Published online 9 August 2019 Nucleic Acids Research, 2019, Vol. 47, No. 17 9231–9242 doi: 10.1093/nar/gkz687 Adenovirus major core protein condenses DNA in clusters and bundles, modulating genome release and capsid internal pressure Natalia Martı́n-González1 , Mercedes Hernando-Pérez2 , Gabriela N. Condezo2 , Marta Pérez-Illana2 , Antonio Šiber3 , David Reguera4,5 , Philomena Ostapchuk6 , Patrick Hearing6 , Carmen San Martı́n 2,* and Pedro J. de Pablo 1,7,* 1 Department of Condensed Matter Physics, Universidad Autónoma de Madrid, Madrid 28049, Spain, 2 Department of Macromolecular Structures, Centro Nacional de Biotecnologı́a (CNB-CSIC), Madrid 28049, Spain, 3 Institute of Physics, Zagreb, Croatia, 4 Departament de Fı́sica de la Matèria Condensada, Facultat de Fı́sica, Universitat de Barcelona, Martı́ i Franqués 1, 08028 Barcelona, Spain, 5 Universitat de Barcelona Institute of Complex Systems (UBICS), 08028 Barcelona, Spain, 6 Department of Molecular Genetics and Microbiology, School of Medicine, Stony Brook University, Stony Brook, NY 11794-5222, USA and 7 Instituto de Fı́sica de la Materia Condensada (IFIMAC), Universidad Autónoma de Madrid, Madrid 28049, Spain Received June 05, 2019; Revised July 10, 2019; Editorial Decision July 21, 2019; Accepted August 06, 2019 ABSTRACT INTRODUCTION Some viruses package dsDNA together with large Regulation of dsDNA condensation is crucial for the man- amounts of positively charged proteins, thought to agement of genetic information. Major players in this reg- help condense the genome inside the capsid with ulation are specialized proteins which bend, wrap DNA, or no evidence. Further, this role is not clear because form bridges between dsDNA strands (1). In eukaryotes, these viruses have typically lower packing fractions wrapping by H2A, H2B, H3 and H4 histone octamers pro- vides the first organization level of chromatin, with ∼10 nm than viruses encapsidating naked dsDNA. In addi- diameter nucleosomes forming a beads-on-string structure. tion, it has recently been shown that the major ade- Another histone, H1, is a bridger that clasps linker DNA at novirus condensing protein (polypeptide VII) is dis- the nucleosome exit, stabilizing the structure (2). In bacte- pensable for genome encapsidation. Here, we study ria, nucleoid-associated proteins have mainly bending and the morphology and mechanics of adenovirus parti- bridging roles (3). The highest known condensation of nu- cles with (Ad5-wt) and without (Ad5-VII-) protein VII. cleic acids is the packing of viral genomes (4). In tailed bac- Ad5-VII- particles are stiffer than Ad5-wt, but DNA- teriophages and herpesviruses, naked dsDNA is stored in- counterions revert this difference, indicating that side the capsid at very high volume fractions approximately VII screens repulsive DNA-DNA interactions. Con- in the form of a spool, with polyamines and cations help- sequently, its absence results in increased internal ing to relieve the electrostatic repulsion between dsDNA pressure. The core is slightly more ordered in the strands (4,5). In polyomaviruses, dsDNA is packed in com- plex with cell histones, forming a chromatin-like structure absence of VII and diffuses faster out of Ad5-VII– (6). Adenoviruses and some of their structural relatives (as- than Ad5-wt fractured particles. In Ad5-wt unpacked farviruses, chloroviruses, mimiviruses, marseilliviruses) en- cores, dsDNA associates in bundles interspersed code and package positively charged proteins with similari- with VII-DNA clusters. These results indicate that pro- ties to cell histones (7–10). It has traditionally been thought tein VII condenses the adenovirus genome by com- that the main role of these proteins is to screen DNA-DNA bining direct clustering and promotion of bridging by electrostatic repulsion so that the viral genomes can be con- other core proteins. This condensation modulates densed to fit the limited space available inside the capsid. the virion internal pressure and DNA release from However, since these viruses have lower packing fractions disrupted particles, which could be crucial to keep than tailed phages (∼0.3 versus ∼0.5), the actual need for the genome protected inside the semi-disrupted cap- genome condensation agents is not that clear (11). Further- sid while traveling to the nuclear pore. * To whom correspondence should be addressed. Tel: +34 914974410; Email: p.j.depablo@uam.es Correspondence may also be addressed to Carmen San Martı́n. Tel: +34 915855450 ; Email: carmen@cnb.csic.es C The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com 