can facilitate molecular virologists to develop new vaccine strains against the foot-and-mouth disease virus, revolutionizing vaccinology via reverse genetic engineering and synthetic biology. [ABSTRACT FROM AUTHOR]

From the first isolation of the cystovirus bacteriophage Φ6 from Pseudomonas syringae 50 years ago, we have progressed to a better understanding of the structure and transformations of many parts of the virion. The three-layered virion, encapsulating the tripartite double-stranded RNA (dsRNA) genome, breaches the cell envelope upon infection, generates its own transcripts, and coopts the bacterial machinery to produce its proteins. The generation of a new virion starts with a procapsid with a contracted shape, followed by the packaging of single-stranded RNA segments with concurrent expansion of the capsid, and finally replication to reconstitute the dsRNA genome. The outer two layers are then added, and the fully formed virion released by cell lysis. Most of the procapsid structure, composed of the proteins P1, P2, P4, and P7 is now known, as well as its transformations to the mature, packaged nucleocapsid. The outer two layers are less well-studied. One additional study investigated the binding of the host protein YajQ to the infecting nucleocapsid, where it enhances the transcription of the large RNA segment that codes for the capsid proteins. Finally, I relate the structural aspects of bacteriophage Φ6 to those of other dsRNA viruses, noting the similarities and differences. [ABSTRACT FROM AUTHOR]

The foot-and-mouth disease virus is a highly contagious and economically devastating virus of cloven-hooved animals, including cattle, buffalo, sheep, and goats, causing reduced animal productivity and posing international trade restrictions. For decades, chemically inactivated vaccines have been serving as the most effective strategy for the management of foot-and-mouth disease. Inactivated vaccines are commercially produced in cell culture systems, which require successful propagation and adaptation of field isolates, demanding a high cost and laborious time. Cell culture adaptation is chiefly indebted to amino acid substitutions in surface-exposed capsid proteins, altering the necessity of RGD-dependent receptors to heparan sulfate macromolecules for virus binding. Several amino acid substations in VP1, VP2, and VP3 capsid proteins of FMDV, both at structural and functional levels, have been characterized previously. This literature review combines frequently reported amino acid substitutions in virus capsid proteins, their critical roles in virus adaptation, and functional characterization of the substitutions. Furthermore, this data can facilitate molecular virologists to develop new vaccine strains against the foot-and-mouth disease virus, revolutionizing vaccinology via reverse genetic engineering and synthetic biology.

From the first isolation of the cystovirus bacteriophage Φ6 from Pseudomonas syringae 50 years ago, we have progressed to a better understanding of the structure and transformations of many parts of the virion. The three-layered virion, encapsulating the tripartite double-stranded RNA (dsRNA) genome, breaches the cell envelope upon infection, generates its own transcripts, and coopts the bacterial machinery to produce its proteins. The generation of a new virion starts with a procapsid with a contracted shape, followed by the packaging of single-stranded RNA segments with concurrent expansion of the capsid, and finally replication to reconstitute the dsRNA genome. The outer two layers are then added, and the fully formed virion released by cell lysis. Most of the procapsid structure, composed of the proteins P1, P2, P4, and P7 is now known, as well as its transformations to the mature, packaged nucleocapsid. The outer two layers are less well-studied. One additional study investigated the binding of the host protein YajQ to the infecting nucleocapsid, where it enhances the transcription of the large RNA segment that codes for the capsid proteins. Finally, I relate the structural aspects of bacteriophage Φ6 to those of other dsRNA viruses, noting the similarities and differences.

Foot-and-mouth disease is endemic in livestock in large parts of Africa and Asia, where it is an important driver of food insecurity and a major obstacle to agricultural development and the international trade in animal products. Virtually all commercially available vaccines are inactivated whole-virus vaccines produced in cell culture, but the adaptation of a field isolate of the virus to growth in culture is laborious and time-consuming. This is of particular concern for the development of vaccines to newly emerging virus lineages, where long lead times from virus isolate to vaccine can delay the implementation of effective control programs. High antigen yields in production cells are also necessary to make vaccines affordable for less developed countries in endemic areas. Therefore, a rational approach to cell culture adaptation that combines prior knowledge of common adaptive mutations and reverse genetics techniques is urgently required. This review provides an overview of amino acid exchanges in the viral capsid proteins in the context of adaptation to cell culture. [ABSTRACT FROM AUTHOR]

