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Out of the three core proteins in human adenovirus, protein V is believed to connect the inner capsid surface to the outer genome layer. Here, we explored mechanical properties and in vitro disassembly of particles lacking protein V (Ad5-ΔV). Ad5-ΔV particles were softer and less brittle than the wild-type ones (Ad5-wt), but they were more prone to release pentons under mechanical fatigue. In Ad5-ΔV, core components did not readily diffuse out of partially disrupted capsids, and the core appeared more condensed than in Ad5-wt. These observations suggest that instead of condensing the genome, protein V antagonizes the condensing action of the other core proteins. Protein V provides mechanical reinforcement and facilitates genome release by keeping DNA connected to capsid fragments that detach during disruption. This scenario is in line with the location of protein V in the virion and its role in Ad5 cell entry.

Vaults are ubiquitous ribonucleoprotein particles involved in a diversity of cellular processes, with promising applications as nanodevices for delivery of multiple cargos. The vault shell is assembled by the symmetrical association of multiple copies of the major vault protein that, initially, generates half vaults. The pairwise, anti-parallel association of two half vaults produces whole vaults. Here, using a combination of vault recombinant reconstitution and structural techniques, we characterized the molecular determinants for the vault opening process. This process commences with a relaxation of the vault waist, causing the expansion of the inner cavity. Then, local disengagement of amino-terminal domains at the vault midsection seeds a conformational change that leads to the aperture, facilitating access to the inner cavity where cargo is hosted. These results inform a hitherto uncharacterized step of the vault cycle and will aid current engineering efforts leveraging vault for tailored cargo delivery., This work was supported by the Spanish Ministry for Science and Innovation (grants BIO2017-83906-P and PID2020-117976GB-I00), the Generalitat de Catalunya (grant 2017 SGR 01192), and the CSIC (grant 20202CEX003).

Understanding adenovirus assembly and disassembly poses many challenges due to the virion complexity. A distinctive feature of adenoviruses is the large amount of virus-encoded proteins packed together with the dsDNA genome. Cryo-electron microscopy (cryo-EM) structures are broadening our understanding of capsid variability along evolution, but little is known about the organization of the non-icosahedral nucleoproteic core and its influence in adenovirus function. Atomic force microscopy (AFM) probes the biomechanics of virus particles, while simultaneously inducing and monitoring their disassembly in real time. Synergistic combination of AFM with EM shows that core proteins play unexpected key roles in maturation and entry, and uncoating dynamics are finely tuned to ensure genome release at the appropriate time and place for successful infection.

The assembly of 3-dimensional covalent organic frameworks on the surface of carbon nanotubes is designed and successfully developed for the first time via the hybridization of imine-based covalent organic frameworks (COF-300) and oxidized MWCNTs by one-pot chemical synthesis. The resulting hybrid material ox-MWCNTs@COF exhibits a conformal structure that consists of a uniform amorphous COF layer covering the ox-MWCNT surface. The measurements of individual hybrid nanotube mechanical strength performed with atomic force microscopy provide insights into their stability and resistance. The results evidence a very robust hybrid tubular nanostructure that preserves the benefits obtained from COF, such as CO2 adsorption. Further digestion of the organic structure with aniline enables the study of the interplay between the hybrid interface and its nanomechanics. This new hybrid nanomaterial presents exceptional mechanical and electrical properties, merging the properties of the CNT template and COF-300., We thank the Spanish Government (RTI2018-095038-B-I00), “Comunidad de Madrid” and European Structural Funds (S2018/NMT-4367). P. J. P. thanks to “María de Maeztu” Program for Units of Excellence in R&D (MDM-2014-0377), FIS2017-89549-R and Human Frontiers Science Program (HFSPO RGP0012/2018). C. S. M. acknowledges support from the Spanish State Research Agency and European Regional Development Fund [BFU2016-74868-P]. M. H.-P. is a recipient of a Juan de la Cierva postdoctoral contract funded by the Spanish Ministry of Economy and Competitivity. M. P.-I. holds a predoctoral fellowship funded by La Caixa Foundation.

The encapsulation of an organic dye, 10-phenylphenothiazine (

The encapsulation of an organic dye, 10-phenylphenothiazine (PTH), in the inner cavity of single-walled carbon nanotubes (SWNTs) as a breaking heterogenization strategy is presented. The PTH@oSWNT material was microscopically and spectroscopically characterized, showing intense photoemission when illuminated with visible light at the nanoscale. Thus, PTH@oSWNT was employed as a heterogeneous photocatalyst in single electron transfer dehalogenation reactions under visible light irradiation. The material showed an enhanced photocatalytic activity, achieving turnover numbers as high as 3200, with complete recyclability and stability for more than eight cycles. Computational calculations confirm that electronic communication between both partners is established because, upon illumination, an electron of the excited PTH is transferred from the πsystem of the molecule to the delocalized π-cloud of the SWNT, thus justifying the enhanced photocatalytic activity, Financial support was provided by the European Research Council (ERC-CoG, contract number: 647550), the Spanish Government (RTI2018-095038-B-I00, PID2019-110091GB-I00), and the “Comunidad de Madrid” and European Structural Funds (S2018/NMT-4367). M.B. wishes to thank the Spanish Government for a Juan de la Cierva contract (IJC2019-042157-I). A.M.-S. acknowledges support by the Comunidad Autónoma de Madrid under grant 2016-T2/IND1660