9232 Nucleic Acids Research, 2019, Vol. 47, No. 17 more, knowledge about the organization and properties of in layers near the confining capsid wall will increase when nucleoprotein complexes inside virus particles is scarce. the strength of protein-DNA interactions is decreased (11). Human adenovirus type 5 (Ad5) is the best charac- Experimentally, atomic force microscopy (AFM) of single terized system encapsidating virus encoded proteins The virus particles in liquid (25) has proved the existence of in- Ad5 icosahedral, pseudoT = 25 capsid is composed of ternal pressure in tailed phages (26,27), where cryo-electron 240 hexon trimers, 12 penton-base pentamers, 12 penton microscopy (cryo-EM) shows the naked genome ordered in base-associated fiber trimers and four minor coat pro- concentric shells (5). Tailed phages release this pressure to teins (IIIa, VI, VIII and IX) (12). This capsid encloses the translocate their genome into the host (28). No concentric non-icosahedral core, formed by a linear, 35 kbp dsDNA shells are observed in Ad5 cryo-EM maps, and a change molecule bound to positively charged viral proteins that in internal pressure related to particle maturation has also constitute ∼50% of the core molecular weight: 150 copies been detected by AFM (18). Here we use AFM and cryo- of protein V (41 kDa), 500–800 copies of protein VII (22 EM to compare the mechanics and core organization of kDa), and 100–300 copies of protein ␮ (9 kDa) (13–15). Ad5-wt and Ad5-VII– particles (Figure 1A), to understand Previous studies have shown that cores released from Ad5 how protein VII influences the architecture and physical particles disrupted with pyridine contain polypeptides V, properties of adenovirus particles. VII, and ␮, and have a thick (15–30 nm) fibrous appear- ance when observed at the electron microscope after neg- MATERIALS AND METHODS ative staining or metal shadowing (16). After high ionic strength treatment of these cores, essentially only protein Virus specimens VII remains attached to the DNA, forming tangled struc- For Ad5-wt particles we used the E1-E3 deleted, GFP ex- tures with ∼200 beads of 9.5 nm diameter located at highly pressing Ad5/attP vector which is structurally wildtype variable distances (∼10–130 nm) (16). These observations (29). For empty capsids, we used light density particles suggested a chromatin-like organization for the adenovirus produced by the Ad5 delayed packaging mutant FC31 genome, with protein VII acting as a wrapper, while protein (FC31-L3), which lack genome and core proteins but have ␮ could have a bridging function to form the thicker fibers a complete icosahedral shell (30). Both were propagated at (17,18). Currently, the only model for the core architecture 37◦ C in HEK293 cells and harvested at 36 and 56 h post- in situ (in the crowded conditions within the intact virion) is infection, respectively. The parent virus Ad5-VII-loxP con- based on cryo-electron tomography combined with molec- tains loxP sites flanking the protein VII open reading frame, ular dynamics simulations, and proposes an organization of and was generated and propagated as described (24). Ad5- the DNA-protein complex as a fluid of soft repulsive parti- VII– particles were produced using the Cre66 cell line, a cles (17). Cre recombinase expressing cell line derived from HEK-293 The Ad5 particle becomes fully infectious only after mat- cells (a gift from Dr Stefan Kochaneck, University Ulm, uration, which involves cleavage of several capsid and core Germany; unpublished), maintained in Dulbecco’s modi- proteins by the viral protease (19). Cleavage of proteins fied Eagle’s medium supplemented with 10% HyClone Fe- VII and ␮ results in decreased core condensation and in- talclone III serum, penicillin, streptomycin, and 0.25 mg/ml creased internal pressure, priming the virion for sequential of Geneticin (Life Technologies). Cre66 cells (6 × 108 to- uncoating and facilitating genome release (20–23). How- tal cells) were infected at 200 CsCl-purified Ad5-VII-loxP ever, it has recently been shown that the hypothesized con- virus particles/cell for 1 h at 37◦ C. Infected cells were har- densing role of protein VII, the most abundant core pro- vested 3 days after infection when full cytopathic effect was tein in Ad5 (60%∼70% of the core protein mass), is not re- evident. The cells were pelleted by centrifugation, medium quired for genome encapsidation. An Ad5 construct where removed, and cell pellets flash frozen in liquid nitrogen for the VII gene was excised from the genome using the