Seeing and touching adenovirus: Complementary approaches for understanding assembly and disassembly of a complex virion

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 infectio

Exploring nucleic acid condensation and release from individual parvovirus particles with different physicochemical cues

In the infection cycle, viruses release their genome in the host cell during uncoating. Here we use a variety of physicochemical procedures to induce and monitor the in vitro uncoating of ssDNA from individual Minute Virus of Mice (MVM) particles. Our experiments revealed two pathways of genome release: i) filamentous ssDNA appearing around intact virus particles when using gradual mechanical fatigue and heating at moderate temperature (50 °C). ii) thick structures of condensed ssDNA appearing when the virus particle is disrupted by mechanical nanoindentations, denaturing agent guanidinium chloride and high temperature (70 °C). We propose that in the case of filamentous ssDNA, when the capsid integrity is conserved, the genome is externalized through one channel of the capsid pores. However, the disruption of virus particles revealed a native structure of condensed genome. The mechanical analysis of intact particles after DNA strands ejection confirm the stabilization role of ssDNA in MV

Mechanical disassembly of human picobirnavirus like particles indicates that cargo retention is tuned by the RNA-coat protein interaction

Here we investigate the cargo retention of individual human picobirnavirus (hPBV) virus-like particles (VLPs) which differ in the N-terminal of their capsid protein (CP): (i) hPBV CP contains the full-length CP sequence; (ii) hPBV Δ45-CP lacks the first 45 N-terminal residues; and (iii) hPBV Ht-CP is the full-length CP with a N-terminal 36-residue tag that includes a 6-His segment. Consequently, each VLP variant holds a different interaction with the ssRNA cargo. We used atomic force microscopy (AFM) to induce and monitor the mechanical disassembly of individual hPBV particles. First, while Δ45-CP particles that lack ssRNA allowed a fast tip indentation after breakage, CP and Ht-CP particles that pack heterologous ssRNA showed a slower tip penetration after being fractured. Second, mechanical fatigue experiments revealed that the increased length in 8% of the N-terminal (Ht-CP) makes the virus particles to crumble ∼10 times slower than the wild type N-terminal CP, indicating enhanced RNA cargo retention. Our results show that the three differentiated N-terminal topologies of the capsid result in distinct cargo release dynamics during mechanical disassembly experiments because of the different interaction with RNAFIS2017-89549-R, FIS2017-90701-REDT, PID2021-126608OB-I00, PID2020-113287RB-I0

Monitoring SARS-COV-2 surrogate TGEV individual virions structure survival under harsh physicochemical environments

Effective airborne transmission of coronaviruses via liquid microdroplets requires a virion structure that must withstand harsh environmental conditions. Due to the demanding biosafety requirements for the study of human respiratory viruses, it is important to develop surrogate models to facilitate their investigation. Here we explore the mechanical properties and nanostructure of transmissible gastroenteritis virus (TGEV) virions in liquid milieu and their response to different chemical agents commonly used as biocides. Our data provide two-fold results on virus stability: First, while particles with larger size and lower packing fraction kept their morphology intact after successive mechanical aggressions, smaller viruses with higher packing fraction showed conspicuous evidence of structural damage and content release. Second, monitoring the structure of single TGEV particles in the presence of detergent and alcohol in real time revealed the stages of gradual degradation of the virus structure in situ. These data suggest that detergent is three orders of magnitude more efficient than alcohol in destabilizing TGEV virus particles, paving the way for optimizing hygienic protocols for viruses with similar structure, such as SARS-CoV-

Nanotribology and electrical properties of carbon nanotubes hybridized with covalent organic frameworks