9 pags, 6 figs. -- Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.actbio.2021.08.019., The adenovirus (AdV) icosahedral capsid encloses a nucleoprotein core formed by the dsDNA genome bound to numerous copies of virus-encoded, positively charged proteins. For an efficient delivery of its genome, AdV must undergo a cascade of dismantling events from the plasma membrane to the nuclear pore. Throughout this uncoating process, the virion moves across potentially disruptive environments whose influence in particle stability is poorly understood. In this work we analyze the effect of acidic conditions on AdV particles by exploring their mechanical properties, genome accessibility and capsid disruption. Our results show that under short term acidification the AdV virion becomes softer and its genome less accessible to an intercalating dye, even in the presence of capsid openings. The AFM tip penetrates deeper in virions at neutral pH, and mechanical properties of genome-less particles are not altered upon acidification. Altogether, these results indicate that the main effect of acidification is the compaction of the nucleoproteic core, revealing a previously unknown role for chemical cues in AdV uncoating. STATEMENT OF SIGNIFICANCE: Studying the behavior of virus particles under changing environmental conditions is key to understand cell entry and propagation. One such change is the acidification undergone in certain cell compartments, which is thought to play a role in the programmed uncoating of virus genomes. Mild acidification in the early endosome has been proposed as a trigger signal for human AdV uncoating. However, the actual effect of low pH in AdV stability and entry is not well defined. Understanding the consequences of acidification in AdV structure and stability is also relevant to define storage conditions for therapeutic vectors, or design AdV variants resistant to intestinal conditions for oral administration of vaccines., We thank M. G. Mateu (CBMSO-CSIC-UAM) for careful read-ing and insightful comments on early drafts, M. Castellanos and L. A. Campos (CNB-CSIC) for advice with fluorescence measurements and analyses, and M.I. Laguna (CNB-CSIC) for expert technical help. This work was supported by grants from the Spanish Ministry of Economy, Industry and Competitiveness (FIS2017-89549-R ; "Maria de Maeztu" Program for Units of Excellence in R&D MDM-2014-0377; and FIS2017-90701-REDT) and from the Human Frontiers Science Program (HFSPO RGP0012/2018) to P.J.P.; as well as grants PID2019-104098GB-I00/AEI/10.13039/501100011033 and BFU2016-74868-P, co-funded by the Spanish State Research Agency and the European Regional Development Fund , and 2019AEP045 from the Agencia Estatal CSIC to C.S.M. The CNB-CSIC is further supported by a Severo Ochoa Excellence grant (SEV 2017-0712) . MM was funded by grant RTI2018-099985-B-I0 0, (MICINN/FEDER, UE) and the Ciber of Respiratory Diseases (CIBERES) , an initiative from the Spanish Institute of Health Carlos III (ISCIII) . M.H.-P. was a recipient of a Juan de la Cierva Incorporation postdoctoral contract funded by the Spanish State Research Agency. M.P.-I. held a predoctoral contract from La Caixa Foundation (ID 10 0 010434, under agreement LCF/BQ/SO16/52270 032) . J. G. is a re-cipient of a FPI predoctoral contract (BES-2017-079868) funded by the Spanish State Research Agency.

Icosahedral virus capsids are closed shells built up with a hexagonal lattice of proteins, which incorporate pentamers at their fivefold vertices. Human adenovirus particles lose pentamers (pentons) during infection under a variety of physicochemical cues, including mechanical pulling of molecular motors and the viscous drag of the cytoplasm. By combining atomic force microscopy experiments with survival analysis and Markovian transition state theory, we investigate the sequence of adenovirus penton disassembly that reveals the aging of the virus structure. We show evidence that the lifetime of pentons gradually decreases, accompanied by capsid softening as neighboring pentons are lost. This cooperative dismantling process, which involves first-neighbor penton-penton distances of at least 45 nm, leads to a 50% increase in the virus disassembling rate of the virus particle. Theory and experiments fit remarkably well, allowing us to obtain the spontaneous escape rate and the energy barrier of penton disassembly (∼30 kBT). The observed increase in the penton's loss rate reveals long-range structural correlations within the capsid. Our estimations suggest that the mechanical cues arising from the strokes of protein motors carrying the virus to the nucleus could help penton disassembly and warrant the timely delivery of weak-enough capsids for adenovirus infection., Agencia Estatal de Investigación and European Regional Development Fund (BFU2016-74868-P and PID2019–104098 GB-I00/AEI/10.13039/501100011033); the Ministerio de Economía y Competitividad of Spain (BFU2013-41249-P and BIO2015-68990-REDT); and the Agencia Estatal CSIC (2019AEP045). The CNB-CSIC is further supported by a Severo Ochoa Excellence grant (SEV 2017-0712) and Regional Madrid Government (PEJ16/MED/TL-0935). P. J.d.P. acknowledges support by grants from the Spanish Ministry of Economy, Industry and Competitiveness projects (FIS2017- 89549-R; “Maria de Maeztu” Program for Units of Excellence in R&D MDM-2014-0377; and FIS2017-90701- REDT) Spanish Ministry of Econonomy, Industry and Competitiveness FIS2017-86007-C3-1-P

We developed the Fluctuating Nonlinear Spring (FNS) model to describe the dynamics of mechanical deformation of biological particles, such as virus capsids. The theory interprets the force-deformation spectra in terms of the "Hertzian stiffness" (non-linear regime of a particle's small-amplitude deformations), elastic constant (large-amplitude elastic deformations), and force range in which the particle's fracture occurs. The FNS theory enables one to quantify the particles' elasticity (Young's moduli for Hertzian and bending deformations), and the limits of their strength (critical forces, fracture toughness) and deformability (critical deformations) as well as the probability distributions of these properties, and to calculate the free energy changes for the particle's Hertzian, elastic, and plastic deformations, and eventual fracture. We applied the FNS theory to describe the protein capsids of bacteriophage P22, Human Adenovirus, and Herpes Simplex virus characterized by deformations before fracture that did not exceed 10-19% of their size. These nanoshells are soft (~1-10-GPa elastic modulus), with low ~50-480-kPa toughness - a regime of material behavior that is not well understood, and with the strength increasing while toughness decreases with their size. The particles' fracture is stochastic, with the average values of critical forces, critical deformations, and fracture toughness comparable with their standard deviations. The FNS theory predicts 0.7-MJ/mol free energy for P22 capsid maturation, and it could be extended to describe uniaxial deformation of cylindrical microtubules and ellipsoidal cellular organelles.

