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

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

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

The influence of topography on vertical velocity of air in relation to severe storms near the Southern Andes Mountains

On the basis of 180 storms which took place between 2004 and 2011 over the province of Mendoza (Argentina) near to the Andes Range at southern mid-latitudes, we consider those registered in the northern and central crop areas (oases). The regions affected by these storms are currently protected by an operational hail mitigation project. Differences with previously reported storms detected in the southern oasis are highlighted. Mendoza is a semiarid region situated roughly between 32S and 37S at the east of the highest Andes top. It forms a natural laboratory where different sources of gravity waves, mainly mountain waves, occur. In this work, we analyze the effects of flow over topography generating mountain waves and favoring deep convection. The joint occurrence of storms with hail production and mountain waves is determined from mesoscale numerical simulations, radar and radiosounding data. In particular, two case studies that properly represent diverse structures observed in the region are considered in detail. A continuous wavelet transform is applied to each variable and profile to detect the main oscillation modes present. Simulated temperature profiles are validated and compared with radiosounding data. Each first radar echo, time and location are determined. The necessary energy to lift a parcel to its level of free convection is tested from the Convective Available Potential Energy and Convection Inhibition. This last parameter is compared against the mountain waves' vertical kinetic energy. The time evolution and vertical structure of vertical velocity and equivalent potential temperature suggest in both cases that the detected mountain wave amplitudes are able to provide the necessary energy to lift the air parcel and trigger convection. A simple conceptual scheme linking the dynamical factors taking place before and during storm development is proposed.Fil: de la Torre, Alejandro. Universidad Austral. Facultad de Ingeniería; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Pessano, H.. Universidad Tecnologica Nacional. Facultad Regional San Rafael; ArgentinaFil: Hierro, Rodrigo Federico. Universidad Austral. Facultad de Ingeniería; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Santos, J. R.. Universidad Nacional de Cuyo. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Llamedo Soria, Pablo Martin. Universidad Austral. Facultad de Ingeniería; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Alexander, Pedro Manfredo. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Física; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin