Mechanical Disassembly of Single Virus Particles Reveals Kinetic Intermediates Predicted by Theory

AbstractNew experimental approaches are required to detect the elusive transient intermediates predicted by simulations of virus assembly or disassembly. Here, an atomic force microscope (AFM) was used to mechanically induce partial disassembly of single icosahedral T = 1 capsids and virions of the minute virus of mice. The kinetic intermediates formed were imaged by AFM. The results revealed that induced disassembly of single minute-virus-of-mice particles is frequently initiated by loss of one of the 20 equivalent capsomers (trimers of capsid protein subunits) leading to a stable, nearly complete particle that does not readily lose further capsomers. With lower frequency, a fairly stable, three-fourths-complete capsid lacking one pentamer of capsomers and a free, stable pentamer were obtained. The intermediates most frequently identified (capsids missing one capsomer, capsids missing one pentamer of capsomers, and free pentamers of capsomers) had been predicted in theoretical studies of reversible capsid assembly based on thermodynamic-kinetic models, molecular dynamics, or oligomerization energies. We conclude that mechanical manipulation and imaging of simple virus particles by AFM can be used to experimentally identify kinetic intermediates predicted by simulations of assembly or disassembly

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

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

Quantitative nanoscale electrostatics of viruses

Electrostatics is one of the fundamental driving forces of the interaction between biomolecules in solution. In particular, the recognition events between viruses and host cells are dominated by both specific and non-specific interactions and the electric charge of viral particles determines the electrostatic force component of the latter. Here we probe the charge of individual viruses in liquid milieu by measuring the electrostatic force between a viral particle and the Atomic Force Microscope tip. The force spectroscopy data of co-adsorbed 29 bacteriophage proheads and mature virions, adenovirus and minute virus of mice capsids is utilized for obtaining the corresponding density of charge for each virus. The systematic differences of the density of charge between the viral particles are consistent with the theoretical predictions obtained from X-ray structural data. Our results show that the density of charge is a distinguishing characteristic of each virus, depending crucially on the nature of the viral capsid and the presence/absence of the genetic material.MINECO of Spain through project FIS2011-29493, FIS2014-59562-R, and the Spanish Interdisciplinary Network on the Biophysics of Viruses (Biofivinet, FIS2011-16090-E). CSM acknowledges funding from BFU2013- 41249-P, and Biofivinet. MGM acknowledges funding from the Spanish Government (BIO2012-37649), Comunidad de Madrid (S-505/MAT-0303), and by an institutional grant from Fundación Areces to the Centro de Biología MolecularPeer Reviewe

Monitoring dynamics of human adenovirus disassembly induced by mechanical fatigue

The standard pathway for virus infection of eukaryotic cells requires disassembly of the viral shell to facilitate release of the viral genome into the host cell. Here we use mechanical fatigue, well below rupture strength, to induce stepwise disruption of individual human adenovirus particles under physiological conditions, and simultaneously monitor disassembly in real time. Our data show the sequence of dismantling events in individual mature (infectious) and immature (noninfectious) virions, starting with consecutive release of vertex structures followed by capsid cracking and core exposure. Further, our experiments demonstrate that vertex resilience depends inextricably on maturation, and establish the relevance of penton vacancies as seeding loci for virus shell disruption. The mechanical fatigue disruption route recapitulates the adenovirus disassembly pathway in vivo, as well as the stability differences between mature and immature virionsWe acknowledge funding by grants from the Ministry of Science and Innovation of Spain, PIB2010US-00233, FIS2011-29493, Consolider CSD2010-00024 and CAM project and the Comunidad de Madrid No. S2009/MAT-1467 to P. J. P.; BFU2010-16382/BMC to C.S.M.; and FIS2011-16090-E to C.S.M. and P.J.P. S.J.F acknowledges funding from the National Institutes of Health, USA (GM037705 and AI1058172). A.J.P.-B. holds a Juan de la Cierva postdoctoral contract from the Ministry of Science and Innovation of Spain; A.O.-E. and R.M.-C. are recipients of predoctoral fellowships from the Ministry of Education and the Instituto de Salud Carlos III of Spain, respectivel

Mechanical elasticity as a physical signature of conformational dynamics in a virus particle