[Display omitted] • PQBP1 facilitates cGAS-mediated responses to HIV-1 by bridging the viral capsid and cGAS. • The primary interaction mode between PQBP1 and the HIV-1 capsid involves charge-complementary interactions. • The primary interaction is insufficient for stable capsid binding, and is likely bolstered by contributions from other regions of PQBP1. Human immunodeficiency virus type 1 (HIV-1) stimulates innate immune responses upon infection, including cyclic GMP-AMP synthase (cGAS) signaling that results in type I interferon production. HIV-1-induced activation of cGAS requires the host cell factor polyglutamine binding protein 1 (PQBP1), an intrinsically disordered protein that bridges capsid recognition and cGAS recruitment. However, the molecular details of PQBP1 interactions with the HIV-1 capsid and their functional implications remain poorly understood. Here, we show that PQBP1 binds to HIV-1 capsids through charge complementing contacts between acidic residues in the N-terminal region of PQBP1 and an arginine ring in the central channel of the HIV-1 CA hexamer that makes up the viral capsid. These studies reveal the molecular details of PQBP1′s primary interaction with the HIV-1 capsid and suggest that additional elements are likely to contribute to stable capsid binding. [ABSTRACT FROM AUTHOR]

Although virus capsids appear as rigid, symmetric particles in experimentally determined structures; biochemical studies suggest a significant degree of structural flexibility in the particles. We carried out all-atom simulations on the icosahedral capsid of an insect virus, Flock House Virus, which show intriguing differences in the degree of flexibility of quasi-equivalent capsid subunits consistent with previously described biological behaviour. The flexibility of all the β and γ subunits of the protein and RNA fragments is analysed and compared. Both γ A subunit and RNA fragment exhibit higher flexibility than the γ B and γ C subunits. The capsid shell is permeable to the bidirectional movement of water molecules, and the movement is heavily influenced by the geometry of the capsid shell along specific symmetry axes. In comparison to the symmetry axes along I5 and I3, the I2 axis exhibits a slightly higher water content. This enriched water environment along I2 could play a pivotal role in facilitating the structural transitions necessary for RNA release, shedding some light on the intricate and dynamic processes underlying the viral life cycle. Our study suggests that the physical characterization of whole virus capsids is the key to identifying biologically relevant transition states in the virus life cycle and understanding the basis of virus infectivity. Molecular dynamics simulation of an icosahedral T=3 virus. Chemically similar subunits in an icosahedral asymmetric unit displayed variations in flexibility. Water density along symmetry axes within the shell influenced by capsid geometry. Physical properties of capsid like local flexibility and geometry may be crucial for virus infectivity. [Display omitted] • We carried out Molecular dynamics simulation of a whole icosahedral T = 3 virus. • Chemically similar subunits in an icosahedral asymmetric unit displayed variations in flexibility. • Water density along symmetry axes within the shell was influenced by capsid geometry. • Physical properties of capsid like local flexibility and geometry may be crucial for virus infectivity. [ABSTRACT FROM AUTHOR]

Supramolecular protein complexes are the corner stone of biological processes; they are essential for many biological functions. Unraveling the interactions responsible for the (dis)assembly of these complexes is required to understand nature and to exploit such systems in future applications. Virus capsids are well-defined assemblies of hundreds of proteins and form the outer shell of non-enveloped viruses. Due to their potential as a drug carriers or nano-reactors and the need for virus inactivation strategies, assessing the intactness of virus capsids is of great interest. Current methods to evaluate the (dis)assembly of these protein assemblies are experimentally demanding in terms of instrumentation, expertise and time. Here we investigate a new strategy to monitor the disassembly of fluorescently labeled virus capsids. To monitor surfactant-induced capsid disassembly, we exploit the complex photophysical interplay between multiple fluorophores conjugated to capsid proteins. The disassembly of the capsid changes the photophysical interactions between the fluorophores, and this can be spectrally monitored. The presented data show that this low complexity method can be used to study and monitor the disassembly of supramolecular protein complexes like virus capsids. However, the range of labeling densities that is suitable for this assay is surprisingly narrow.

The hepatitis B virus (HBV) capsid is an attractive drug target, relevant to combating viral hepatitis as a major public health concern. Among small molecules known to interfere with capsid assembly, the phenylpropenamides, including AT130, represent an important antiviral paradigm based on disrupting the timing of genome packaging. Here, all-atom molecular dynamics simulations of an intact AT130-bound HBV capsid reveal that the compound increases spike flexibility and improves recovery of helical secondary structure in the spike tips. Regions of the capsid-incorporated dimer that undergo correlated motion correspond to established sub-domains that pivot around the central chassis. AT130 alters patterns of correlated motion and other essential dynamics. A new conformational state of the dimer is identified, which can lead to dramatic opening of the intradimer interface and disruption of communication within the spike tip. A novel salt bridge is also discovered, which can mediate contact between the spike tip and fulcrum even in closed conformations, revealing a mechanism of direct communication across these sub-domains. Altogether, results describe a dynamical connection between the intra- and interdimer interfaces and enable mapping of allostery traversing the entire core protein dimer.