Cre- storage. In all cases, particles were purified by equilibrium lox recombination system has an assembly efficiency similar centrifugation in two consecutive CsCl gradients, desalted to the wild type virus (Ad5-wt), although the particles pro- on a Bio-Rad 10 DC column, and stored in HBS buffer (20 duced (Ad5-VII-) are not infectious due to a defect in entry mM HEPES, pH 7.8, 150 mM NaCl) supplemented with (24). The actual cause for this entry defect is not clear. It has 10% glycerol for long term storage at –80◦ C. been hypothesized that, since the absence of VII represents a large loss of mass inside the virion, the entry defect may re- Atomic force microscopy sult from lack of internal pressurization, thereby hindering the start of the uncoating cascade. An alternative hypothe- Virus samples to be used for AFM assays were dialyzed sis is that the absence of VII would allow the genome to in- against HBS at 4◦ C to remove glycerol, frozen in single-use, teract with positively charged proteins on the inner capsid 5 ␮l aliquots and stored at -20◦ C. Stock particle concen- surface, obstructing genome release (24). Therefore, more trations were 2.3 × 1011 virus particles (vp)/ml for Ad5- work is required to ascertain the actual role of core protein wt, 2.7 × 1011 vp/ml for empty particles, and 3.1 × 1011 VII in adenovirus assembly and disassembly. vp/ml for Ad5-VII-. For AFM measurements, single-use Theoretical models simulating the interactions of ds- aliquots were thawed, diluted in 20 ␮l of HBS with 5 mM DNA and proteins within the capsid confinement predict NiCl2 (Ni-HBS), and adsorbed onto freshly cleaved mica that the presence of condensing proteins decreases inter- for 20 min at 4◦ C. After five washes with Ni-HBS, 200 ␮l nal capsid pressure, despite the decrease in free volume of buffer were deposited on the mica surface to avoid desic- caused by their presence. These models also envisage that cation. The AFM tip was prewetted with 30 ␮l of Ni-HBS the natural tendency for the capsid contents to organize so the total volume was about 260 ␮l. For experiments in Nucleic Acids Research, 2019, Vol. 47, No. 17 9233 Figure 1. Ad5-wt and Ad5-VII– viral particles. (A) Schematic representation showing the core composition of the two specimens studied (modified from (15)). (B) Height distribution of all the viral particles imaged with AFM. The inset shows AFM topography images of Ad5-wt (left), Ad5-VII– (center) particles, and the topographical profiles (right) corresponding to the line between the red (Ad5-wt) and blue (Ad5-VII-) arrows. the presence of spermidine, 2.3 ␮l of a 100 mM solution of single indentation assays, to avoid virus collapse at once. of spermidine (Sigma Aldrich S.L) in water were added to While the first indentation just removes a few capsomers to the sample drop, to obtain a final concentration of 1 mM crack open the capsid, subsequent indentations probe and spermidine. The AFM (Nanotec Electrónica S.L, Madrid, modify the core in a controlled fashion as explained else- Spain) was operated in jumping mode plus (31) in liquid, where (18). A typical multiple indentation assay is described with RC800PSA cantilevers (Olympus, Tokyo, Japan) with in Supplementary Figure S1. nominal spring constant of 0.05 N/m. The cantilever spring To analyze the structure of the core contents, we per- constants were calibrated with the Sader method (32). AFM formed mechanical fatigue assays (21) consisting in the con- exploration at low force (∼60 pN) was carried out to ensure tinuous imaging of a single virus particle at low force (∼70 a homogeneously dispersed population of particles with a 3- pN). Imaging in jumping mode plus (31) implies that the fold icosahedral symmetry orientation on the mica surface virus is probed once at every pixel, inducing low energy im- before starting measurements (Figure 1B, inset). This ori- pacts between the tip apex and the virus particle (34). This entation maximizes the stable attachment of particles to the fatigue induces a gradual disassembly of the capsid, includ- mica substrate for the mechanical characterization, because ing capsomer disruption and core exposure (18). Obtaining the icosahedral particles are resting on a triangular facet consecutive images of the same particle allows us to monitor (21). For single indentation assays, viral particles were de- these morphological changes until the virion collapses, ex- formed with the AFM tip until breakage by means of a force pelling a mixture of fibered and blob structures. We devised versus z-piezo displacement experiment that is transformed a procedure to estimate the size of the fibers and blobs lay- into a force-indentation curve (FIC) (33). From these data ing on the substrate after particle disruption, based on sta- the spring constant was obtained (Figure 2, inset left). In tistical measures of height in specific regions of the AFM multiple indentation assays the particles were probed with image (Supplementary Figures S4 and S5). consecutive indentations that reach lower forces that those 9234 Nucleic Acids Research, 2019, Vol. 47, No. 17 Figure 2. Mechanical properties of intact capsids. Stiffness (spring constants) measured for each specimen used in this work. Top right inset: Topography image of an adenovirus particle after performing a single indentation. Spring constant values are obtained from the force-indentation curve (FIC) (center left inset). Cryo-electron microscopy and image processing metry was imposed throughout the refinement process. The ◦ initial reference for 3D classification was a non-human ade- Purified virus particles were dialyzed for 1 h at 4 C against novirus cryo-EM map (unpublished), low-pass filtered to 60 phosphate buffered saline (PBS, 137 mM NaCl, 2.7 mM Å resolution. The class yielding the best resolution (23 060 KCl, 10 mM Na2 HPO4 , 1.8 mM KH2 PO4 at pH 7.4) to particles for wt, 21 762 particles for VII-) was individu- remove the storage glycerol. Stock particle concentrations ally refined using the original 752 px boxed particles and were 8.6 × 1011 vp/ml for Ad5-wt and 1.5 × 1012 vp/ml the map obtained during the 3D classification as a refer- for Ad5-VII-. The Ad5-VII- sample was concentrated 11 ence, yielding a final map at 5.5 Å resolution for Ad5-wt times by spinning at 4◦ C in a 100 000 MWCO Amicon Ul- and 5.0 Å for Ad5-VII-. Resolution was estimated accord- tra centrifugal filter (Millipore) before application to glow ing to the gold-standard FSC = 0.143 criterion as imple- discharged Quantifoil R2/4 300 mesh Cu/Rh grids and vit- mented in RELION auto-refine and postprocess routines rification in liquid ethane using a Leica CPC plunger. For (41). The actual sampling for both maps was estimated by the Ad5-wt control, particle concentration was increased by comparison with a human adenovirus near atomic resolu- consecutively incubating the grid on 10 drops of the sample tion model (PDB ID: 6B1T) (42) in UCSF Chimera (43), before the final blotting and plunging in liquid ethane (35). yielding a value of 1.37 Å/px. Xmipp was used to calculate Cryo-EM images (4395 for wt, 1640 for VII-) were recorded histograms and radial average profiles, after both 3D maps in a 200 kV Talos Arctica microscope (FEI) equipped with a were low pass filtered to 5.5 Å resolution and normalized to Falcon II detector, with a total dose of 50 e– /Å2 distributed gray values with average 0 and standard deviation 1 within a over 32 frames, at a nominal pixel size of 1.4 Å and a de- region comprising the hexon shell (radii 322–427 Å). Radial focus range between –0.5 and –3 ␮m. All image processing average profiles were further rescaled to a common value and 3D reconstruction tasks were performed within the Sci- range in the [−1, 1] interval. pion framework (36). Frames were aligned using Motion- cor2 and weighted according to the electron dose received before averaging (37). The contrast transfer function (CTF) was estimated using CTFFIND4 (38). Particles (26 661 for RESULTS AND DISCUSSION wt, 27 909 for VII-) were semi-automatically picked from Absence of protein VII stiffens the Ad5 intact particle micrographs corrected for the phase oscillations of the CTF (phase-flipped), extracted into 752 × 752 pixel boxes, nor- To investigate the role of protein VII in the physical proper- malized and resized to a sampling rate of 2.8 Å/px (376 px ties of Ad5, we first analyzed the mechanics of Ad5-wt and box size), using Xmipp (39). All 2D and 3D classifications Ad5-VII– intact particles by single nanoindentation assays and refinements were performed using RELION (40). 