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Mechanics of Viral Chromatin Reveals the Pressurization of Human Adenovirus Alvaro Ortega-Esteban, Gabriela Condezo, Ana J. Perez-Berná, Miguel Chillón, Sarah J. Flint, David Reguera, Carmen San Martín, and Pedro J de Pablo ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.5b03417 • Publication Date (Web): 22 Oct 2015 Downloaded from http://pubs.acs.org on October 27, 2015
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Mechanics of Viral Chromatin Reveals the Pressurization of Human Adenovirus Alvaro Ortega-Esteban1, Gabriela Condezo2, Ana J. Pérez-Berná2†, Miguel Chillón3, Sarah J. Flint4, David Reguera5, Carmen San Martín2*, Pedro J. de Pablo1,6*
1
Departamento de Física de la Materia Condensada, Universidad Autónoma de Madrid, 28049 Madrid (Spain) 2
Department of Macromolecular Structure and NanoBioMedicine Initiative, Centro Nacional de Biotecnología (CNB-CSIC). Darwin 3, 28049 Madrid (Spain)
3
Institut Català de Recerca i Estudis Avançats (ICREA), CBATEG-Department of Biochemistry and Molecular Biology, Universitat Autonoma Barcelona, Bellaterra Barcelona, 08010, Spain
4
Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA
5
Departament de Física Fonamental, Facultat de Física, Universitat de Barcelona, Martí i Franqués 1, 08028 Barcelona, Spain 6
Instituto de Física de la Materia Condensada IFIMAC. Universidad Autónoma de Madrid, 28049 Madrid (Spain)
†
Present address: ALBA Synchrotron Light Source, MISTRAL Beamline Experiments Division, 08290 Cerdanyola del Vallès, Barcelona, Spain
*Corresponding authors:
[email protected],
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Abstract 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 its genome resulting in a confined DNA-protein condensate (core). Cleavage of these proteins during maturation is thought to decrease 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 unbranched 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.
Keywords: Viral mini-chromosome, DNA-protein condensate, DNA decompaction, virus core, physical virology, Atomic Force Microscopy, nanoindentation, force curve
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Condensation of nucleic acids in biological systems reaches its maximal efficiency in viral capsids.1 Bacteriophage and some eukaryotic viruses pack their protein-free dsDNA genomes in a spool-like architecture within the shell. The confined genome builds up an outwards pressure, which facilitates genome ejection for DNA translocation into the host.2-4 Other dsDNA viruses pack their genome with the help of cellular histones which bend DNA strands into nucleosomes, in a similar way to eukaryotic chromatin.5 Human adenovirus, a pathogen and a therapeutic tool,6 condenses its dsDNA genome using positively charged, histone-like proteins of viral origin, forming a ~50 MDa DNA-protein core within the 95 nm diameter icosahedral shell.7 The adenovirus “mini-chromosome” is a beaded structure formed by the 35 kbp dsDNA genome associated with core proteins VII (~500 copies), V (150 copies) and Mu (µ) (~300 copies), in ~230 nucleosome-like units per viral particle.8-11 To achieve its full infectious potential, adenovirus requires a maturation process whereby a viral protease cleaves several capsid and core proteins, making the virions metastable and primed for uncoating.12 Adenovirus uncoating in the cell occurs in a stepwise manner,13 beginning at the plasma membrane where receptor binding induces loss of protruding fibers,14 and continuing in the early endosome with release of a few vertex capsomers (pentons) and peripheral core components.12, 15, 16 The partially disrupted particle escapes the endosome and travels to the nuclear pore, where final disassembly occurs and its genome is released into the nucleus (see fig. S1 in Supporting Information -SI-).17 The human adenovirus type 2 (HAdV-C2) thermosensitive mutant ts1 does not package the protease and is stalled at the immature state (fig. 1, ts1), although genome packaging proceeds normally.18 The presence of uncleaved precursor proteins makes ts1 particles more stable than wild type (fig. 1, wt), impairing