9 pags., 7 figs., 1 tab., Adenovirus minor coat protein VI contains a membrane-disrupting peptide that is inactive when VI is bound to hexon trimers. Protein VI must be released during entry to ensure endosome escape. Hexon:VI stoichiometry has been uncertain, and only fragments of VI have been identified in the virion structure. Recent findings suggest an unexpected relationship between VI and the major core protein, VII. According to the high-resolution structure of the mature virion, VI and VII may compete for the same binding site in hexon; and noninfectious human adenovirus type 5 particles assembled in the absence of VII (Ad5-VII-) are deficient in proteolytic maturation of protein VI and endosome escape. Here we show that Ad5-VII- particles are trapped in the endosome because they fail to increase VI exposure during entry. This failure was not due to increased particle stability, because capsid disruption happened at lower thermal or mechanical stress in Ad5-VII- compared to wild-type (Ad5-wt) particles. Cryoelectron microscopy difference maps indicated that VII can occupy the same binding pocket as VI in all hexon monomers, strongly arguing for binding competition. In the Ad5-VII- map, density corresponding to the immature amino-terminal region of VI indicates that in the absence of VII the lytic peptide is trapped inside the hexon cavity, and clarifies the hexon:VI stoichiometry conundrum. We propose a model where dynamic competition between proteins VI and VII for hexon binding facilitates the complete maturation of VI, and is responsible for releasing the lytic protein from the hexon cavity during entry and stepwise uncoating, We thank the Centro Nacional de Biotecnología and the Centro de Investigaciones Biológicas cryoelectron microscopy facility for data acquisition.This work was supported by Grant BFU2016-74868-P, cofunded by the Spanish State Research Agency and the European Regional Development Fund (to C.S.M.); as well as by grants from the Spanish Ministry of Economy, Industry and Competitiveness BIO2015-68990-REDT (the Spanish Adenovirus Network, AdenoNet) (to C.S.M.), FIS2017-89549-R, “María de Maeztu” Program for Units of Excellence in R&D (MDM-2014-0377), and FIS2017-90701-REDT (to P.J.d.P.). P.H. was funded by National Institutes of Health Grants CA122677 and AI102577. U.F.G. and M.S. were funded by the Swiss National Science Foundation (SNSF 31003A_179256/1) and the Swiss National Science Foundation program Sinergia (CRSII5_170929/1). M.M. was funded by Grant BFU2015-70052R (Spanish Ministry of Economy, Industry and Competitiveness and European Regional Development Fund) and the Centro de Investigación Biomédica en Red de Enfermedades Respiratorias, an initiative from the Spanish Institute of Health Carlos III. M.H.-P. is a recipient of a Juan de la Cierva postdoctoral contract funded by the Spanish State Research Agency. M.P.-I. holds a predoctoral contract from La Caixa Foundation.

© 2020 Martín-González, Vieira Gonçalves, Condezo, San Martín, Rubiano, Fallis, Rubino, Ijaz, Maillard and De Pablo., Hygiene and disinfection practices play an important role at preventing spread of viral infections in household, industrial and clinical settings. Although formulations based on >70% ethanol are virucidal, there is a currently a need to reformulate products with much lower alcohol concentrations. It has been reported that zinc can increase the virucidal activity of alcohols, although the reasons for such potentiation is unclear. One approach in developing virucidal formulations is to understand the mechanisms of action of active ingredients and formulation excipients. Here, we investigated the virucidal activity of alcohol (40% w/v) and zinc sulfate (0.1% w/v) combinations and their impact on a human adenovirus (HAdV) using, nucleic acid integrity assays, atomic force microscopy (AFM) and transmission electron microscopy (TEM). We observed no difference in virucidal activity (5 log10 reduction in 60 min) against between an ethanol only based formulation and a formulation combining ethanol and zinc salt. Furthermore, TEM imaging showed that the ethanol only formulation produced gross capsid damage, whilst zinc-based formulation or formulation combining both ethanol and zinc did not affect HAdV DNA. Unexpectedly, the addition of nickel salt (5 mM NiCl2) to the ethanol-zinc formulation contributed to a weakening of the capsid and alteration of the capsid mechanics exemplified by AFM imaging, together with structural capsid damage. The addition of zinc sulfate to the ethanol formulation did not add the formulation efficacy, but the unexpected mechanistic synergy between NiCl2 and the ethanol formulation opens an interesting perspective for the possible potentiation of an alcohol-based formulation. Furthermore, we show that AFM can be an important tool for understanding the mechanistic impact of virucidal formulation., Reckitt Benckiser funded a PhD Studentship (LV) administered by Cardiff University. CSM and GNC were supported by grants PID2019-104098GB-I00/AEI/10.13039/501100011033 and BFU2016-74868-P, co-funded by the Spanish State Research Agency and the European Regional Development Fund, as well as BFU2013-41249-P and BIO2015-68990-REDT (the Spanish Adenovirus Network, AdenoNet) from the Spanish Ministry of Economy, Industry and Competitiveness; and the Agencia Estatal CSIC (2019AEP045). The CNB-CSIC was further supported by a Severo Ochoa Excellence grant (SEV 2017-0712). PJP acknowledges grants from the Spanish Ministry of Economy, Industry and Competitiveness projects (FIS2017- 89549-R; Maria de Maeztu Program for Units of Excellence in R&D MDM-2014-0377; and FIS2017-90701- REDT) and Human Frontiers Science Program (HFSPO RGP0012/2018).

The capsid of P22 bacteriophage undergoes a series of structural transitions during maturation that guide it from spherical to icosahedral morphology. The transitions include the release of scaffold proteins and capsid expansion. Although P22 maturation has been investigated for decades, a unified model that incorporates thermodynamic and biophysical analyses is not available. A general and specific model of icosahedral capsid maturation is of significant interest to theoreticians searching for fundamental principles as well as virologists and material scientists seeking to alter maturation to their advantage. To address this challenge, we have combined the results from orthogonal biophysical techniques including differential scanning fluorimetry, atomic force microscopy, circular dichroism, and hydrogen-deuterium exchange mass spectrometry. By integrating these results from single particle and population measurements, an energy landscape of P22 maturation from procapsid through expanded shell to wiffle ball emerged, highlighting the role of metastable structures and the thermodynamics guiding maturation. The propagation of weak quaternary interactions across symmetric elements of the capsid is a key component for stability in P22. A surprising finding is that the progression to wiffle ball, which lacks pentamers, shows that chemical and thermal stability can be uncoupled from mechanical rigidity, elegantly demonstrating the complexity inherent in capsid protein interactions and the emergent properties that can arise from icosahedral symmetry. On a broader scale, this work demonstrates the power of applying orthogonal biophysical techniques to elucidate assembly mechanisms for supramolecular complexes and provides a framework within which other viral systems can be compared.