2D (Materials and Methods). AFM topography images that re- classification was used to discard low quality particles, and solve individual protein capsomers revealed no substantial run for 25 iterations, with 50 classes, angular sampling 5 differences between Ad5-wt and Ad5-VII– particles (Figure and regularization parameter T = 2. Classification in 3D 1B, inset). Height measurements of all particles included in was run for 40 iterations, with three classes, starting with this study (Ad5-wt, Ad5-VII– and empty shells) provided an angular sampling of 3.7◦ and sequentially decreasing to values ∼86 nm (Figure 1B) consistent with the expected 0.5, and regularization parameter T = 4. Icosahedral sym- capsid diameter and indicating that none of them suffered Nucleic Acids Research, 2019, Vol. 47, No. 17 9235 measurable deformations induced by the interaction with parallel. In this approximation, the spring constants of the the substrate (21,44,45). Ad5 core in the presence or absence of protein VII would be The spring constants of Ad5-wt and empty particles, as core kwt = kwt − kE = 0.14 N/m, and kVII− core = kVII− − kE = measured by single indentation assays (Figure 2), were kwt 0.22 N/m, predicting that the core in Ad5-wt would be = 0.58 ± 0.02 N/m (mean ± SEM; N = 53) and kE = 0.44 ± softer than in Ad5-VII– when confined within the icosahe- 0.03 N/m (mean ± SEM; N = 54) in agreement with previ- dral shell. In contrast, one could expect that in the absence ously published values showing that the presence of the core of VII, the core would be softer because it has less molecu- stiffens Ad5 particles (18). For Ad5-VII-, the elastic con- lar mass. In fact, we found this tendency when probing di- stant was higher, kVII– = 0.66 ± 0.04 N/m (mean ± SEM; rectly the mechanics of the opened cores (Figure 3B). This N = 33) (Figure 2). Therefore, Ad5-VII– particles are stiffer behavior is reminiscent of our previous observation on the than Ad5-wt. mechanics of Ad5 mature and immature particles, where we Two possible reasons for stiffening in Ad5 have been de- found that upon maturation the core becomes softer, but the scribed. First, upon maturation cleavage of proteins VII and capsid becomes stiffer due to an increase in internal pressure ␮, the screening of DNA-DNA electrostatic repulsion by (18). the core proteins is decreased, resulting in an increase of in- To investigate whether particle stiffening in Ad5-VII– ternal pressure and a stiffer particle (18). Second, a point was also related to changes in genome condensation and mutation in capsid protein VI that partially hinders its mat- internal pressurization, we modified the factors govern- uration cleavage also produced an increase in particle stiff- ing the energetics of confined DNA (DNA bending and ness (46). Since it has been observed that the Ad5-VII– par- DNA-DNA electrostatic repulsion) by incubation with the ticles also present deficient proteolytic cleavage of protein condensing agent spermidine (18,27,51). Spermidine is a VI (24), we next investigated the mechanical properties of trivalent polyamine that screens the phosphate negative the Ad5-VII– core, to clarify the cause of the stiffening ob- charges, induces dsDNA condensation in solution and acts served. inside virus capsids changing the DNA conformation in- side phages (52,53). We have previously shown that sper- sp midine decreases the Ad5-wt elastic constant to kwt = Absence of protein VII softens the Ad5 core 0.49 ± 0.02 N/m (mean ± SEM, N = 36), but does not After the first deformation induced by the AFM tip has change the elasticity of empty particles, indicating that this fractured the capsid shell (Figure 2, top right inset), succes- polyamine can diffuse through the capsid shell to mod- sive indentations probe directly the viral core through the ify the electrostatic interaction between DNA strands (18). opened crack (18). Figure 3A presents the typical evolution We now find that upon incubation with spermidine, the sp of Ad5-wt and Ad5-VII– particles in a multiple indenta- Ad5-VII– particle spring constant decreased to kVII− = tion experiment. The first indentation (FIC#0) on the intact 0.55 ± 0.03 N/m (mean ± SEM, N = 36), which is sim- shell (Figure 3A#0) evidences a linear behavior according ilar to that of Ad5-wt (Figure 2). This result indicates that to the thin shell theory (33). This initial indentation opens genome condensation induced by spermidine softens the a crack at the icosahedral facet with a similar size to the Ad5-VII– particle, and therefore informs about the role of apex of the AFM tip (∼20 nm) (Figure 3A#1), allowing di- protein VII in screening DNA negative charges. rect access of the tip to the core. As previously observed for Together, our data on core and particle stiffness show that Ad5 mature and immature particles (18), subsequent FICs the absence of protein VII loosens the core compaction, de- present a non-linear Hertzian behaviour (Supplementary creasing the rigidity of the virus core (Figure 3B) and in- Figure S1) in contrast with the linear deformation found in creasing the pressure within intact particles (Figure 2). We