The assembly of alphavirus nucleocapsid cores requires electrostatic interactions between the positively charged N-terminus of the capsid protein (CP) and the encapsidated polyanionic cargo. This system differs from many other viruses that can self-assemble particles in the absence of cargo, or form “empty” particles. We hypothesized that the introduction of a mutant, anionic CP could replace the need for charged cargo during assembly. In this work, we produced a CP mutant, Minus 38 (M38), where all N-terminal charged residues are negatively-charged. When wild-type (WT) and M38 CPs were mixed, they assembled into core-like particles (CLPs). These “empty” particles were of similar size and morphology to WT CLPs assembled with DNA cargo, but did not contain nucleic acid. When DNA cargo was added to the assembly mixture, the amount of M38 CP that was assembled into CLPs decreased, but was not fully excluded from the CLPs, suggesting that M38 competes with DNA to interact with WT CPs. The composition of CLPs can be tuned by altering the order of addition of M38 CP, WT CP, and DNA cargo. The ability to produce alphavirus CLPs that contain a range of amounts of encapsidated cargo, including none, introduces a new platform for packaging cargo for delivery or imaging purposes.

The hepatitis B virus capsid represents a promising therapeutic target. Experiments suggest the capsid must be flexible to function; however, capsid structure and dynamics have not been thoroughly characterized in the absence of icosahedral symmetry constraints. Here, all-atom molecular dynamics simulations are leveraged to investigate the capsid without symmetry bias, enabling study of capsid flexibility and its implications for biological function and cryo-EM resolution limits. Simulation results confirm flexibility and reveal a propensity for asymmetric distortion. The capsid’s influence on ionic species suggests a mechanism for modulating the display of cellular signals and implicates the capsid’s triangular pores as the location of signal exposure. A theoretical image reconstruction performed using simulated conformations indicates how capsid flexibility may limit the resolution of cryo-EM. Overall, the present work provides functional insight beyond what is accessible to experimental methods and raises important considerations regarding asymmetry in structural studies of icosahedral virus capsids.

[Display omitted] • Cryo-EM of biocomplexes shows information on their local equilibrium dynamics. • Mechanical stiffening and impaired equilibrium dynamics of a virus particle are linked. The biological function of macromolecular complexes depends not only on large-scale transitions between conformations, but also on small-scale conformational fluctuations at equilibrium. Information on the equilibrium dynamics of biomolecular complexes could, in principle, be obtained from local resolution (LR) data in cryo-electron microscopy (cryo-EM) maps. However, this possibility had not been validated by comparing, for a same biomolecular complex, LR data with quantitative information on equilibrium dynamics obtained by an established solution technique. In this study we determined the cryo-EM structure of the minute virus of mice (MVM) capsid as a model biomolecular complex. The LR values obtained correlated with crystallographic B factors and with hydrogen/deuterium exchange (HDX) rates obtained by mass spectrometry (HDX-MS), a gold standard for determining equilibrium dynamics in solution. This result validated a LR-based cryo-EM approach to investigate, with high spatial resolution, the equilibrium dynamics of biomolecular complexes. As an application of this approach, we determined the cryo-EM structure of two mutant MVM capsids and compared their equilibrium dynamics with that of the wild-type MVM capsid. The results supported a previously suggested linkage between mechanical stiffening and impaired equilibrium dynamics of a virus particle. Cryo-EM is emerging as a powerful approach for simultaneously acquiring information on the atomic structure and local equilibrium dynamics of biomolecular complexes. [ABSTRACT FROM AUTHOR]

A diferencia de lo que ocurre con diversos virus icosahédricos, la estructura a alta resolución de la cápside de los virus flexuosos de plantas pertenecientes a la familia Potyviviridae no ha podido ser determinada aún. Los potyvirus son un grupo de gran importancia económica en la agricultura al afectar cultivos como papa, tomate, tabaco, papaya y caña de azúcar, entre muchos otros; por lo cual la comprensión de su estructura puede arrojar información valiosa para lograr un conocimiento más detallado de sus mecanismos biológicos, con miras al diseño de estrategias de control. En este trabajo se presenta un modelo de la estructura tridimensional de la región central de la proteína de la cápside del virus A de la papa (PVA), utilizando una combinación de herramientas de predicción de estructura secundaria y docking. El modelo presentado tiene dimensiones compatibles con la estructura de baja resolución obtenida en otros estudios mediante microscopía electrónica y será de gran utilidad en el diseño de experimentos de mutagénesis dirigida, enfocados en el estudio del ensamblaje de la partícula viral y como base para modelar la estructura de otras especies potyvirales de importancia actual en Colombia como el virus Y de la papa (PVY), virus de la malformación de las hojas del tomate de árbol (TaLMV) y el virus de la mancha anular de la papaya (PRSV).In contrast to icosahedric viruses, a high-resolution structure of flexuous viruses of plants belonging to the family Potyviridae has yet to be obtained. Potyviruses are economically important in agriculture due to their ability to infect crops such as potato, tomato, tobacco, papaya, and sugar cane, among many others; thus an understanding of its three-dimensional structure might provide valuable insight into biological mechanisms, with the aim of designing control strategies. In this study, we present a model for the three-dimensional structure of the core region of Potato virus A (PVA), using a combination tool to predict the secondary structure and docking. The model presented has dimensions compatible with the low resolution structure in other studies using electron microscopy and will be highly useful in the design of experiments of directed mutagenesis aimed at understanding potyvirus assembly and as a basis for modelling the structure of other important related virus species of importance in Colombia, such as Potato virus Y (PVY), Tamarillo leaf malformation virus (TaLMV), and Papaya ringspot virus (PRSV).