Microscopes are used to characterize small specimens with the help of probes, such as photons and electrons in optical and electron microscopies, respectively. In atomic force microscopy (AFM) the probe is a nanometric tip located at the end of a microcantilever which palpates the specimen under study as a blind person manages a white cane to explore the surrounding. In this way, AFM allows obtaining nanometric resolution images of individual protein shells, such as viruses, in liquid milieu. Beyond imaging, AFM also enables the manipulation of single protein cages, and the characterization of every physico-chemical property able of inducing any measurable mechanical perturbation to the microcantilever that holds the tip. Here we describe several AFM approaches to study individual protein cages, including imaging and spectroscopic methodologies for extracting mechanical and electrostatic properties. In addition, AFM allows discovering and testing the self-healing capabilities of protein cages because occasionally they may recover fractures induced by the AFM tip. Beyond the protein shells, AFM also is able of exploring the genome inside, obtaining, for instance, the condensation state of dsDNA and measuring its diffusion when the protein cage breaks.

Microscopes are used to characterize small objects with the help of probes that interact with the specimen, such as photons and electrons in optical and electron microscopies, respectively. In atomic force microscopy (AFM), the probe is a nanometric tip located at the end of a microcantilever which palpates the specimen under study just as a blind person manages a walking stick. In this way, AFM allows obtaining nanometric resolution images of individual protein shells, such as viruses, in a liquid milieu. Beyond imaging, AFM also enables not only the manipulation of single protein cages, but also the characterization of every physicochemical property capable of inducing any measurable mechanical perturbation to the microcantilever that holds the tip. In the present revision, we start revising some recipes for adsorbing protein shells on surfaces. Then, we describe several AFM approaches to study individual protein cages, ranging from imaging to spectroscopic methodologies devoted to extracting physical information, such as mechanical and electrostatic properties. We also explain how a convenient combination of AFM and fluorescence methodologies entails monitoring genome release from individual viral shells during mechanical unpacking.

Despite their diversity, most double-stranded-RNA (dsRNA) viruses share a specialized T=1 capsid built from dimers of a single protein that provides a platform for genome transcription and replication. This ubiquitous capsid remains structurally undisturbed throughout the viral cycle, isolating the genome to avoid triggering host defense mechanisms. Human picobirnavirus (hPBV) is a dsRNA virus frequently associated with gastroenteritis, although its pathogenicity is yet undefined. Here, we report the cryo-electron microscopy (cryo-EM) structure of hPBV at 2.6-Å resolution. The capsid protein (CP) is arranged in a single-shelled, ∼380-Å-diameter T=1 capsid with a rough outer surface similar to that of dsRNA mycoviruses. The hPBV capsid is built of 60 quasisymmetric CP dimers (A and B) stabilized by domain swapping, and only the CP-A N-terminal basic region interacts with the packaged nucleic acids. hPBV CP has an α-helical domain with a fold similar to that of fungal partitivirus CP, with many domain insertions in its C-terminal half. In contrast to dsRNA mycoviruses, hPBV has an extracellular life cycle phase like complex reoviruses, which indicates that its own CP probably participates in cell entry. Using an in vitro reversible assembly/disassembly system of hPBV, we isolated tetramers as possible assembly intermediates. We used atomic force microscopy to characterize the biophysical properties of hPBV capsids with different cargos (host nucleic acids or proteins) and found that the CP N-terminal segment not only is involved in nucleic acid interaction/packaging but also modulates the mechanical behavior of the capsid in conjunction with the cargo., This work was supported by grants from the Spanish Ministry of Economy and Competitivity (BFU2017-88736-R) and the Comunidad Autónoma de Madrid (P2018/NMT-4389) to J.R.C.

Adenovirus minor coat protein VI contains a membrane-disrupting peptide which is inactive when VI is bound to hexon trimers. Protein VI must be released during entry to ensure endosome escape. Hexon:VI stoichiometry has been uncertain, and only fragments of VI have been identified in the virion structure. Recent findings suggest an unexpected relationship between VI and the major core protein, VII. According to the high resolution structure of the mature virion, VI and VII may compete for the same binding site in hexon; and non-infectious human adenovirus type 5 particles assembled in the absence of VII (Ad5-VII-) are deficient in proteolytic maturation of protein VI and endosome escape. Here we show that Ad5-VII- particles are trapped in the endosome because they fail to increase VI exposure during entry. This failure was not due to increased particle stability, because capsid disruption happened at lower thermal or mechanical stress in Ad5-VII- compared to wildtype (Ad5-wt) particles. Cryo-EM difference maps indicated that VII can occupy the same binding pocket as VI in all hexon monomers, strongly arguing for binding competition. In the Ad5-VII- map, density corresponding to the immature amino-terminal region of VI indicates that in the absence of VII the lytic peptide is trapped inside the hexon cavity, and clarifies the hexon:VI stoichiometry conundrum. We propose a model where dynamic competition between proteins VI and VII for hexon binding facilitates the complete maturation of VI, and is responsible for releasing the lytic protein from the hexon cavity during entry and stepwise uncoating.Significance StatementCorrect assembly of an adenovirus infectious particle involves the highly regulated interaction of more than ten different proteins as well as the viral genome. Here we examine the interplay between two of these proteins: the major core protein VII, involved in genome condensation, and the multifunctional minor coat protein VI. Protein VI binds to the inner surface of adenovirus hexons (trimers of the major coat protein) and contains a lytic peptide which must be released during entry to ensure endosome rupture. We present data supporting a dynamic competition model between proteins VI and VII for hexon binding during assembly. This competition facilitates the release of the lytic peptide from the hexon cavity and ensures virus escape from the early endosome.

Atomic force microscopy (AFM) probes surface-adsorbed samples at the nanoscale by using a sharp stylus of nanometric size located at the end of a micro-cantilever. This technique can also work in a liquid environment and offers unique possibilities to study individual protein assemblies, such as viruses, under conditions that resemble their natural liquid milieu. Here, I show how AFM can be used to explore the topography of viruses and protein cages, including that of structures lacking a well-defined symmetry. AFM is not limited for imaging and allows the manipulation of individual viruses with force spectroscopy approaches, such as single indentation and mechanical fatigue assays. These pushing experiments deform the protein cages to obtain their mechanical information and can be used to monitor the structural changes induced by maturation or the exposure to different biochemical environments, such as pH variation. We discuss how studying capsid rupture and self-healing events offers insight into virus uncoating pathways. On the other hand, pulling tests can provide information about the virus-host interaction established between the viral fibers and the cell membrane.