In this study we test the hypothesis that mechanically elastic regions in a virus particle (or large biomolecular complex) must coincide with conformationally dynamic regions, because both properties are intrinsically correlated. Hypothesis-derived predictions were subjected to verification by using 19 variants of the minute virus of mice capsid. The structural modifications in these variants reduced, preserved, or restored the conformational dynamism of regions surrounding capsid pores that are involved in molecular translocation events required for virus infectivity. The mechanical elasticity of the modified capsids was analyzed by atomic force microscopy, and the results corroborated every prediction tested: Any mutation (or chemical cross-linking) that impaired a conformational rearrangement of the pore regions increased their mechanical stiffness. On the contrary, any mutation that preserved the dynamics of the pore regions also preserved their elasticity. Moreover, any pseudo-reversion that restored the dynamics of the pore regions (lost through previous mutation) also restored their elasticity. Finally, no correlation was observed between dynamics of the pore regions and mechanical elasticity of other capsid regions. This study (i) corroborates the hypothesis that local mechanical elasticity and conformational dynamics in a viral particle are intrinsically correlated; (ii) proposes that determination by atomic force microscopy of local mechanical elasticity, combined with mutational analysis, may be used to identify and study conformationally dynamic regions in virus particles and large biomolecular complexes; (iii) supports a connection between mechanical properties and biological function in a virus; (iv) shows that viral capsids can be greatly stiffened by protein engineering for nanotechnological applications.MICINN; Fundación Ramón ArecesPeer Reviewe

Nuevas localidades de especies interesantes en Doñana y la costa de Huelva (Sw España)

New floristic records of species for Doñana and Huelva (SW Spain) Palabras clave. Flora, Doñana, Calystegia soldanella, Herniaria cinerea, Trigonella monspeliaca, Viola lactea, Wolffia arrhiza, especies amenazadas.Key words. Flora, Doñana, Calystegia soldanella, Herniaria cinerea, Trigonella monspeliaca, Viola lactea, Wolffia arrhiza, threatened species

Thrombospondin-1/CD47 interaction regulates Th17 and treg differentiation in psoriasis

Accumulating evidence on the role of Thrombospondin-1 (TSP-1) in the immune response has emerged during the last years. In spite of the importance of TSP-1 not only as anti-angiogenic factor but also as an immunomodulatory molecule, studies on the role of TSP-1 in psoriasis have been neglected. TSP-1 and CD47 expression were analyzed in skin samples from psoriasis patients and control subjects using RT-PCR and immunofluorescence. Expression of these molecules was also evaluated in peripheral blood CD4+ T cells, moDCs, and circulating primary DCs. The functional role of TSP-1/CD47 signaling axis in psoriasis was assessed in Th17 and Treg differentiation assays. Additionally, small interfering RNA assays specific to TSP-1 were performed in CD4+ T cells and monocyte derived DC to specifically evaluate the function of this protein. Lesional skin of psoriasis patients expressed lower TSP-1 and CD47 mRNA levels compared to non-lesional skin or skin from controls. Immunofluorescence staining revealed decreased expression of CD47 in CD45+ dermal cells from psoriasis samples compared to control subjects. Peripheral CD4+ T cells and circulating primary DCs from psoriasis also expressed lower levels of CD47 compared to controls. Although no significant differences were detected in TSP-1 expression in CD4+ T cells and moDCs between patients and controls, TSP-1 expression in psoriasis patients inversely correlated with disease activity evaluated by the Psoriasis Area and Index Activity. Furthermore, exogenous TSP-1 inhibited Th17 differentiation and stimulated the differentiation of CD4+ T cells toward Treg cells. Furthermore, RNA interference specific for TSP-1 confirmed the role of this molecule as a negative regulator of T cell activation. Because of the impact of TSP-1/CD47 signaling axis in Th17 and Treg differentiation, a dysregulated expression of these molecules in the immune cells from psoriasis patients may favor the exacerbated inflammatory response in this diseaseInstituto de Salud Carlos III (AES 2017): PI17/01972 to ED. Janssen; Spanish Ministry of Economy and Competitiveness (MINECO): Plan Nacional de Salud SAF2017-82886-R, Centro de Investigación Biomédica en Red de Enfermedades Cardiovasculares (CIBERCV); Proyecto Integrado de Excelencia PIE13/00041, Instituto de Salud Carlos III to FS-M, Instituto de Salud Carlos III PI16/02166, Universidad Autónoma de Madrid-Banco Santander (grant 2017/EEUU/03), and Red Temática de Excelencia en Investigación en Hipoxia (SAF 2017-90794-REDT) to MJC. This research has been co-financed by Fondo Europeo de Desarrollo Regional (FEDER

A protein with simultaneous capsid scaffolding and dsRNA-binding activities enhances the birnavirus capsid mechanical stability

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 platformsThis 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