proper uncoating and aborting infection.12, 19, 20 Core proteins VII and µ are among those cleaved during maturation.21 Concomitant with these cleavages, the viral core decompacts, and penton release and genome detachment from the capsid shell are facilitated.12, 20, 22 The reason why vertex proteins detach more easily from mature than immature particles is unclear, since no additional structural elements corresponding to the uncleaved capsid precursors were observed in direct interaction with pentons in ts1 cryo-EM reconstructions.22, 23 Here we use the nano-dissection capabilities of the Atomic Force Microscope (AFM) to gain direct access to single adenovirus mini-chromosomes in mature and immature particles, and directly probe their mechanical properties. The interplay of core and capsid mechanics reveals that core decompaction occurring during maturation pressurizes adenovirus to facilitate its metastabilization for proper uncoating and infection. RESULTS AND DISCUSSION Mechanics of intact adenovirus capsids We used Atomic Force Microscopy (AFM) to investigate the mechanics of three kinds of adenovirus particles representing different assembly states: ts1 (genome containing, immature), wt (genome containing, mature) and FC31-L3 (empty). Particles attached to 3 ACS Paragon Plus Environment
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freshly cleaved mica substrates were scanned as previously described.20 Since our experiments require successive manipulation events of the same particle, we focused on those showing a 3-fold symmetry orientation, which maximizes their area of contact with
Figure 1. Adenovirus particles used in this study. The left hand side column shows typical images taken with the AFM in buffer for the mature, wildtype virion (wt); the immature ts1 mutant (ts1); the empty particles (FC31-L3); and the wildtype virion in the presence of 1 mM spermidine (wt+sp). The right hand side column shows cartoon representations for each kind of particle. 4 ACS Paragon Plus Environment
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the substrate. Figure 1 shows that the three types of particles are topographically similar, as expected, presenting a height close to the nominal diameter (fig. S2),20 and with well resolved individual capsomers. The fact that empty particles show the same height as the full ones indicates that the adsorption forces are low enough to preserve their integrity. Once a single particle was identified on the substrate, we conducted single indentation experiments (Methods) to extract both its rigidity (spring constant) and breaking force (fig. 2). Nanoindentations performed on intact particles, without evident capsomer vacancies, provided the mechanical properties of capsids. Intact particles exhibited the typical linear behavior corresponding to a shell-like deformation, until the elastic limit (breaking force) was reached (fig. S3).24, 25 We studied 77 wt, 20 ts1, and 61 FC31-L3 particles. Analysis of the curves (fig. 2) 0.60
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0.55 5 0.50 4 0.45
Force (nN)
Spring Constant (N/m)
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3 0.40
0.35
2
FC31-L3 (empty)
wt (mature)
ts1 (immature)
wt+sp (spermidine)
Figure 2. Mechanical properties of intact adenovirus particles. Black symbols and line represent the spring constant, and the red symbols and line represent the breaking force, corresponding to the left and right axes, respectively.
indicates that empty (FC31-L3) particles are the softest (kFC31-L3 = 0.41±0.01 N/m, average ± standard error of the mean -SEM-), while wt particles are the stiffest (kwt = 0.56±0.02 N/m). Interestingly, ts1 particles exhibit an intermediate value kts1 = 0.49±0.04 N/m. Breaking forces reflect the same tendency, with 2.6 ± 0.1 nN for FC31-L3, 2.9 ± 0.2 nN for ts1, and 5.0 ± 0.1 nN for wt (fig. 2) (See SI). These results indicate that the presence of the 5 ACS Paragon Plus Environment
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genome and associated proteins increases the rigidity of the adenovirus particle, but the maximum stiffness is attained after proteolytic maturation.