Nanomanipulation of molecular materials such as carbon nanotubes (CNTs) or new covalent organic frameworks (COFs) is key not only for the study of their fundamental physicochemical properties, but also for building and probing nanodevices. Therefore, we have investigated the tribological properties of oxidized MWCNTs (ox-MWCNTs) and their hybridization with COF building blocks (ox-MWCNTs@COF) adsorbed on a mica surface. We used the AFM tip to apply torsional forces on individual nanotubes. Depending on the manipulation parameters, the lateral displacements of the AFM tip slide and/or bend nanotubes enabling the direct quantification of the nanotube-mica adhesion. We found striking changes in the behaviour of the lateral force needed to manipulate each carbon nanotube variant which indicates an increased adhesion of ox-MWCNTs@COF with respect to ox-MWCNTs (∼10x). In addition, the use of the AFM tip as a mobile electrode enabled the measurement of electrical transport through individual nanotubes that revealed a rectifying behaviour of the ox-MWCNTs@COF with high resistivity, which was in contrast with the near ohmic performance of ox-MWCNTsP. J.d.P. acknowledges support by grants from the Ministerio de Ciencia e Innovacion (FIS2017- 89549-R; “Maria de Maeztu” Program for Units of Excellence in R&D MDM2014-0377; and FIS2017-90701- REDT) and the Human Frontiers Science Program (HFSPO RGP0012/ 2018). R. M. ackowledges support by grant PID2019-110637RB-10

Cementing proteins provide extra mechanical stabilization to viral cages

The study of virus shell stability is key not only for gaining insights into viral biological cycles but also for using viral capsids in materials science. The strength of viral particles depends profoundly on their structural changes occurring during maturation, whose final step often requires the specific binding of ‘decoration’ proteins (such as gpD in bacteriophage lambda) to the viral shell. Here we characterize the mechanical stability of gpD-free and gpD-decorated bacteriophage lambda capsids. The incorporation of gpD into the lambda shell imparts a major mechanical reinforcement that resists punctual deformations. We further interrogate lambda particle stability with molecular fatigue experiments that resemble the sub-lethal Brownian collisions of virus shells with macromolecules in crowded environments. Decorated particles are especially robust against collisions of a few kBT (where kB is the Boltzmann’s constant and T is the temperature ~300 K), which approximate those anticipated from molecular insults in the environmentWe acknowledge the MINECO of Spain (PIB2010US-00233, FIS3011-29493, Consolider CSD2010-00024, CAMprojectNo.S3009/MAT-1467), and the US National Science Foundation (MCB-1158107) for their financial support of this researc

Trombo recurrente sobre dispositivo de cierre percutáneo de orejuela izquierda: una situación difícil de manejar

Left atrial appendage occlusion offers an alternative to anticoagulation in complex patients with atrial fibrillation, and its use has grown in the last years. However, device-related thrombus is a complication of the technique, which it has been estimated in 3% to 4% of cases according to literature records. We describe the case of a 85-year-old patient with history of myelodysplastic syndrome who presented thrombosis on a WatchmanTM-type appendage closure device.El cierre percutáneo de orejuela izquierda ofrece una alternativa a la anticoagulación en determinados pacientes con fibrilación auricular, cuyo uso se ha expandido en los últimos años. Sin embargo, la trombosis relacionada con el dispositivo supone una complicación de la intervención, que se ha estimado en un 3 a 4% de los casos según los registros publicados. Describimos el caso de una paciente de 85 años con historia de síndrome mielodisplásico que presenta trombosis recurrente sobre dispositivo WatchmanTM

Adenovirus core protein V reinforces the capsid and enhances genome release from disrupted particles