Charge detection mass spectrometry (CDMS) is a single-molecule technique particularly well-suited to measuring the mass and charge distributions of heterogeneous, MDa-sized ions. In this work, CDMS has been used to analyze the assembly products of two coat protein variants of bacteriophage P22. The assembly products show broad mass distributions extending from 5 to 15 MDa for A285Y and 5 to 25 MDa for A285T coat protein variants. Because the charge of large ions generated by electrospray ionization depends on their size, the charge can be used to distinguish hollow shells from more compact structures. A285T was found to form T = 4 and T = 7 procapsids, and A285Y makes a small number of T = 3 and T = 4 procapsids. Owing to the decreased stability of the A285Y and A285T particles, chemical cross-linking was required to stabilize them for electrospray CDMS. Graphical Abstract [ABSTRACT FROM AUTHOR]

Viruses can be conceptualized as self-replicating multiprotein assemblies, containing coding nucleic acids. Viruses have evolved to exploit host cellular components including enzymes to ensure their replicative life cycle. New findings indicate that also viral capsid proteins recruit host factors to accelerate their assembly. These assembly machines are RNA-containing multiprotein complexes whose composition is governed by allosteric sites. In the event of viral infection, the assembly machines are recruited to support the virus over the host and are modified to achieve that goal. Stress granules and processing bodies may represent collections of such assembly machines, readily visible by microscopy but biochemically labile and difficult to isolate by fractionation. We hypothesize that the assembly of protein multimers such as encountered in neurodegenerative or other protein conformational diseases, is also catalyzed by assembly machines. In the case of viral infection, the assembly machines have been modified by the virus to meet the virus’ need for rapid capsid assembly rather than host homeostasis. In the case of the neurodegenerative diseases, it is the monomers and/or low n oligomers of the so-called aggregated proteins that are substrates of assembly machines. Examples for substrates are amyloid β peptide (Aβ) and tau in Alzheimer's disease, α-synuclein in Parkinson's disease, prions in the prion diseases, Disrupted-in-schizophrenia 1 (DISC1) in subsets of chronic mental illnesses, and others. A likely continuum between virus capsid assembly and cell-to-cell transmissibility of aggregated proteins is remarkable. Protein aggregation diseases may represent dysfunction and dysregulation of these assembly machines analogous to the aberrations induced by viral infection in which cellular homeostasis is pathologically reprogrammed. In this view, as for viral infection, reset of assembly machines to normal homeostasis should be the goal of protein aggregation therapeutics. A key basis for the commonality between viral and neurodegenerative disease aggregation is a broader definition of assembly as more than just simple aggregation, particularly suited for the crowded cytoplasm. The assembly machines are collections of proteins that catalytically accelerate an assembly reaction that would occur spontaneously but too slowly to be relevant in vivo . Being an enzyme complex with a functional allosteric site, appropriated for a non-physiological purpose (e.g. viral infection or conformational disease), these assembly machines present a superior pharmacological target because inhibition of their active site will amplify an effect on their substrate reaction. Here, we present this hypothesis based on recent proof-of-principle studies against Aβ assembly relevant in Alzheimer's disease. [ABSTRACT FROM AUTHOR]

Norovirus infects different animals, including humans, mice, dogs, and cats. Here, we show an X-ray crystal structure of a feline GIV.2 norovirus capsid-protruding (P) domain to 2.35 Å resolution. The feline GIV.2 P domain was reminiscent of human norovirus P domains, except for a novel P2 subdomain α-helix and an extended P1 subdomain interface loop. These new structural features likely obstructed histo-blood group antigens, which are attachment factors for human norovirus, from binding at the equivalent sites on the feline GIV.2 P domain. Additionally, an ELISA showed that the feline GIV.2 was antigenically distinct from a human GII.10 norovirus. [ABSTRACT FROM AUTHOR]

Understanding how virus capsids assemble around their nucleic acid (NA) genomes could promote efforts to block viral propagation or to reengineer capsids for gene therapy applications. We develop a coarse-grained model of capsid proteins and NAs with which we investigate assembly dynamics and thermodynamics. In contrast to recent theoretical models, we find that capsids spontaneously ‘overcharge’; that is, the negative charge of the NA exceeds the positive charge on capsid. When applied to specific viruses, the optimal NA lengths closely correspond to the natural genome lengths. Calculations based on linear polyelectrolytes rather than base-paired NAs underpredict the optimal length, demonstrating the importance of NA structure to capsid assembly. These results suggest that electrostatics, excluded volume, and NA tertiary structure are sufficient to predict assembly thermodynamics and that the ability of viruses to selectively encapsidate their genomic NAs can be explained, at least in part, on a thermodynamic basis.

