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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 genus Ebolavirus from the family Filoviridae is composed of five species including Sudan ebolavirus, Reston ebolavirus, Bundibugyo ebolavirus, Taï Forest ebolavirus, and Ebola virus (previously known as Zaire ebolavirus). These viruses have a large non-segmented, negative-strand RNA of approximately 19 kb that encodes for glycoproteins (i.e., GP, sGP, ssGP), nucleoproteins, virion proteins (i.e., VP 24, 30,40) and an RNA dependent RNA polymerase. These viruses have become a global health concern because of mortality, their rapid dissemination, new outbreaks in West-Africa, and the emergence of a new condition known as “Post-Ebola virus disease syndrome” that resembles inflammatory and autoimmune conditions such as rheumatoid arthritis, systemic lupus erythematosus and spondyloarthritis with uveitis. However, there are many gaps in the understanding of the mechanisms that may induce the development of such autoimmune-like syndromes. Some of these mechanisms may include a high formation of neutrophil extracellular traps, an uncontrolled “cytokine storm”, and the possible formation of auto-antibodies. The likely appearance of autoimmune phenomena in Ebola survivors suppose a new challenge in the management and control of this disease and opens a new field of research in a special subgroup of patients. Herein, the molecular biology, pathogenesis, clinical manifestations, and treatment of Ebola virus disease are reviewed and some strategies for control of disease are discussed. © 2019 The Authors

Arboviral diseases caused by dengue (DENV), Zika (ZIKV) and chikungunya (CHIKV) viruses represent a major public health problem worldwide, especially in tropical areas where millions of infections occur every year. The aim of this research was to identify candidate molecules for the treatment of these diseases among the drugs currently available in the market, through in silico screening and subsequent in vitro evaluation with cell culture models of DENV and ZIKV infections. Numerous pharmaceutical compounds from antibiotics to chemotherapeutic agents presented high in silico binding affinity for the viral proteins, including ergotamine, antrafenine, natamycin, pranlukast, nilotinib, itraconazole, conivaptan and novobiocin. These five last compounds were tested in vitro, being pranlukast the one that exhibited the best antiviral activity. Further in vitro assays for this compound showed a significant inhibitory effect on DENV and ZIKV infection of human monocytic cells and human hepatocytes (Huh-7 cells) with potential abrogation of virus entry. Finally, intrinsic fluorescence analyses suggest that pranlukast may have some level of interaction with three viral proteins of DENV: envelope, capsid, and NS1. Due to its promising results, suitable accessibility in the market and reduced restrictions compared to other pharmaceuticals; the anti-asthmatic pranlukast is proposed as a drug candidate against DENV, ZIKV, and CHIKV, supporting further in vitro and in vivo assessment of the potential of this and other lead compounds that exhibited good affinity scores in silico as therapeutic agents or scaffolds for the development of new drugs against arboviral diseases. © 2019 Elsevier B.V. Universidad Tecnológica de Pereira, UTP: TRFCI-1P2016 National Institutes of Health, NIH National Institutes of Health, NIH: R01 AI24493 Department of Science, Information Technology and Innovation, Queensland Government, DSITI: 811-2018 Universidad Autónoma de Bucaramanga, UNAB The authors wish to thank the Administrative Department of Science, Technology and Innovation of Colombia [Grant: Colciencias No. 811-2018 ], Universidad Nacional Autónoma de México [Grant: Programa de Becas Posdoctorales en la UNAM 2016 ], Universidad Tecnológica de Bolívar [Grant: TRFCI-1P2016 ] and the National Institutes of Health [NIH grant R01 AI24493 ] for their financial support. Appendix A A continuación se relacionan los compuestos químicos y su número de registro CAS (Chemical Abstracts Service) antrafenine, 55300-29-3; conivaptan, 168626-94-6, 210101-16-9; ergotamine, 113-15-5, 52949-35-6; itraconazole, 84625-61-6; natamycin, 52882-37-8, 7681-93-8; nilotinib, 641571-10-0; novobiocin, 1476-53-5, 303-81-1, 39301-00-3, 4309-70-0; pranlukast, 103177-37-3

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.