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 entryThis 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.d.P., as well as grant PID2019-104098GB-I00/AEI/10.13039/501100011033, cofunded by the Spanish State Research Agency and the European Regional Development Fund to C.S.M. The CNB-CSIC was further supported by a Spanish State Research Agency Severo Ochoa Excellence grant (SEV 2017-0712). C.S.M. is a member of the CSIC funded consortium LifeHub (CSIC grant number: 202120E47). M.H.-P. was a recipient of a Juan de la Cierva postdoctoral contract and currently holds a Ramón y Cajal position (RyC2021-030929-I) funded by the Spanish State Research Agency. M.H.-P. acknowledges funding from Ayudas a Proyectos de I+D para Jóvenes Doctores de la Universidad Autónoma de Madrid 2021, SI3/PJI/2021-00216 supported by the Community of Madrid and the University Autónoma de Madrid, and TED2021-129937B-I00 from Spanish Ministry of Science and Innovation. Financial support of the Community of Madrid and the European Union through the European Regional Development Fund (ERDF), financed as part of the Union response to Covid-19 pandemic (VIRMAT-REACT), is also acknowledged. Access to the cryo-EM CNB-CSIC facility was carried out via the CRIOMECORR project (ESFRI-2019-01- CSIC-16). Work in the Greber laboratory was supported by a grant to UFG from Schweizerischer Nationalfonds (Swiss National Science Foundation, 31003A_179256/1). Author contributions: N.M.-G. performed AFM experiments and analyzed the data. M.H.-P. and C.S.M. carried out cryoEM analyses. A.G.-G. and M.B. produced the viral samples and carried out thermostability analyses. U.F.G., C.S.M., and P.J.d.P. designed research. N.M.-G. wrote the manuscript, with input from U.F.G., C.S.M., and P.J.d.P. All authors read and edited the manuscript. Competing interests: The authors declare that they have no competing interests. Data and materials availability: The Ad5-ΔV cryo-EM map is deposited at the Electron Microscopy Data Bank (EMDB; www.ebi.ac.uk/pdbe/emdb) with accession number EMD-15694. All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Material

Improving the Lateral Resolution of Quartz Tuning Fork-Based Sensors in Liquid by Integrating Commercial AFM Tips into the Fiber End

The use of quartz tuning fork sensors as probes for scanning probe microscopy is growing in popularity. Working in shear mode, some methods achieve a lateral resolution comparable with that obtained with standard cantilevered probes, but only in experiments conducted in air or vacuum. Here, we report a method to produce and use commercial AFM tips in electrically driven quartz tuning fork sensors operating in shear mode in a liquid environment. The process is based on attaching a standard AFM tip to the end of a fiber probe which has previously been sharpened. Only the end of the probe is immersed in the buffer solution during imaging. The lateral resolution achieved is about 6 times higher than that of the etched microfiber on its own

Temporal response to drought stress in several Prunus rootstocks and wild species

Prunus species are important crops in temperate regions. In these regions, drought periods are predicted to occur more frequently due to climate change. In this sense, to reduce the impact of climate warming, obtaining new tolerant/resistant cultivars and rootstocks is a mandatory goal in Prunus breeding. Therefore, the current study assembled three Prunus species including almond, (P. dulcis Mill D.A. Webb), apricot (P. armeniaca L.) and peach (P. persica L.) to model the temporal effects of drought. A hybrid peach × almond and a wild almond-relative species Prunus webbii were also included in the study. Physiological traits associated with photosynthetic activity, leaf water status, and chlorophyll content were assessed under three watering treatments. Results showed that effects of time, genotype, and treatment interact significantly in all traits. In addition, results confirmed that P. webbii have a greater tolerance to drought than commercial rootstocks. However, “Real Fino” apricot showed the fastest recovery after re-irrigation while being one of the most affected cultivars. In addition, from the better response to these watering treatments by the almond genotypes, two different trends were observed after re-irrigation treatment that clearly differentiate the response of the almond cultivar “Garrigue” from the rest of Prunus genotypes. A better characterization of the short-term drought response in Prunus, an accurate and more efficient evaluation of the genotype effect was obtained from the use of mixed models considering appropriate variance–covariance structures. Although the advantages of these approaches are rarely used in Prunus breeding, these methodologies should be undertaken in the future by breeders to increase efficiency in developing new breeding materials.info:eu-repo/semantics/publishedVersio