The output of icosahedral symmetry-adapted functions (ISAF) reconstruction algorithm is a virus capsid, where the density function of every outside point is theoretically zero. However, original ISAF algorithm takes reconstructed map as a solid sphere and density function of every point in the whole sphere is calculated. The density functions outside virus capsid may not be always zero, which may sometimes produce noise. In this study, Fourier shell correlation (FSC) is used for proving that density function calculation of original ISAF algorithm can reduce reconstruction precision. Subsequently, the range of virus capsid is determined with radial density function curve and the original ISAF algorithm is improved by calculating density function inside virus capsid. The experimental results on Cryo-electron microscopy (Cryo-EM) data of cytoplasmic polyhedrosis virus (CPV) indicate that the improved ISAF algorithm can generate more details of virus capsid and achieve a higher resolution. [ABSTRACT FROM AUTHOR]

Several challenges need to be addressed when developing viruses for clinical applications in gene therapy, vaccination, or viral oncolysis, including specific and efficient target cell transduction, virus delivery via the blood stream, and evasion of pre-existing immunity. With rising frequency, these goals are tackled by generating chimeric viruses containing nucleic acid fragments or proteins from two or more different viruses, thus combining different beneficial features of the parental viruses. These chimeras have boosted the development of virus-based treatment regimens for major inherited and acquired diseases, including cancer. Using adenoviruses as the paradigm and prominent examples from other virus families, we review the technological and functional advances in therapeutic virus chimera development and recent successful applications that can pave the way for future therapies. [ABSTRACT FROM AUTHOR]

A simple model for describing the non-quasi-equivalent icosahedral virus capsid composed of 72 pentameric capsomeres is developed. By means of six-step operations, a new 4-gons polyhedron P is obtained which contains 72 pentagons, 80 trigons and 210 quadrilaterals. More importantly, it bears icosahedral symmetry. The rationality of the existence of the 4-gons polyhedron P is further discussed. The results show that this model can be used to represent the capsids of papovaviruses. [ABSTRACT FROM AUTHOR]

Viral shells are self-assembled protein nanocontainers with remarkable material properties. They combine simplicity of construction with toughness and complex functionality. These properties make them interesting for bionanotechnology. To date we know little about how virus structure determines assembly pathways and shell mechanics. We have here used atomic force microscopy to study structural failure of the shells of the bacteriophage F29. We observed rigidity patterns following the symmetry of the capsid proteins. Under prolonged force exertion, we observed fracture along well-defined lines of the 2D crystal lattice. The mechanically most stable building block of the shells was a trimer. Our approach of "reverse engineering" the virus shells thus made it possible to identify stable structural intermediates. Such stable intermediates point to a hierarchy of interactions among equal building blocks correlated with distinct next-neighbor interactions. The results also demonstrate that concepts from macroscopic materials science, such as fracture, can be usefully employed in molecular engineering. [ABSTRACT FROM AUTHOR]

Chemically and/or genetically engineered viruses, viral capsids and viral-like particles carry the promise of important and diverse applications in biomedicine, biotechnology and nanotechnology. Potential uses include new vaccines, vectors for gene therapy and targeted drug delivery, contrast agents for molecular imaging and building blocks for the construction of nanostructured materials and electronic nanodevices. For many of the contemplated applications, the improvement of the physical stability of viral particles may be critical to adequately meet the demanding physicochemical conditions they may encounter during production, storage and/or medical or industrial use. The first part of this review attempts to provide an updated general overview of the fast-moving, interdisciplinary virus engineering field; the second part focuses specifically on the modification of the physical stability of viral particles by protein engineering, an emerging subject that has not been reviewed before. [ABSTRACT FROM AUTHOR]