Some viruses package dsDNA together with large amounts of positively charged proteins, thought to help condense the genome inside the capsid with no evidence. Further, this role is not clear because these viruses have typically lower packing fractions than viruses encapsidating naked dsDNA. In addition, it has recently been shown that the major adenovirus condensing protein (polypeptide VII) is dispensable for genome encapsidation. Here, we study the morphology and mechanics of adenovirus particles with (Ad5-wt) and without (Ad5-VII-) protein VII. Ad5-VII- particles are stiffer than Ad5-wt, but DNA-counterions revert this difference, indicating that VII screens repulsive DNA-DNA interactions. Consequently, its absence results in increased internal pressure. The core is slightly more ordered in the absence of VII and diffuses faster out of Ad5-VII- than Ad5-wt fractured particles. In Ad5-wt unpacked cores, dsDNA associates in bundles interspersed with VII-DNA clusters. These results indicate that protein VII condenses the adenovirus genome by combining direct clustering and promotion of bridging by other core proteins. This condensation modulates the virion internal pressure and DNA release from disrupted particles, which could be crucial to keep the genome protected inside the semi-disrupted capsid while traveling to the nuclear pore., Spanish Ministry of Economy and Competitivity [FIS2017-89549-R; “María de Maeztu” Program for Units of Ex-cellence in R&D MDM-2014-0377; and FIS2017-90701-REDT] and Human Frontiers Science Program [HFSPORGP0012/2018] to P.J.P. Spanish State Research Agency and European Regional Development Fund [BFU2016-74868-P] and Spanish Ministry of Economy, Industry and Competitivity [BIO2015-68990-REDT, the Spanish Aden-ovirus Network, AdenoNet] to C.S.M. MINECO/FEDER,UE [FIS2015-67837-P] to D.R. National Institutes ofHealth [CA122677 and AI102577]

Atomic force microscopy (AFM) probes surface-adsorbed samples at the nanoscale by using a sharp stylus of nanometric size located at the end of a micro-cantilever. This technique can also work in a liquid environment and offers unique possibilities to study individual protein assemblies, such as viruses, under conditions that resemble their natural liquid milieu. Here, I show how AFM can be used to explore the topography of viruses and protein cages, including that of structures lacking a well-defined symmetry. AFM is not limited for imaging and allows the manipulation of individual viruses with force spectroscopy approaches, such as single indentation and mechanical fatigue assays. These pushing experiments deform the protein cages to obtain their mechanical information and can be used to monitor the structural changes induced by maturation or the exposure to different biochemical environments, such as pH variation. We discuss how studying capsid rupture and self-healing events offers insight into virus uncoating pathways. On the other hand, pulling tests can provide information about the virus-host interaction established between the viral fibers and the cell membrane.

Microscopes are used to characterize small objects with the help of probes that interact with the specimen, such as photons and electrons in optical and electron microscopies, respectively. In atomic force microscopy (AFM) the probe is a nanometric tip located at the end of a micro cantilever which palpates the specimen under study as a blind person manages a walking stick. In this way AFM allows obtaining nanometric resolution images of individual protein shells, such as viruses, in liquid milieu. Beyond imaging, AFM also enables not only the manipulation of single protein cages, but also the characterization of every physicochemical property able of inducing any measurable mechanical perturbation to the microcantilever that holds the tip. In this chapter we start revising some recipes for adsorbing protein shells on surfaces. Then we describe several AFM approaches to study individual protein cages, ranging from imaging to spectroscopic methodologies devoted for extracting physical information, such as mechanical and electrostatic properties. We also explain how a convenient combination of AFM and fluorescence methodologies entails monitoring genome release from individual viral shells during mechanical unpacking.

Microscopes are used to characterize small objects with the help of probes that interact with the specimen, such as photons and electrons in optical and electron microscopies, respectively. In atomic force microscopy (AFM) the probe is a nanometric tip located at the end of a micro cantilever which palpates the specimen under study as a blind person manages a walking stick. In this way AFM allows obtaining nanometric resolution images of individual protein shells, such as viruses, in liquid milieu. Beyond imaging, AFM also enables not only the manipulation of single protein cages, but also the characterization of every physicochemical property able of inducing any measurable mechanical perturbation to the microcantilever that holds the tip. In this chapter we start revising some recipes for adsorbing protein shells on surfaces. Then we describe several AFM approaches to study individual protein cages, ranging from imaging to spectroscopic methodologies devoted for extracting physical information, such as mechanical and electrostatic properties. We also explain how a convenient combination of AFM and fluorescence methodologies entails monitoring genome release from individual viral shells during mechanical unpacking.

We use the nano-dissection capabilities of atomic force microscopy to induce structural alterations on individual virus capsids in liquid milieu. We fracture the protein shells either with single nanoindentations or by increasing the tip-sample interaction force in amplitude modulation dynamic mode. The normal behavior is that these cracks persist in time. However, in very rare occasions they self-recuperate to retrieve apparently unaltered virus particles. In this work, we show the topographical evolution of three of these exceptional events occurring in T7 bacteriophage capsids. Our data show that single nanoindentation produces a local recoverable fracture that corresponds to the deepening of a capsomer. In contrast, imaging in dynamic mode induced cracks that separate the virus morphological subunits. In both cases, the breakage patterns follow intratrimeric loci. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s10867-018-9492-9) contains supplementary material, which is available to authorized users.

Viruses package their genome in a robust protein coat to protect it during transmission between cells and organisms. In a reaction termed uncoating, the virus is progressively weakened during entry into cells. At the end of the uncoating process the genome separates, becomes transcriptionally active, and initiates the production of progeny. Here, we triggered the disruption of single human adenovirus capsids with atomic force microscopy and followed genome exposure by single-molecule fluorescence microscopy. This method allowed the comparison of immature (noninfectious) and mature (infectious) adenovirus particles. We observed two condensation states of the fluorescently labeled genome, a feature of the virus that may be related to infectivity. Beyond tracking the unpacking of virus genomes, this approach may find application in testing the cargo release of bioinspired delivery vehicles.