Mechanics of adenovirus cores An image was acquired immediately after each nanoindentation to monitor structural changes in the viral particle (fig. 3). These images revealed that by keeping the maximum indentation of the AFM tip on the virus just below the breaking force, the protein cage lost some capsomers without collapsing. A few such deformations opened the particle and allowed direct access to the core. Subsequent nanoindentations performed on the opened virus exhibited non-linear deformations with a variety of discontinuities and steps, which might correspond to additional structural changes in the shell-core system. For instance, the intact
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Figure 3. Gaining access to the core. (a) Topographical evolution of a wt particle with the indentation curves shown in (b). (c) Topographical evolution of a ts1 particle with the indentation curves shown in (d). (e) Topographical evolution of a wt particle in the presence of 1 mM spermidine with the indentation curves shown in (f). The numbers indicate the correspondence between each topography image and its indentation curve. Indentation curves are horizontally shifted for the sake of clarity.
wt particle in figure 3a1 is indented for the first time (fig. 3b, curve #1), resulting in minor damage (fig. 3a2) not large enough to allow entry of the tip into the core. However, indentation #3 (fig. 3b, curve 3) generates a large crack, whose topographical profile (fig. 3a4) indicates that the AFM tip is accessing the core. Therefore, indentation #4 in fig. 3b performed at the crack observed in fig. 3a4 is the first indentation directly reaching the core. The successive indentations, from curve #4 henceforth, show a non-linear nature in contrast with the initial indentations #1 to #3. Figures 3a8 to 3a10 demonstrate further particle deterioration until the final collapse. Figure 3c shows a similar experiment performed on a ts1 particle. In this case indentation #3 creates a large crack and the successive curves present a non-linear dependence in contrast with the three initial curves (fig. 3d). Successive deformations also induce the progressive demolition of the particle. The stringent requirements of our cyclic loading experiments allowed evaluating 8 wt and 8 ts1 cores in this way.20 All the particles showed a few initial linear curves until opening the protein cage with a large disruption (curve 3 in figs. 3b and 3d). All subsequent indentations were non-linear. The linear to non-linear transition in the indentation curves likely relates to the way in which the viral particle deforms. Linear indentations indicate the viral shell to be the major responsible for deformation.26 Some non-linear curves (curves 26, fig. 3b) presented a stepped shape linked to the uncontrolled deformation and/or breakage of unstable shell debris remaining on the crack. Interestingly, some monotonic indentations showed a Hertzian-like nature (curves of fig. 3b8 and 3d7).27, 28 These curves were obtained by indentations on cracks with a size similar to the diameter of the tip apex (∼30 nm). In this situation, the AFM tip directly probes the unshielded region of the core. Therefore, we can gain mechanical information on the core by fitting these curves to Hertzian models for obtaining the Young’s modulus. To do so, it is convenient to consider the effect of the sample thickness29, which in our case corresponds to the height of the adenovirus core. (Methods). Figure 4a shows such curves for 8 individual ts1 and 8 wt particles. Figure 4b presents the results of the fitting for the Young’s modulus to be Ewt=0.30±0.04 MPa (average±SEM) and Ets1=1.2±0.1 MPa for the wt and ts1 cores, respectively. Since the cores are adsorbed to the substrate through the remaining lower half of the virus shell, these Young’s modulus values may not be quantitatively precise, but they are useful as relative mechanical parameters for the sake of comparison. In fact, the ratio between Young’s modulus for all types of particle studied is preserved when using models28 where the thickness of the sample is not contemplated (fig. S4). In any case, effects coming from the hard substrate would influence all our measurements in a similar
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way, not affecting the relative comparison between the mechanical properties of our samples .
Figure 4. Core mechanics. (a) FZ curves corresponding to indentations on the core of each type of particle. (b) Young modulus values obtained from fitting the curves in (a) as explained in the text. (c) Cartoon of the proposed model of core decondensation occurring during maturation. Dashed and full red arrows indicate different degrees of repulsion between adjacent regions of the dsDNA genome.