Abstract: Protein–protein interactions play a crucial role in virus assembly and stability. With the view of disrupting capsid assembly and capturing smaller oligomers, interfacial residue mutations were carried out in the coat protein gene of Sesbania Mosaic Virus, a T =3 ss (+) RNA plant virus. A single point mutation of a Trp 170 present at the five-fold interface of the virus to a charged residue (Glu or Lys) arrested assembly of virus like particles and resulted in stable soluble dimers of the capsid protein. The X-ray crystal structure of one of the isolated dimer mutants — rCPΔN65W170K was determined to a resolution of 2.65 Å. Detailed analysis of the dimeric mutant protein structure revealed that a number of structural changes take place, especially in the loop and interfacial regions during the course of assembly. The isolated dimer was “more relaxed” than the dimer found in the T =3 or T =1 capsids. The isolated dimer does not bind Ca2+ ion and consequently four C-terminal residues are disordered. The FG loop, which interacts with RNA in the virus, has different conformations in the isolated dimer and the intact virus suggesting its flexible nature and the conformational changes that accompany assembly. The isolated dimer mutant was much less stable when compared to the assembled capsids, suggesting the importance of inter-subunit interactions and Ca2+ mediated interactions in the stability of the capsids. With this study, SeMV becomes the first icosahedral virus for which X-ray crystal structures of T =3, T =1 capsids as well as a smaller oligomer of the capsid protein have been determined. [Copyright &y& Elsevier]

Abstract: In this study canine parvovirus, CPV, was found to induce disorder in DPPC:cholesterol membranes in acidic conditions. This acidicity-induced fluidizing effect is suggested to originate from the N-terminus of the viral capsid protein VP1. In accordance with the model membrane studies, a fluidizing effect was seen also in the endosomal membranes during CPV infection implying an important functional role of the fluidization in the endocytic entry of the virus. [Copyright &y& Elsevier]

Abstract: Genetic variation in viral structural proteins is often explained by evolutionary escape of strong host defenses through processes such as immune evasion, host switching, and tissue tropism. An understanding of the mechanisms driving evolutionary change in virus surface proteins is key to designing effective intervention strategies to disease emergence. This study investigated the predictability of virus genomic evolution in response to highly specific differences in host receptor structure. The bacteriophage ΦX174 was evolved on three E. coli mutant hosts, each differing only by a single sugar group in the lipopolysaccharides, used for phage attachment. Large phage populations were used in order to maximize the amount of sequence space explored by mutation, and thus the potential for parallel evolution. Repeatability was assessed by genome sequencing of multiple isolates from endpoint populations and by fitness of the endpoint population relative to its ancestor. Evolutionary lines showed similar magnitudes of fitness increase between treatments. Only one mutation, occurring in the internal DNA pilot protein H, was completely repeatable, and it appeared to be a necessary stepping stone toward further adaptive change. Substitutions in the surface accessible major capsid protein F appeared to be involved in capsid stability rather than specific interactions with host receptors, suggesting that non-specific alterations to capsid structure could be an important component of adaptation to novel hosts. 33% of mutations were synonymous and showed evidence of selection on codon usage. Lastly, results supported previous findings that evolving populations of small ssDNA viruses may maintain relatively high levels of genetic variation. [Copyright &y& Elsevier]

Abstract: Initiation of infection by herpes simplex virus (HSV-1) involves a step in which the parental virus capsid docks at a nuclear pore and injects its DNA into the nucleus. Once “uncoated” in this way, the virus DNA can be transcribed and replicated. In an effort to clarify the mechanism of DNA injection, we examined DNA release as it occurs in purified capsids incubated in vitro. DNA ejection was observed following two different treatments, trypsin digestion of capsids in solution, and heating of capsids after attachment to a solid surface. In both cases, electron microscopic analysis revealed that DNA was ejected as a single double helix with ejection occurring at one vertex presumed to be the portal. In the case of trypsin-treated capsids, DNA release was found to correlate with cleavage of a small proportion of the portal protein, UL6, suggesting that UL6 cleavage may be involved in making the capsid permissive for DNA ejection. In capsids bound to a solid surface, DNA ejection was observed only when capsids were warmed above 4 °C. The proportion of capsids releasing their DNA increased as a function of incubation temperature with nearly all capsids ejecting their DNA when incubation was at 37 °C. The results demonstrate heterogeneity among HSV-1 capsids with respect to their sensitivity to heat-induced DNA ejection. Such heterogeneity may indicate a similar heterogeneity in the ease with which capsids are able to deliver DNA to the infected cell nucleus. [Copyright &y& Elsevier]

Abstract: Most double-stranded RNA viruses have a characteristic capsid consisting of 60 asymmetric coat protein dimers in a so-called T = 2 organization, a feature probably related to their unique life cycle. These capsids organize the replicative complex(es) that is actively involved in genome transcription and replication. Available structural data indicate that their RNA-dependent RNA polymerase (RDRP) is packaged as an integral capsid component, either as a replicative complex at the pentameric vertex (as in reovirus capsids) or as a fusion protein with the coat protein (as in some totivirus). In contrast with members of the family Reoviridae, there are two well-established capsid arrangements for dsRNA fungal viruses, exemplified by the totiviruses L-A and UmV and the chrysovirus PcV. Whereas L-A and UmV have a canonical T = 2 capsid, the PcV capsid is based on a T = 1 lattice composed of 60 capsid proteins. We used cryo-electron microscopy combined with three-dimensional reconstruction techniques and hydrodynamic analysis to determine the structure at 13.8 Å resolution of Helminthosporium victoriae 190S virus (Hv190SV), a totivirus isolated from a filamentous fungus. The Hv190SV capsid has a smooth surface and is based on a T = 2 lattice with 60 equivalent dimers. Unlike the RDRP of some other totiviruses, which are expressed as a capsid protein-RDRP fusion protein, the Hv190SV RDRP is incorporated into the capsid as a separate, nonfused protein, free or non-covalently associated to the capsid interior. [Copyright &y& Elsevier]