Even though viruses evolve mainly in liquid milieu, their horizontal transmission routes often include episodes of dry environment. Along their life cycle, some insect viruses, such as viruses from the Dicistroviridae family, withstand dehydrated conditions with presently unknown consequences to their structural stability. Here, we use atomic force microscopy to monitor the structural changes of viral particles of Triatoma virus (TrV) after desiccation. Our results demonstrate that TrV capsids preserve their genome inside, conserving their height after exposure to dehydrating conditions, which is in stark contrast with other viruses that expel their genome when desiccated. Moreover, empty capsids (without genome) resulted in collapsed particles after desiccation. We also explored the role of structural ions in the dehydration process of the virions (capsid containing genome) by chelating the accessible cations from the external solvent milieu. We observed that ion suppression helps to keep the virus height upon desiccation. Our results show that under drying conditions, the genome of TrV prevents the capsid from collapsing during dehydration, while the structural ions are responsible for promoting solvent exchange through the virion wall. TEM images were collected at the Servicio General de Microscopia Analitica y de Alta Resolucion en Biomedicina (SGIKER, UPV/EHU). PJP thanks FIS2014-59562-R, FIS2015-71108-REDT, Fundacion BBVA and 'Maria de Maeztu' Program for Units of Excellence in R&D (MDM-2014-0377), and FIS2017-89549-R. DMAG thanks the Servicio de Microscopia Analitica y de Alta Resolucion en Biomedicina of SGIker (UPV/EHU) for TEM measurements. GAM is a staff member of CONICET. This work was partially supported by a grant to DMAG from the Ministerio de Ciencia e Innovacion (BFU2012-36241), and Gobierno Vasco (Elkartek KK-2017/00008), Spain. SMGD thanks the Fundacion Biofisica Bizkaia, Spain, for traveling support to visit PJP's lab. GAM and DMAG acknowledge a grant from the CYTED (216RT0506). GAM is recipient of grants from the Agencia Nacional de Promocion Cientifica y Tecnica (PICT No 2014-1536 and 2015-0665), Argentina.

In Atomic Force Microscopy (AFM) the probe is a nanometric tip located at the end of a microcantilever which palpates the specimen under study as a blind person uses a white cane. In this way AFM allows obtaining nanometric resolution images of individual protein shells, such as viruses, in liquid milieu. Beyond imaging, AFM also enables the manipulation of single protein cages, and the characterization a variety physicochemical properties able of inducing any measurable mechanical perturbation to the microcantilever that holds the tip. In this chapter we start revising some recipes for adsorbing protein shells on surfaces. Then we describe several AFM approaches to study individual protein cages, ranging from imaging to spectroscopic methodologies devoted to extracting physical information, such as mechanical and electrostatic properties.

In Atomic Force Microscopy (AFM) the probe is a nanometric tip located at the end of a microcantilever which palpates the specimen under study as a blind person uses a white cane. In this way AFM allows obtaining nanometric resolution images of individual protein shells, such as viruses, in liquid milieu. Beyond imaging, AFM also enables the manipulation of single protein cages, and the characterization a variety physicochemical properties able of inducing any measurable mechanical perturbation to the microcantilever that holds the tip. In this chapter we start revising some recipes for adsorbing protein shells on surfaces. Then we describe several AFM approaches to study individual protein cages, ranging from imaging to spectroscopic methodologies devoted to extracting physical information, such as mechanical and electrostatic properties.

Vault particles are naturally occurring proteinaceous cages with promising application as molecular containers. The use of vaults as functional transporters requires a profound understanding of their structural stability to guarantee the protection and controlled payload delivery. Previous results performed with bulk techniques or at non-physiological conditions have suggested pH as a parameter to control vault dynamics. Here we use Atomic Force Microscopy (AFM) to monitor the structural evolution of individual vault particles while changing the pH in real time. Our experiments show that decreasing the pH of the solution destabilize the barrel region, the central part of vault particles, and leads to the aggregation of the cages. Additional analyses using Quartz-Crystal Microbalance (QCM) and Differential Scanning Fluorimetry (DSF) are consistent with our single molecule AFM experiments. The observed topographical defects suggest that low pH weakens the bonds between adjacent proteins. We hypothesize that the observed effects are related to the strong polar character of the protein-protein lateral interactions. Overall, our study unveils the mechanism for the influence of a biologically relevant range of pHs on the stability and dynamics of vault particles., This work was supported by grants from the Spanish Ministry of Economy and Competitiveness (FIS2014- 59562-R, Fundación BBVA and “María de Maeztu” Program for Units of Excellence in R&D (MDM-2014-0377) to PJP; BIO2014-54588-P and MDM-2014-0435 to NV). PJP and NV acknowledge to the Spanish Excellence Network of Physical Virology FIS2015-71108-REDT. BB and RK received support from the National Institutes of Health (R01 AI081961).

Virus-like particles (VLPs) provide engineering platforms for the design and implementation of protein-based nanostructures. These capsids are comprised of protein subunits whose precise arrangement and mutual interactions determine their stability, responsiveness to destabilizing environments, and ability to undergo morphological transitions. The precise interplay between subunit contacts and the overall stability of the bulk capsid population remains poorly resolved. Approaching this relationship requires a combination of techniques capable of accessing nanoscale properties, such as the mechanics of individual capsids, and bulk biochemical procedures capable of interrogating the stability of the VLP ensemble. To establish such connection, a VLP system is required where the subunit interactions can be manipulated in a controlled fashion. The P22 VLP is a promising platform for the design of nanomaterials and understanding how nanomanipulation of the particle affects bulk behavior. By contrasting single-particle atomic force microscopy and bulk chemical perturbations, we have related symmetry-specific anisotropic mechanical properties to the bulk ensemble behavior of the VLPs. Our results show that the expulsion of pentons at the vertices of the VLP induces a concomitant chemical and mechanical destabilization of the capsid and implicates the capsid edges as the points of mechanical fracture. Subsequent binding of a decoration protein at these critical edge regions restores both chemical and mechanical stability. The agreement between our single molecule and bulk techniques suggests that the same structural determinants govern both destabilizing and restorative mechanisms, unveiling a phenomenological coupling between the chemical and mechanical behavior of self-assembled cages and laying a framework for the analysis and manipulation of other VLPs and symmetric self-assembled structures.

Nucleic acids are the natural cargo of viruses and key determinants that affect viral shell stability. In some cases the genome structurally reinforces the shell, whereas in others genome packaging causes internal pressure that can induce destabilization. Although it is possible to pack heterologous cargoes inside virus-derived shells, little is known about the physical determinants of these artificial nanocontainers’ stability. Atomic force and three-dimensional cryo-electron microscopy provided mechanical and structural information about the physical mechanisms of viral cage stabilization beyond the mere presence/absence of cargos. We analyzed the effects of cargo–shell and cargo–cargo interactions on shell stability after encapsulating two types of proteinaceous payloads. While bound cargo to the inner capsid surface mechanically reinforced the capsid in a structural manner, unbound cargo diffusing freely within the shell cavity pressurized the cages up to ∼30 atm due to steric effects. Strong cargo–cargo coupling reduces the resilience of these nanocompartments in ∼20% when bound to the shell. Understanding the stability of artificially loaded nanocages will help to design more robust and durable molecular nanocontainers.