intact particles (Figure 2, left chart inset). During the first can estimate the magnitude of the change in internal pres- indentation after capsid cracking (Figure 3A, #1), Ad5-wt sure using the continuous elasticity prediction for the spring cores are not deformed beyond 40 nm at 3 nN, while Ad5- constant of a pressurized thin spherical shell indented by a VII– cores undergo larger indentations, up to ∼60 nm, at point force (54): forces as low as 2 nN (Figure 3B). This difference indicates that it is easier for the AFM tip to penetrate/deform Ad5-  2 1 VII– than Ad5-wt cores. That is, in the absence of protein π τ −1 2 k1 = k0   (1) VII the Ad5 core is softer and less condensed. 2 arctanh 1 − τ −2  12 Absence of protein VII increases the Ad5 internal pressure Here, τ = p R1 /k0 is a dimensionless parameter com- It is generally accepted that viral genomes modulate the me- paring the relative strength of pressure p against the elastic chanics of virus particles by increasing their stiffness (47– constant of the unpressurized shell k0 , and R1 is the middle 50). If the genome is assumed to be a solid material fill- radius of the adenovirus capsid shell ( R1 = 38 nm). Tak- ing the cavity of the capsid, the deformation of the capsid- ing k0 = kwt = 0.58 N/m, and k1 = kVII− = 0.66 N/m, genome ensemble can be simplified by considering genome and solving Equation (1) for p, we obtain an estimate of 3.4 and capsid as individual springs associated in parallel. The ± 0.7 MPa for the increase of pressure due to the lack of AFM tip roughly compress the shell (kE ) and the core by protein VII. This variation of pressure is similar to that gen- the same amount d. Thus the total force on the AFM tip be- erated during maturation (3 ± 1 MPa) (18), consistent with comes –(kE d + kcore d) = –(kE + kcore ) d, yielding and equiv- both effects resulting from changes in genome compaction alent spring constant kE + kcore as in the case of springs in by the core proteins. An increase in internal pressure is also 9236 Nucleic Acids Research, 2019, Vol. 47, No. 17 Figure 3. Mechanical properties of the core. (A) Images showing the evolution Ad5-wt and Ad5-VII– particles during multiple indentation assays. Images are colored by height, as indicated by the color bar at the right hand side. FICs can be found in Supplementary Figure S1. (B) FIC curves corresponding to the first indentation performed on the core of 8 Ad5-wt (red) and 12 Ad5-VII– (blue) particles. (C) Evolution of the core (as indicated by the minimum height in the crater, grey crosses) for eight Ad5-VII– and six Ad5-wt particles. Insets: Examples of topographical profiles obtained through the crater opened by the indentation. consistent with the behavior predicted by theoretical mod- the evolution of this parameter along six consecutive inden- els for weaker protein-DNA interaction regimes (11). tations for eight Ad5-VII– and six Ad5-wt particles (Figure 3C) showed that, indeed, the core components are leaving Absence of protein VII facilitates genome diffusion out of the faster the Ad5-VII– than the Ad5-wt cracked particles. Ad5 capsid To further explore this aspect, we also compared how soon unpacked DNA was observed when particle disrup- In multiple indentation experiments, elongated structures tion was induced by mechanical fatigue assays (M&M). Me- with a height compatible with dsDNA strands (55) (Figure chanical fatigue caused by repeated AFM imaging of the 3A, yellow) appeared on the substrate surrounding Ad5- same particle at very low forces (∼100 pN) promotes grad- VII– particles after the first fracture of the shell (Figure ual disruption of the capsid, while allowing real time mon- 3A#1, top), but not in Ad5-wt (Figure 3A#1, bottom). Ma- itoring of the disassembly pathway from the initially intact terial consistent with unpacked DNA seemed to be more to the final collapsed state (Figure 4A and videos SM1 and abundant in Ad5-VII– particles that in Ad5-wt for the SM2) (21). Complete particle disruption by mechanical fa- equivalent indentation (Figure 3A). In a previous work we tigue requires a different number of images for each individ- showed that combining AFM with fluorescence microscopy ual particle. Thus, we quantify the time required to first ob- in a similar unpacking experiment resulted in light emission serve dsDNA expelled from the capsid during a fatigue ex- in the presence of the dsDNA accesibility reporter YOYO1 periment as a percentage of the total particle life time. That (20). This qualitative observation suggests that it is easier is, 0% experiment