Abstract: The structure–function relationship is critical to understanding the biologically relevant functions of protein structures. Various experimental techniques and numerical modeling methods, normal mode analysis (NMA) in particular, have been employed to gain insight into this relationship. Experimental methods are often unable to provide all the desired information and comprehensive modeling techniques are often too computationally expensive. The authors build upon and optimize their cluster normal mode analysis (cNMA) tool, which uses embedded rigid-bodies and harmonic potentials to capture the biologically significant, low-frequency, oscillations of protein structures. cNMA represents atomic details with a scalable number of degrees-of-freedom, which can be chosen independent of structure size. This representation overcomes the otherwise quadratic order memory requirements and cubic order computational complexity associated with traditional all-atom NMA. cNMA is two orders of magnitude faster than traditional all-atom NMA when clustering by residue (very high resolution) and in the more traditional application using a fixed number of clusters, cNMA computationally scales as , which is two orders of complexity faster than all-atom NMA. cNMA is presented and very large example structures with up to atoms are analyzed on a notebook PC in the time scale of minutes/hours. The resulting mode shapes help identify biologically significant, conformational pathways. [Copyright &y& Elsevier]

Phenomena such as protein folding, crystallisation, self-assembly, and the observation of magic number clusters in molecular beams are all the result of non-random searches. Analysis of the underlying potential energy surface may provide a unifying framework to explain how such events occur as the result of a guided exploration of the landscape. In particular, icosahedral shells composed of 12 pentagonal pyramids are found to be thermodynamically favourable and kinetically accessible when the pyramids are not too spiky and not too flat. Hence, viruses with icosahedral capsids not only minimise the genetic material required to encode the repeated subunits, but may also utilise the favourable properties of a potential energy surface that effectively directs self-assembly. [ABSTRACT FROM AUTHOR]

To function adequately, many if not all proteins involved in macromolecular assemblies show conformational polymorphism as an intrinsic feature. This general strategy has been described for many essential cellular processes. Here we describe this structural polymorphism in a viral protein, the coat protein of Rabbit hemorrhagic disease virus (RHDV), which is required during virus capsid assembly. By combining genetic, structure modeling, and cryo-electron microscopy and image processing analysis, we have established the mechanism that allows RHDV coat protein to switch among quasi-equivalent conformational states to achieve the appropriate curvature for the formation of a closed shell.The RHDV capsid structure is based on a T = 3 lattice, containing 180 copies of identical subunits, similar to those of other caliciviruses. The quasi-equivalent interactions between the coat proteins are achieved by the N-terminal region of a subset of subunits, which faces the inner surface of the capsid shell. Mutant coat protein lacking this N-terminal sequence assembles into T = 1 capsids. Our results suggest that the polymorphism of the RHDV T = 3 capsid might bear resemblance to that of plant virus T = 3 capsids. [Copyright &y& Elsevier]

Although double-stranded (ds) RNA viruses are a rather diverse group, they share general architectural principles and numerous functional features. All dsRNA viruses, from the mammalian reoviruses to the bacteriophage φ6, including fungal viruses, share a specialized capsid involved in transcription and replication of the dsRNA genome, and release of the viral plus strand RNA. This ubiquitous capsid consists of 120 protein subunits in a so-called T=2 organization. The stringent requirements of dsRNA metabolism may explain the similarities observed in capsid architecture among a broad spectrum of dsRNA viruses. We have used cryo-electron microscopy combined with three-dimensional reconstruction techniques and complementary biophysical techniques, to determine the structure at 26 A˚ resolution of the Penicillium chrysogenum virus (PcV) capsid. In contrast to all previous studies of dsRNA viruses, PcV capsid is an authentic T=1 capsid with 60 equivalent protein subunits. This T=1 capsid is built with the largest structural protein (110 kDa). Structural comparison between viral particles and capsids devoid of RNA show changes along the inner surface of the capsid, mostly located around the icosahedral 5 and 3-fold axis. Considering that there may be numerous interactions between the inner surface of the protein shell and the underlying RNA, the genome could have an important role in the conformation of the structural subunits. The empty capsid structure suggests a mechanism for transcript release from actively transcribing particles. Furthermore, sequence analysis of the PcV coat protein revealed that both halves of the protein share numerous regions of similar amino acid residues. These results open new perspectives when considering the structural organization of dsRNA virus capsids. [Copyright &y& Elsevier]