14 pags, 6 figs, 5 tabs, Incluye material complementario en la web del editor, Adenovirus assembly concludes with proteolytic processing of several capsid and core proteins. Immature virions containing precursor proteins lack infectivity because they cannot properly uncoat, becoming trapped in early endosomes. Structural studies have shown that precursors increase the network of interactions maintaining virion integrity. Using different biophysical techniques to analyze capsid disruption in vitro, we show that immature virions are more stable than the mature ones under a variety of stress conditions and that maturation primes adenovirus for highly cooperative DNA release. Cryoelectron tomography reveals that under mildly acidic conditions mimicking the early endosome, mature virions release pentons and peripheral core contents. At higher stress levels, both mature and immature capsids crack open. The virus core is completely released from cracked capsids in mature virions, but it remains connected to shell fragments in the immature particle. The extra stability of immature adenovirus does not equate with greater rigidity, because in nanoindentation assays immature virions exhibit greater elasticity than the mature particles. Our results have implications for the role of proteolytic maturation in adenovirus assembly and uncoating. Precursor proteins favor assembly by establishing stable interactions with the appropriate curvature and preventing premature ejection of contents by tightly sealing the capsid vertices. Upon maturation, core organization is looser, particularly at the periphery, and interactions preserving capsid curvature are weakened. The capsid becomes brittle, and pentons are more easily released. Based on these results, we hypothesize that changes in core compaction during maturation may increase capsid internal pressure to trigger proper uncoating of adenovirus., This work was also supportedby Ministry of Science and Innovation of Spain Grants BFU2010-16382/BMC (to C. S. M.), MAT2008-02533, PIB2010US-00233, and FIS2011-29493 (to P. J. P.), FIS2010-10552-E and FIS2011-16090-E (to C. S. M. and P. J. P.), and BFU2009-10052 (to M. M.), and by Local Madrid Government Grant P2009/MAT-1467 (to P. J. P.)

Using AFM nanoindentation experiments, DNA-full phi29 phage capsids are shown to be stiffer than when empty. The presence of counterions softens full viruses in a reversible manner, indicating that pressure originates from the confined DNA. A finite element analysis of the experiments provides an estimate of the pressure of ∼40 atm inside the capsid, which is similar to theoretical predictions.

In the present work, we investigate wetting phenomena on freshly prepared nanostructured porous silicon (nPS) with tunable properties. Surface roughness and porosity of nPS can be tailored by controlling fabrication current density in the range 40-120 mA/cm(2). The length scale of the characteristic surface structures that compose nPS allows the application of thermodynamic wettability approaches. The high interaction energy between water and surface is determined by measuring water contact angle (WCA) hysteresis, which reveals Wenzel wetting regime. Moreover, the morphological analysis of the surfaces by atomic force microscopy allows predicting WCA from a semiempiric model adapted to this material.

The organization of virus-like particles (VLPs) on surfaces is a relevant matter for both fundamental and biomedical sciences. In this work, the authors have tailored surfaces with different surface tension components aiming at finding a relationship with the affinity of the different geometric/surface features of icosahedral P22 VLPs. The surfaces have been prepared by titanate assisted organosilanization with glycidyloxy, amino, and perfluoro silanes. Vibrational and photoelectron spectroscopies have allowed identifying the different functional groups of the organosilanes on the surfaces. Atomic force microscopy (AFM) showed that, irrespective of the organosilane used, the final root mean square roughness remains below 1 nm. Contact angle analyses confirm the effective formation of a set of surface chemistries exhibiting different balance among surface tension components. The study of the adsorption of P22 VLPs has involved the analysis of the dynamics of virus immobilization by fluorescence microscopy and the interpretation of the final VLP orientation by AFM. These analyses give rise to statistical distributions pointing to a higher affinity of VLPs toward perfluorinated surfaces, with a dominant fivefold conformation on this hydrophobic surface, but threefold and twofold symmetries dominating on hydrophilic surfaces. These results can be explained in terms of a reinforced hydrophobic interaction between the perfluorinated surface and the dominating hydrophobic residues present at the P22 pentons.

We report the growth of isolated single walled carbon nanotubes (SWCNTs) on a silicon surface by chemical vapor deposition, in the temperature range from 800 to 950 degrees C using two different iron catalyst precursors, Fe(NO3)3 x 9H2O and Fe(CO)5. The results show that while for the first catalyst precursor temperature is the key factor in determining nanotube length, for the second it is the density of catalyst precursor on the surface. Solutions of Fe(CO)5 adsorbed on silicon oxide result in a suitable catalyst precursor to obtain SWCNTs of controllable diameter and with clean surfaces.

In a previous study, we showed that the DNA molecule within a spherical virus (the minute virus of mice) plays an architectural role by anisotropically increasing the mechanical stiffness of the virus. A finite element model predicted that this mechanical reinforcement is a consequence of the interaction between crystallographically visible, short DNA patches and the inner capsid wall. We have now tested this model by using protein engineering. Selected amino acid side chains have been truncated to specifically remove major interactions between the capsid and the visible DNA patches, and the effect of the mutations on the stiffness of virus particles has been measured using atomic force microscopy. The mutations do not affect the stiffness of the empty capsid; however, they significantly reduce the difference in stiffness between the DNA-filled virion and the empty capsid. The results ( i ) reveal that intermolecular interactions between individual chemical groups contribute to the mechanical properties of a supramolecular assembly and ( ii ) identify specific protein–DNA interactions as the origin of the anisotropic increase in the rigidity of a virus. This study also demonstrates that it is possible to control the mechanical properties of a protein nanoparticle by the rational application of protein engineering based on a mechanical model.