Hollow protein containers are widespread in nature, and include virus capsids as well as eukaryotic and bacterial complexes. Protein cages are studied extensively for applications in nanotechnology, nanomedicine and materials science. Their inner and outer surfaces can be modified chemically or genetically, and the internal cavity can be used to template, store and/or arrange molecular cargos. Virus capsids and virus-like particles (VLP, noninfectious particles) provide versatile platforms for nanoscale bioengineering. Study of capsid protein self-assembly into monodispersed particles, and of VLP structure and biophysics is necessary not only to understand natural processes, but also to infer how these platforms can be redesigned to furnish novel functional VLP. Here we address the assembly dynamics of infectious bursal disease virus (IBDV), a complex icosahedral virus. IBDV has a ~70 nm-diameter T = 13 capsid with VP2 trimers as the only structural subunits. During capsid assembly, VP2 is synthesized as a precursor (pVP2) whose C terminus is cleaved. The pVP2 C terminus has an amphipathic helix that controls VP2 polymorphism. In the absence of the VP3 scaffolding protein, necessary for control of assembly, 466/456-residue pVP2 intermediates bearing this helix assemble into VLP only when expressed with an N-terminal His6 tag (the HT-VP2-466 protein). HT-VP2-466 capsids are optimal for genetic insertion of proteins (cargo space ~78 000 nm3). We established an in vitro assembly/disassembly system of HT-VP2-466-based VLP for heterologous nucleic acid packaging and/or encapsulation of drugs and other molecules. HT-VP2-466 (empty) capsids were disassembled and reassembled by dialysis against low-salt/basic pH and high-salt/acid pH buffers, respectively, thus illustrating the reversibility in vitro of IBDV capsid assembly. HT-VP2-466 VLP also packed heterologous DNA by non-specific confinement during assembly. These and previous results establish the bases for biotechnological applications based on the IBDV capsid and its ability to incorporate exogenous proteins and nucleic acids.

The ability of adenoviruses to infect a broad range of species and tissues has led to a widespread interest in their biological functioning. However, there remains a big gap in our understanding of their assembly and maturation pathways. Here, we present AFM (Atomic Force Microscopy) nanoindentation and fatigue studies1,2 of adenovirus capsids3 at different stages of maturation. Surprisingly, we find that the intermediate (no DNA) immature capsid is mechanically indistinguishable as compared with the mature (DNA filled), suggesting a major stabilizing role of the scaffold protein.3 However, these capsids have distinctly different disassembly pathways, as indicated by a mechanically-induced fatigue analysis. Additionally, we observed that mutation of the protease cleavage site of the precursor protein VI yields a maturation-intermediate capsid, G33A, which has reduced infectivity and releases half as many pentons as the WT capsid. The presented results strongly indicate that the reduced infectivity results from a reduction in protein VI exposure, partially inhibiting lysis of the endosome and leading to abortive infection.

The ability of adenoviruses to infect a broad range of species and tissues has led to a widespread interest in their biological functioning. However, there remains a big gap in our understanding of their assembly and maturation pathways. Here, we present AFM (Atomic Force Microscopy) nanoindentation and fatigue studies1,2 of adenovirus capsids3 at different stages of maturation. Surprisingly, we find that the intermediate (no DNA) immature capsid is mechanically indistinguishable as compared with the mature (DNA filled), suggesting a major stabilizing role of the scaffold protein.3 However, these capsids have distinctly different disassembly pathways, as indicated by a mechanically-induced fatigue analysis. Additionally, we observed that mutation of the protease cleavage site of the precursor protein VI yields a maturation-intermediate capsid, G33A, which has reduced infectivity and releases half as many pentons as the WT capsid. The presented results strongly indicate that the reduced infectivity results from a reduction in protein VI exposure, partially inhibiting lysis of the endosome and leading to abortive infection.