The structure of the capsid of bacteriophage HK97 has been solved at various stages of maturity by crystallography and cryo-electron microscopy, and has been reported previously in the literature. Typically the capsid assembles through polymerization and maturation processes. Maturation is composed of proteolytic cleavages to the precursor capsid (called Prohead II), expansion triggered by DNA packaging (in which the largest conformational changes of the capsid appear), and covalent cross-links of neighboring subunits to create the mature capsid called Head II. We apply a coarse-grained elastic network interpolation (ENI) to generate a feasible pathway for conformational change from Prohead II to Head II. The icosahedral symmetry of the capsid structure offers a significant computational advantage because it is not necessary to consider the whole capsid structure but only an asymmetric unit consisting of one hexamer plus an additional subunit from an adjacent pentamer. We also analyze normal modes of the capsid structure using an elastic network model which is also subject to symmetry constraints. Using our model, we can visualize the smooth evolution of capsid expansion and revisit in more detail several interesting geometric changes recognized in early experimental works such as rigid body motion of two compact domains (A and P) with two refolding extensions (N-arm and E-loop) and track the approach of the two particular residues associated with isopeptide bonds that make hexagonal cross-links in Head II. The feasibility of the predicted pathway is also supported by the results of our normal mode analysis. [Copyright &y& Elsevier]

Supramolecular protein complexes are the corner stone of biological processes; they are essential for many biological functions. Unraveling the interactions responsible for the (dis)assembly of these complexes is required to understand nature and to exploit such systems in future applications. Virus capsids are well-defined assemblies of hundreds of proteins and form the outer shell of non-enveloped viruses. Due to their potential as a drug carriers or nano-reactors and the need for virus inactivation strategies, assessing the intactness of virus capsids is of great interest. Current methods to evaluate the (dis)assembly of these protein assemblies are experimentally demanding in terms of instrumentation, expertise and time. Here we investigate a new strategy to monitor the disassembly of fluorescently labeled virus capsids. To monitor surfactant-induced capsid disassembly, we exploit the complex photophysical interplay between multiple fluorophores conjugated to capsid proteins. The disassembly of the capsid changes the photophysical interactions between the fluorophores, and this can be spectrally monitored. The presented data show that this low complexity method can be used to study and monitor the disassembly of supramolecular protein complexes like virus capsids. However, the range of labeling densities that is suitable for this assay is surprisingly narrow. [ABSTRACT FROM AUTHOR]

The hepatitis B virus (HBV) capsid is an attractive drug target, relevant to combating viral hepatitis as a major public health concern. Among small molecules known to interfere with capsid assembly, the phenylpropenamides, including AT130, represent an important antiviral paradigm based on disrupting the timing of genome packaging. Here, all-atom molecular dynamics simulations of an intact AT130-bound HBV capsid reveal that the compound increases spike flexibility and improves recovery of helical secondary structure in the spike tips. Regions of the capsid-incorporated dimer that undergo correlated motion correspond to established sub-domains that pivot around the central chassis. AT130 alters patterns of correlated motion and other essential dynamics. A new conformational state of the dimer is identified, which can lead to dramatic opening of the intradimer interface and disruption of communication within the spike tip. A novel salt bridge is also discovered, which can mediate contact between the spike tip and fulcrum even in closed conformations, revealing a mechanism of direct communication across these sub-domains. Altogether, results describe a dynamical connection between the intra- and interdimer interfaces and enable mapping of allostery traversing the entire core protein dimer. [ABSTRACT FROM AUTHOR]

The assembly of alphavirus nucleocapsid cores requires electrostatic interactions between the positively charged N-terminus of the capsid protein (CP) and the encapsidated polyanionic cargo. This system differs from many other viruses that can self-assemble particles in the absence of cargo, or form "empty" particles. We hypothesized that the introduction of a mutant, anionic CP could replace the need for charged cargo during assembly. In this work, we produced a CP mutant, Minus 38 (M38), where all N-terminal charged residues are negatively-charged. When wild-type (WT) and M38 CPs were mixed, they assembled into core-like particles (CLPs). These "empty" particles were of similar size and morphology to WT CLPs assembled with DNA cargo, but did not contain nucleic acid. When DNA cargo was added to the assembly mixture, the amount of M38 CP that was assembled into CLPs decreased, but was not fully excluded from the CLPs, suggesting that M38 competes with DNA to interact with WT CPs. The composition of CLPs can be tuned by altering the order of addition of M38 CP, WT CP, and DNA cargo. The ability to produce alphavirus CLPs that contain a range of amounts of encapsidated cargo, including none, introduces a new platform for packaging cargo for delivery or imaging purposes. [ABSTRACT FROM AUTHOR]