The assembly and regulation of viral capsid proteins into highly ordered macromolecular complexes is essential for viral replication. Here, we utilize crystal structures of the capsid protein from the smallest and simplest known viruses capable of autonomously replicating in animal cells, circoviruses, to establish structural and mechanistic insights into capsid morphogenesis and regulation. The beak and feather disease virus, like many circoviruses, encode only two genes: a capsid protein and a replication initiation protein. The capsid protein forms distinct macromolecular assemblies during replication and here we elucidate these structures at high resolution, showing that these complexes reverse the exposure of the N-terminal arginine rich domain responsible for DNA binding and nuclear localization. We show that assembly of these complexes is regulated by single-stranded DNA (ssDNA), and provide a structural basis of capsid assembly around single-stranded DNA, highlighting novel binding interfaces distinct from the highly positively charged N-terminal ARM domain., Circoviruses are the simplest viruses known to autonomously replicate in vertebrates. Here the authors present three structures for distinct macromolecular assemblies of the capsid protein from the beak and feather disease virus that provides insights into the regulation of viral capsid assembly.

Tight confinement of naked genomes within some viruses results in high internal pressure that facilitates their translocation into the host. Adenovirus, however, encodes histone-like proteins that associate with its genome resulting in a confined DNA-protein condensate (core). Cleavage of these proteins during maturation decreases core condensation and primes the virion for proper uncoating via unidentified mechanisms. Here we open individual, mature and immature adenovirus cages to directly probe the mechanics of their chromatin-like cores. We find that immature cores are more rigid than the mature ones, unveiling a mechanical signature of their condensation level. Conversely, intact mature particles demonstrate more rigidity than immature or empty ones. DNA-condensing polyamines revert the mechanics of mature capsid and cores to near-immature values. The combination of these experiments reveals the pressurization of adenovirus particles induced by maturation. We estimate a pressure of ∼30 atm by continuous elasticity, which is corroborated by modeling the adenovirus mini-chromosome as a confined compact polymer. We propose this pressurization as a mechanism that facilitates initiating the stepwise disassembly of the mature particle, enabling its escape from the endosome and final genome release at the nuclear pore.

Viral capsids are metastable structures that perform many essential processes; they also act as robust cages during the extracellular phase. Viruses can use multifunctional proteins to optimize resources (e.g., VP3 in avian infectious bursal disease virus, IBDV). The IBDV genome is organized as ribonucleoproteins (RNP) of dsRNA with VP3, which also acts as a scaffold during capsid assembly. We characterized mechanical properties of IBDV populations with different RNP content (ranging from none to four RNP). The IBDV population with the greatest RNP number (and best fitness) showed greatest capsid rigidity. When bound to dsRNA, VP3 reinforces virus stiffness. These contacts involve interactions with capsid structural subunits that differ from the initial interactions during capsid assembly. Our results suggest that RNP dimers are the basic stabilization units of the virion, provide better understanding of multifunctional proteins, and highlight the duality of RNP as capsidstabilizing and genetic information platforms, This work was supported by grants from the Spanish Ministry of Economy and Competitivity (FIS2011-29493 to PJP, BFU2011-29038 to JLC and BFU2014-55475R to JRC) and Comunidad Autónoma de Madrid (S2013/MIT-2850 to JLC and S2013/MIT-2807 to JRC)

The compound [Mn(mu-ox)(4atr)2]n (1) (ox = oxalato and 4atr = 4-amine-1,2,4-triazole) has been synthesized and characterized by FT-IR spectroscopy, thermal analysis, variable-temperature magnetic measurements, and X-ray single-crystal diffraction methods. The crystal structure of compound 1 consists of one-dimensional linear chains in which trans-[Mn(4atr)2]2+ units are sequentially bridged by centrosymmetric bis-bidentate oxalato ligands. Cryomagnetic measurements show an overall antiferromagnetic behavior of the compound. Isolated chains of this polymer have been obtained by sonication of 1 in ethanol or treatment of the polymer with NaOH and morphologically characterized on highly oriented pyrolitic graphite and mica surfaces by atomic force microscopy and scanning tunneling microscopy. The procedures employed to obtain single chains of this coordination polymer open a route for future nanotechnological applications of these types of materials.

Human rights and the environment are inherently linked. This connection is even stronger in cases of indigenous peoples rights, the close ties they have with the land is the fundamental basis of their cultural, spiritual, and material life. Although inherent, this link between human rights and the environment is not commonly reflected in the relevant conventions of different regional human rights systems. In turn, they do not provide for any special protection to indigenous peoples. However, jurisprudential developments evidence a clear trend towards the recognition of the said relationship. Arguably, the Inter-American System for the Protection of Human Rights is coming to take a leading role in this evolving process. Moreover, its jurisprudence has become an important referent regarding the rights of indigenous peoples and environmental protection. This paper explores the issue of indigenous peoples rights and the environment at the global level. Then, it analyses the main jurisprudential developments regarding human rights and environmental protection at the regional level, with particular emphasis on the indigenous jurisprudence of the Inter-American System.

The stability and strength of viral nanoparticles are crucial to fulfill the functions required through the viral cycle as well as using capsids for biomedical and nanotechnological applications. The mechanical properties of viral shells obtained through Atomic Force Microscopy (AFM) and continuum elasticity theory, such as stiffness or Young's modulus, have been interpreted very often in terms of stability. However, viruses are normally subjected to chemical rather than to mechanical aggression. Thus, a correct interpretation of mechanics in terms of stability requires an adequate linkage between the ability of viral cages to support chemical and mechanical stresses. Here we study the mechanical fragility and chemical stability of bacteriophage T7 in two different maturation states: the early proheads and the final mature capsids. Using chemical stress experiments we show that proheads are less stable than final mature capsids. Still, both particles present similar anisotropic stiffness, indicating that a continuum elasticity description in terms of Young's modulus is not an adequate measure of viral stability. In combination with a computational coarse-grained model we demonstrate that mechanical anisotropy of T7 emerges out of the discrete nature of the proheads and empty capsids. Even though they present the same stiffness, proheads break earlier and have fractures ten times larger than mature capsids, in agreement with chemical stability, thus demonstrating that fragility rather than stiffness is a better indicator of viral cages' stability.

In this chapter we compile a battery of biophysical and imaging methods suitable to investigate adenovirus structural stability, structure, and assembly. Some are standard methods with a long history of use in virology, such as embedding and sectioning of infected cells, negative staining, or immunoelectron microscopy, as well as extrinsic fluorescence. The newer cryo-electron microscopy technique, which combined with advanced image processing tools has recently yielded an atomic resolution picture of the complete virion, is also described. Finally, we detail the procedure for imaging and interacting with single adenovirus virions using the atomic force microscope in liquid conditions. We provide examples of the kind of data obtained with each technique.