Interplay between adenovirus shell and core mechanics Virus studies at single particle level have elicited new biophysical discoveries on the interplay between structure and function.30 In particular, it is generally accepted that genomes play an important role in modulating the mechanics of virus particles.25, 31-34 In herpesvirus and bacteriophages, cleavage of proteins and naked dsDNA packaging during 8 ACS Paragon Plus Environment
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maturation increases virion rigidity and stability.35, 36 It is usually assumed that capsid and nucleic acid deformations contribute as independent springs in parallel, so that the genome mechanical properties are estimated by directly subtracting the empty-shell spring-constant from that of the full-shell. Our experiments provide a unique approach by studying both the combined and independent mechanics of a viral shell and its confined genome. Intact adenovirus full particles (ts1 and wt) present both higher spring constants and breaking forces than the empty ones (FC31-L3) (fig. 2), revealing a mechanical reinforcement induced by the viral chromatin. The consideration of shell and core as two springs in parallel4 would give a rigidity value for the ts1 core kcts1=kts1-kFC31-L3=0.08 N/m. Likewise, the spring constant of the wt core could be obtained like kcwt=kwt-kFC31-L3=0.15 N/m. Thus, the mature viral chromatin would resemble a stiffer spring than the immature core when confined inside intact shells. However, the Young’s modulus derived from our direct mechanical studies on cores accessed through cracks in open particles indicate the opposite situation, with Ewt< Ets1. That is, although the presence of the genome induces particle stiffening as in other viruses, in adenovirus this stiffening becomes maximal in the mature particle, which however has a softer, less compact core than the immature one. The higher rigidity of the wt particle is not correlated with a higher rigidity of its core, but rather the opposite. Understanding the physical nature of the virus core requires discerning how chromatin decondensation relates to mechanics.37 We can consider the Young’s modulus as a relative parameter for evaluating the compaction degree of the viral core. Thus in ts1 (immature), the core is more rigid (more condensed) than in wt (mature), because Ets1>Ewt. This is a direct observation of the decompaction transition undergone by the adenovirus minichromosome during maturation.12, 22 Another proof is the structural evolution of cores along fatigue cycles.20 Successive topographical profiles show that the wt particles undergo a gradual loss of the core (fig. S5 wt) after shell breakage, although the remaining shell wall survives for a while (fig. 3a). In contrast, in ts1 particles (fig. 3c), successive indentations partially peel the viral shell away at the top, but the core (fig. S5 ts1) does not experience the drastic loss of material shown by wt.20 Stiffening of virus particles associated with the presence of genetic material may have two different origins. On the one hand, structural changes in the shell provided by genomecapsid interaction confer a mechanical stiffening in a similar way that beams buttress the structure of a building, as previously demonstrated for the Minute Virus of Mice.32 On the other hand, if the genome were confined at high pressure inside the capsid, it would generate an outwards force that could stiffen the shell. In viruses packing naked dsDNA, such as bacteriophage lambda or phi29, high pressures of ∼10s of atmospheres arise due to DNA bending, DNA-DNA electrostatic repulsion4, 38 and entropy loss due the confinement of dsDNA chains.39 In adenovirus, the reversed mechanics of intact particles (kwt> kts1) and exposed cores (Ewt< Ets1), substantiates the pressurization of virions during maturation. Recondensation of mature cores with spermidine
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To assess our pressurization hypothesis, we assayed the possibility that recondensation of dsDNA within mature adenovirus particles would reduce their rigidity.4 Spermidine is a trivalent polyamine commonly used to induce DNA condensates in solution1 that also actuates inside virus particles.4, 40 We analyzed the mechanics of mature adenovirus in the presence of spermidine (fig. 1, wt+sp). In these conditions, the spring constant of wt particles decreased from kwt=0.56 ± 0.02 N/m to kwtSP = 0.49 ± 0.02 N/m (fig. 2). Noticeably, the rigidity of wt particles in the presence of spermidine is very similar, within the error, to that of ts1. Probing the mechanics of wt adenovirus cores after opening the shell in the presence of spermidine (figs. 3 and 4) yielded a Young’s modulus of 0.71±0.07 MPa (average±SEM) for 8 particles (fig. 4b), that is, above that of wt without spermidine but below that of ts1. This result indicates that spermidine induced stiffening and condensation in the mature core, which was in turn reflected in lower particle rigidity. As a control, we observed that the rigidity of empty FC31-L3 shells did not change in the presence of spermidine (data not shown). Altogether, our data strongly suggest that core decompaction during maturation induces an internal pressure within the adenovirus particle. Moreover, the experiments in the presence of spermidine reveal that this internal pressure has a multifold origin that includes electrostatic repulsion between the negatively charged dsDNA strands even in the presence of positively charged proteins, as well as DNA bending and entropic components.39, 41 In contrast with previous studies of viruses packing naked dsDNA, proteins heavily cover the adenovirus genome. Our results indicate that maturation affects the above mentioned factors by changing the interactions between DNA and the condensing proteins VII and µ after cleavage by the viral protease. Previous structural studies indicated that cores released from mildly disrupted, mature adenovirus particles contain polypeptides V, VII and µ and have a thick (15-30 nm) fibrous appearance.11 Under more stringent conditions, µ is lost and essentially only polypeptide VII remains, forming a necklace structure with 9.5 nm beads interspersed on the dsDNA molecule at highly variable distances (10-130 nm).10, 11 Disrupted immature adenovirus particles, containing the precursor version of µ, released thicker fibers than the mature ones.22 Architectural proteins involved in DNA condensation throughout nature have been categorized by their role as wrappers, benders, or bridgers.42 A possible model to encompass all this information would have polypeptide VII acting as a wrapper in adenovirus genome condensation forming the nucleosome-like beads. The immature version of protein µ would act as a bridger keeping together two dsDNA chains to form the thick fiber, somehow compensating their mutual repulsion. This compensation would be lost by the disruption of the bridging action of precursor µ upon its cleavage by the viral protease (fig. 4c).21 Estimation of the adenovirus internal pressure We can estimate the magnitude of the internal pressure in adenovirus, irrespective of its origin, using the continuous elasticity prediction for the elastic constant of a pressurized thin spherical shell indented by a point force:43
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π k = k 2
τ − 1
arctanh 1 −
. 1 τ
Here τ = p /k is a dimensionless parameter comparing the relative strength of pressure p against the elastic constant of the unpressurized shell k , and the middle radius of the effective adenovirus ( = 38 nm for adenovirus). Taking k = k = 0.49 N/m, and k = k $ = 0.56 N/m, and solving Eq. (1) for p, we obtain an estimate of 3±1 MPa for the increase in internal pressure upon maturation. This value has been corroborated by Finite Element simulations (SI). We can also rationalize the physical mechanism that builds up this pressure. The obvious approach would be to apply the er inverse spool model used so far for other dsDNA viruses44, 45, which would result in a pressure estimation ∼10 times lower than the one predicted by continuous elasticity modeling. However, the inverse spool model, which provides a reasonable description in viruses packing naked dsDNA, is not applicable in our case, because the adenovirus core is a mixture of dsDNA and proteins. Instead of an ordered spool, this viral chromatin can be better modeled as a linear unbranched polymer with a total length L=11.9 µm made by N segments of an effective Kuhn length b and a persistence length b/2. The Kuhn length defines also the effective radius of the viral chromatin Rg in terms of the usual scaling expression ' ~)* + , where , is a scaling exponent that characterizes its physical behavior. There are evidences, at least for human chromatin, suggesting that short lengths of chromatin behave as a compact or globular state polymer, characterized by a scaling exponent , = 1/3.46, 47
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Figure 5. Entropic pressure of a confined globular polymer. Pressure generated by the confined adenovirus mini-chormosome, modeled as a compact polymer, as a function of its Kuhn length b. For wt virus, a Kuhn length of ) = 480 - 70 /0 is required to reach 3±1 MPa of pressure. In the case of ts1 or wt with spermidine, the mini-chromosome has a much shorter effective Kuhn length leading to a negligible pressure. When this polymer is confined inside a sphere of radius R, its free energy is : 78 ;
= − ?AB = C
DE C