Upon reactivation from latency and during lytic infections in neurons, alphaherpesviruses assemble cytosolic capsids, capsids associated with enveloping membranes, and transport vesicles harboring fully enveloped capsids. It is debated whether capsid envelopment of herpes simplex virus (HSV) is completed in the soma prior to axonal targeting or later, and whether the mechanisms are the same in neurons derived from embryos or from adult hosts. We used HSV mutants impaired in capsid envelopment to test whether the inner tegument proteins pUL36 or pUL37 necessary for microtubule-mediated capsid transport were sufficient for axonal capsid targeting in neurons derived from the dorsal root ganglia of adult mice. Such neurons were infected with HSV1-ΔUL20 whose capsids recruited pUL36 and pUL37, with HSV1-ΔUL37 whose capsids associate only with pUL36, or with HSV1-ΔUL36 that assembles capsids lacking both proteins. While capsids of HSV1-ΔUL20 were actively transported along microtubules in epithelial cells and in the somata of neurons, those of HSV1-ΔUL36 and -ΔUL37 could only diffuse in the cytoplasm. Employing a novel image analysis algorithm to quantify capsid targeting to axons, we show that only a few capsids of HSV1-ΔUL20 entered axons, while vesicles transporting gD utilized axonal transport efficiently and independently of pUL36, pUL37, or pUL20. Our data indicate that capsid motility in the somata of neurons mediated by pUL36 and pUL37 does not suffice for targeting capsids to axons, and suggest that capsid envelopment needs to be completed in the soma prior to targeting of herpes simplex virus to the axons, and to spreading from neurons to neighboring cells., Author summary Human and animal alphaherpesviruses establish lifelong latent infections in neurons of the peripheral nervous system and cause many diseases upon primary infection as well as following reactivation from latency. The highly prevalent human herpes simplex viruses HSV-1 and HSV-2 are responsible for facial and genital herpes, potentially blinding eye infections, and life-threatening encephalitis and meningitis. Here, we asked how these viruses master the bottleneck of being targeted from the neuronal somata to the axons. Our data suggest that only transport vesicles harboring fully matured virus particles can enter axons for spreading infection to the brain or the peripheral organs. Our data imply that the limiting membrane of the transport vesicles must expose viral or host receptors to recruit the microtubule motors required for axonal transport. Inhibiting such viral factors on the surface of the transport vesicles might provide novel therapeutic approaches to prevent the spread of alphaherpesviruses in the nervous system.

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 - rCP Delta N65W170K was determined to a resolution of 2.65 angstrom. 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 chiller 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 choler 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.

The main functions of viral capsids are to protect, transport and deliver their genome. The mechanical properties of capsids are supposed to be adapted to these tasks. Bacteriophage capsids also need to withstand the high pressures the DNA is exerting onto it as a result of the DNA packaging and its consequent confinement within the capsid. It is proposed that this pressure helps driving the genome into the host, but other mechanisms also seem to play an important role in ejection. DNA packaging and ejection strategies are obviously dependent on the mechanical properties of the capsid. This review focuses on the mechanical properties of viral capsids in general and the elucidation of the biophysical aspects of genome packaging mechanisms and genome delivery processes of double-stranded DNA bacteriophages in particular. © 2007 Birkhäuser Verlag.

The simian vacuolating virus (SV40) can efficiently infect a wide range of human cells, which makes this capsid of interest as a capsule for drug delivery. The icosahedral virus capsid is assembled from VP1 pentamers, and this assembly is known to be facilitated by the presence of dsDNA. However, real-time assembly of SV40, and viruses in general, is poorly understood limiting among other things our ability to manipulate capsid re-assembly in vitro for therapeutic purposes. Here, we show real time assembly of VP1 pentamers on dsDNA employing dual-trap optical tweezers (OT) and a new technology, acoustic force spectroscopy (AFS). Real-time shortening of the DNA and modulated stretching curves suggests the assembly of (intermediate) capsid-like structures. Unbinding can be studied by applying relatively low forces to the DNA over a longer time scale. To obtain visual information, atomic force microscopy (AFM) images were captured showing significant cluster formation on DNA supporting the idea that we observe partial assembly. These results reveal that we can indeed track the assembly of viral capsids and visualize intermediate structures. This in turn provides a window into the complex kinetics of viral capsid formation.

The simian vacuolating virus (SV40) can efficiently infect a wide range of human cells, which makes this capsid of interest as a capsule for drug delivery. The icosahedral virus capsid is assembled from VP1 pentamers, and this assembly is known to be facilitated by the presence of dsDNA. However, real-time assembly of SV40, and viruses in general, is poorly understood limiting among other things our ability to manipulate capsid re-assembly in vitro for therapeutic purposes. Here, we show real time assembly of VP1 pentamers on dsDNA employing dual-trap optical tweezers (OT) and a new technology, acoustic force spectroscopy (AFS). Real-time shortening of the DNA and modulated stretching curves suggests the assembly of (intermediate) capsid-like structures. Unbinding can be studied by applying relatively low forces to the DNA over a longer time scale. To obtain visual information, atomic force microscopy (AFM) images were captured showing significant cluster formation on DNA supporting the idea that we observe partial assembly. These results reveal that we can indeed track the assembly of viral capsids and visualize intermediate structures. This in turn provides a window into the complex kinetics of viral capsid formation.

Magnetic nanoparticles have multiple applications in materials science. In particular, virus capsids have been suggested as promising templates for building up nanometric-sized magnetic clusters by taking advantage of their inner cavity as a nanoreactor. In this study we investigate the magnetization of individual cobalt-filled cowpea mosaic virus empty virus-like particles using atomic force microscopy. We also combine the analysis of the effects of dehydration on the structure of virus particles with a comparison of their magnetic signal to that provided by commercially available magnetic nanoparticles of similar size. These two approaches allow the evaluation of the structure of the metallic cluster grown inside the virus capsid. We conclude that, rather than forming solid clusters, cobalt inside viruses forms a discontinuous structure that does not completely fill the virus cavity and reaches about 10% of its volume., The authors acknowledge the MINECO of Spain (PIB2010US-00233, FIS3011-29493, CTQ2012-35513-C02 Consolider CSD2010-00024, CAM project No. S3009/MAT-1467) (P.J.P., M.J., I.B. and R.M.) This work was supported by BB/J004561/1 from the UK Biotechnology and Biological Sciences Research Council (BBSRC) and the John Innes Foundation (to D.J.E. and G.P.L.) and a BBSRC DTG (to A.A.A.A. and P.S.).

Local rules are given for various virus capsid, or protein shell, geometries. Local rules specify local interaction patterns that can direct the self-assembly of coat and scaffolding subunits into different capsid geometries. They provide a mathematical abstraction allowing combinatorial analysis of possible assembly pathways. Local rules and simulations are provided for a geometry called “ T =7” such that a small change in the rules produces an alternate “ T =4” geometry. This is intriguing since evidence has been accumulating that some T =7 capsids are closely related to T =4 capsids.

Addition of affinity tags to bacteriophage particles facilitates a variety of applications, including vaccine construction and diagnosis of bacterial infections. Addition of tags to phage capsids is desirable, as modification of the tails can lead to poor adsorption and loss of infectivity. Although tags can readily be included as fusions to head decoration proteins, many phages do not have decoration proteins as virion components. The addition of a small (10-amino-acid) Strep-tag II (STAG II) to the mycobacteriophage TM4 capsid subunit, gp9, was not tolerated as a genetically homogenous recombinant phage but could be incorporated into the head by growth of wild-type phage on a host expressing the capsid-STAG fusion. Particles with capsids composed of wild-type and STAG-tagged subunit mixtures could be grown to high titers, showed good infectivities, and could be used to isolate phage-bacterium complexes. Preparation of a STAG-labeled fluoromycobacteriophage enabled capture of bacterial complexes and identification of infected bacteria by fluorescence. Fil: Piuri, Mariana. University of Pittsburgh; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Rondon Salazar, Liliana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Urdániz, Estefanía. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Hatfull, Graham F.. University of Pittsburgh; Estados Unidos

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. DOI: http://dx.doi.org/10.7554/eLife.00632.001, eLife digest Viruses are infectious agents made up of proteins and a genome made of DNA or RNA. Upon infecting a host cell, viruses hijack the cell’s gene expression machinery and force it to produce copies of the viral genome and proteins, which then assemble into new viruses that can eventually infect other host cells. Because assembly is an essential step in the viral life cycle, understanding how this process occurs could significantly advance the fight against viral diseases. In many viral families, a protein shell called a capsid forms around the viral genome during the assembly process. However, capsids can also assemble around nucleic acids in solution, indicating that a host cell is not required for their formation. Since capsid proteins are positively charged, and nucleic acids are negatively charged, electrostatic interactions between the two are thought to have an important role in capsid assembly. However, it is unclear how structural features of the viral genome affect assembly, and why the negative charge on viral genomes is actually far greater than the positive charge on capsids. These questions are difficult to address experimentally because most of the intermediates that form during virus assembly are too short-lived to be imaged. Here, Perlmutter et al. have used state of the art computational methods and advances in graphical processing units (GPUs) to produce the most realistic model of capsid assembly to date. They showed that the stability of the complex formed between the nucleic acid and the capsid depends on the length of the viral genome. Yield was highest for genomes within a certain range of lengths, and capsids that assembled around longer or shorter genomes tended to be malformed. Perlmutter et al. also explored how structural features of the virus—including base-pairing between viral nucleic acids, and the size and charge of the capsid—determine the optimal length of the viral genome. When they included structural data from real viruses in their simulations and predicted the optimal lengths for the viral genome, the results were very similar to those seen in existing viruses. This indicates that the structure of the viral genome has been optimized to promote packaging into capsids. Understanding this relationship between structure and packaging will make it easier to develop antiviral agents that thwart or misdirect virus assembly, and could aid the redesign of viruses for use in gene therapy and drug delivery. DOI: http://dx.doi.org/10.7554/eLife.00632.002

Bacteriophage T5 is a virus infecting E. Coli. Its capsid assembly and maturation include severalcritical steps leading to the formation of new virions during the infectious cycle.During my thesis I focused on the expansion and decoration of T5 capsid. Expansion consists inlarge conformationnal reorganizations of the 775 capsid protein subunits, yielding a two-foldincrease of the capsid volume and allowing it to accomodate the full-length genome. I haveascertained physicochemical conditions that trigger in vitro expansion of the capsid, and using atime-resolved SAXS study, I showed that expansion is a higly cooperative two-state process leadingto a remarkably stable final state. I have also carried out a functional study of the decoration proteinpb10, showing that it only binds to expanded proheads. Finally, a low resolution model of pb10 wasdetermined by SAXS.; Le bactériophage T5 est un virus infectant E. Coli. L’assemblage et la maturation de sa capsidecomportent plusieurs étapes critiques pour la formation des virions lors du cycle infectieux.Parmi ces étapes, j’ai étudié les processus d’expansion et de décoration de la capside de T5.L’expansion implique d’importantes réorganisations conformationnelles des 775 sous-unités de laprotéine pb8 composant la capside, conduisant au doublement du volume de la capside qui peutalors contenir le génome du phage. J’ai déterminé des conditions physico-chimiques permettantd’induire l’expansion de la capside in vitro, puis j’ai effectué des expériences de SAXS résoluesdans le temps montrant que l’expansion est un processus hautement coopératif, qui conduit, en uneétape, à un état final remarquablement stable. D’autre part, j’ai réalisé une étude fonctionnelle de laprotéine de décoration pb10, montrant que sa fixation est un marqueur de l’expansion. Enfin l'étudestructurale de pb10 menée par SAXS a permis de déterminer un modèle à basse résolution de sonenveloppe moléculaire.

Capítulo 4 en la obra, Mateu, Mauricio G. (ed.). Structure and Physics of Viruses: An Integrated Textbook. [S.l.]: Springer, 2013, p.117-144. (Subcellular Biochemistry ; 68), For about 30 years X-ray crystallography has been by far the most powerful approach for determining virus structures at close to atomic resolutions. Information provided by these studies has deeply and extensively enriched and shaped our vision of the virus world. In turn, the ever increasing complexity and size of the virus structures being investigated have constituted a major driving force for methodological and conceptual developments in X-ray macromolecular crystallography. Landmarks of new virus structures determinations, such as the ones from the first animal viruses or from the first membrane-containing viruses, have often been associated to methodological breakthroughs in X-ray crystallography. In this chapter we present the common ground of proteins and virus crystallography with an emphasis in the peculiarities of virus studies. For example, the solution of the phase problem, a central issue in X-ray diffraction, has benefited enormously from the presence of non-crystallographic symmetry in virus crystals., This work was supported by grants from the Ministerio de Economia y Competitividad to N.V. (BIO2011-24333) and to I.F. (BFU2009-09268).

Capsid maturation with large-scale subunit reorganization occurs in virtually all viruses that use a motor to package nucleic acid into preformed particles. A variety of ensemble studies indicate that the particles gain greater stability during this process, however, it is unknown which material properties of the fragile procapsids change. Using Atomic Force Microscopy-based nano-indentation, we study the development of the mechanical properties during maturation of bacteriophage HK97, a λ -like phage of which the maturation-induced morphological changes are well described. We show that mechanical stabilization and strengthening occurs in three independent ways: ( i ) an increase of the Young’s modulus, ( ii ) a strong rise of the capsid’s ultimate strength, and ( iii ) a growth of the resistance against material fatigue. The Young’s modulus of immature and mature capsids, as determined from thin shell theory, fit with the values calculated using a new multiscale simulation approach. This multiscale calculation shows that the increase in Young’s modulus isn’t dependent on the crosslinking between capsomers. In contrast , the ultimate strength of the capsids does increase even when a limited number of cross-links are formed while full crosslinking appears to protect the shell against material fatigue. Compared to phage λ , the covalent crosslinking at the icosahedral and quasi threefold axes of HK97 yields a mechanically more robust particle than the addition of the gpD protein during maturation of phage λ . These results corroborate the expected increase in capsid stability and strength during maturation, however in an unexpected intricate way, underlining the complex structure of these self-assembling nanocontainers.

Human immunodeficiency virus type 1 (HIV-1) Gag selects for and mediates genomic RNA (vRNA) encapsidation into progeny virus particles. The host protein, Staufen1 interacts directly with Gag and is found in ribonucleoprotein (RNP) complexes containing vRNA, which provides evidence that Staufen1 plays a role in vRNA selection and encapsidation. In this work, we show that Staufen1, vRNA and Gag are found in the same RNP complex. These cellular and viral factors also colocalize in cells and constitute novel Staufen1 RNPs (SHRNPs) whose assembly is strictly dependent on HIV-1 expression. SHRNPs are distinct from stress granules and processing bodies, are preferentially formed during oxidative stress and are found to be in equilibrium with translating polysomes. Moreover, SHRNPs are stable, and the association between Staufen1 and vRNA was found to be evident in these and other types of RNPs. We demonstrate that following Staufen1 depletion, apparent supraphysiologic-sized SHRNP foci are formed in the cytoplasm and in which Gag, vRNA and the residual Staufen1 accumulate. The depletion of Staufen1 resulted in reduced Gag levels and deregulated the assembly of newly synthesized virions, which were found to contain several-fold increases in vRNA, Staufen1 and other cellular proteins. This work provides new evidence that Staufen1-containing HIV-1 RNPs preferentially form over other cellular silencing foci and are involved in assembly, localization and encapsidation of vRNA. Fil: Abrahamyan, Levon G.. Davis Jewish General Hospital; Canadá Fil: Chatel Chaix, Laurent. Davis Jewish General Hospital; Canadá Fil: Ajamian, Lara. Mc Gill University; Canadá. Davis Jewish General Hospital; Canadá Fil: Milev, Miroslav P.. Mc Gill University; Canadá. Davis Jewish General Hospital; Canadá Fil: Monette, Anne. Mc Gill University; Canadá. Davis Jewish General Hospital; Canadá Fil: Clément, Jean François. Davis Jewish General Hospital; Canadá Fil: Song, Rujun. Mc Gill University; Canadá. Davis Jewish General Hospital; Canadá Fil: Lehmann, Martin. Davis Jewish General Hospital; Canadá Fil: DesGroseillers, Luc. University Of Montreal; Canadá Fil: Laughrea, Michael. Mc Gill University; Canadá. Davis Jewish General Hospital; Canadá Fil: Boccaccio, Graciela Lidia. Fundación Instituto Leloir; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina Fil: Mouland, Andrew J.. Mc Gill University; Canadá. Davis Jewish General Hospital; Canadá

This dissertation presents the synthesis and modeling of multicomponent nanosystems templated by self-assembled virus capsids. The design principles, synthesis, analysis, and future directions for these capsid-based materials are presented.Chapter 1 gives an overview of the literature on the application of virus capsids in constructing nanomaterials. The uses of capsids in three main areas are considered: 1) as templates for inorganic materials or nanoparticles; 2) as vehicles for biological applications like medical imaging and treatment; and 3) as scaffolds for catalytic materials. In light of this introduction, an overview of the material in this dissertation is described.Chapters 2-4 all describe integrated nanosystems templated by bacteriophage MS2, a spherical icosahedral virus capsid. MS2 possesses an interior and exterior surface that can be modified orthogonally using bioconjugation chemistry to create multivalent, multicomponent constructs with precise localization of components attached to the capsid proteins.Chapter 2 describes the use of MS2 to synthesize a photocatalytic construct by modifying the internal surface with sensitizing chromophores and the external surface with a photocatalytic porphyrin. The chromophores absorbed energy that the porphyrin could not, and transferred it to the porphyrin via FRET through the protein shell. The porphyrin was then able to utilize the energy to carry out photocatalysis at new wavelengths.In Chapter 3, porphyrins were installed on the interior surface of MS2 and DNA aptamers specific for Jurkat leukemia T cells on the exterior surface. The dual-modified capsids were able to bind to Jurkat cells, and upon illumination the porphyrins generated singlet oxygen to kill them selectively over non-targeted cells.Chapter 4 explores integrating MS2 with DNA origami in order to arrange the capsids at larger length scales. Capsids modified with fluorescent dyes inside and single-stranded DNA outside were able to bind to origami tiles bearing complementary DNA probes. The tiles could then be used to arrange the capsids in a one-dimensional array with dimensions far exceeding those of individual MS2 particles.In Chapter 5, the use of a different capsid, that of the tobacco mosaic virus (TMV) is described. The defect tolerance of light harvesting systems built using TMV as a scaffold was investigated using a kinetic Monte Carlo model to simulate the energy transfer processes. The results of the simulation were used to understand and explain experimental results obtained from the system.

The main functions of viral capsids are to protect, transport and deliver their genome. The mechanical properties of capsids are supposed to be adapted to these tasks. Bacteriophage capsids also need to withstand the high pressures the DNA is exerting onto it as a result of the DNA packaging and its consequent confinement within the capsid. It is proposed that this pressure helps driving the genome into the host, but other mechanisms also seem to play an important role in ejection. DNA packaging and ejection strategies are obviously dependent on the mechanical properties of the capsid. This review focuses on the mechanical properties of viral capsids in general and the elucidation of the biophysical aspects of genome packaging mechanisms and genome delivery processes of double-stranded DNA bacteriophages in particular. © 2007 Birkhäuser Verlag.

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. © 2004 Elsevier Inc. All rights reserved., This work was supported by Grants 07B/0041/2002 from the Subdirección General de Investigación of the Comunidad Autónoma de Madrid, BMC2002-00996, BMC2000-0555, BIO00-0578-C02-02, and BIO2002-04091-C03-03 from the MCYT, and BIO2002-00517 of CICYT. JRC and JB hold a contract from the “Ramón y Cajal” program (MCYT)