time refers to the initial image when the to exit the disrupted shell for the VII-free genome than for particle is intact, and 100% to the final image when the par- the VII-bound one. An alternative possibility would be that ticle is collapsed. These experiments indicated that dsDNA the VII-free genome has a larger tendency to adsorb to the exits Ad5-VII– particles earlier (30% experiment time) than mica substrate than the VII-bound DNA. Measurements of Ad5-wt (70% experiment time) (Figure 4B). the topographical profile across the crater produced by the Because of the experimental design, at the end of mechan- successive indentations support the first possibility. Because ical fatigue or multiple indentation assays most of the ana- topographical profiles include both the crater and its rims, lyzed particles have released their genome. However, in sin- we used the lowest height inside the crack as an indicator gle indentation assays, only one image is taken after break- of the remaining core contents (Figure 3C, insets). Plotting Nucleic Acids Research, 2019, Vol. 47, No. 17 9237 densed genome in Ad5-VII– that leaves the capsid faster than in Ad5-wt. Protein VII changes the organization of the Ad5 genome in virio and ex virio In virio. To investigate how the absence of protein VII re- flects on the Ad5 core organization inside the intact parti- cle, we compared cryo-EM icosahedrally averaged maps for the Ad5-wt and Ad5-VII– particles (Figure 5). Although the Ad5 core is not icosahedrally ordered, interesting dif- ferences could be observed. In the Ad5-VII– particle, the core density was weaker and more heterogeneous than in Ad5-wt, as can be appreciated from visual inspection of the map cross-sections (Figure 5A) and comparison of gray level statistics. When gray levels in the region correspond- ing to the hexon shell were normalized to the same value range in both maps, in the Ad5-VII– core the average gray level was lower, and the standard deviation larger (Supple- mentary Figure S2 and Table S2). This observation indi- cates that, when protein VII (which accounts for 26–30% of the core mass) is not present, the particle contents are more loosely distributed within the capsid cavity. This looser con- formation is consistent with the changes in core condensa- tion detected with AFM experiments. In the Ad5-VII– map, three weak concentric shells appear near the inner wall of the capsid that are not present in Ad5- wt (Figure 5A). The distance between shells (2.4 and 2.9 nm, Figure 5B) is similar to the distances observed between the concentric layers of naked dsDNA in tailed bacteriophages (56,57). The presence of these weak layers points to an or- ganization of the Ad5-VII– genome somewhat similar to that found in viruses not packaging proteins. This organiza- tion agrees with theoretical models predicting that weaken- ing the interactions between condensing proteins and DNA would result in an increased tendency to form layers be- neath the confining capsid surface (11). Ex virio. More information on the conformation of the Ad5 core nucleoprotein complex can be derived from the observation of unpacked contents at the end of in situ AFM mechanical fatigue assays (videos SM1 and SM2, Supple- mentary Figure S3). Fibered shapes (yellow) and homoge- neously dispersed formations (blobs, red) surround the ma- jor debris located at the center of the images (Figure 6A and Supplementary Figure S3). The characterization of struc- tures with a size similar to the radius of the AFM tip apex is strongly influenced by dilation artefacts in the lateral di- Figure 4. Genome exposure in mechanical fatigue experiments. (A) Topo- mension (58). However, vertical measurements are not af- graphical images of one Ad5-VII– (left) and one Ad5-wt (right) particle fected by these dilation effects. Therefore, we used the height at different times in the experiment (indicated as percentage of the total as a size indicator of these objects to find that the vertical experiment time). (B) Boxplot showing the percentage of experiment time when the genome was observed on the mica surface. thickness of the fibered shapes is compatible with the ds- DNA diameter as measured by AFM (55). A close-up view of these areas reveals that some structures can be resolved as individual filaments (Figure 6B). However, topographical age of the capsid, and released DNA is not always ob- profiles show that in Ad5-wt the filament diameter is larger served, indicating that the genome can remain inside the than in Ad5-VII– (Figure 6C, inset). A statistical analysis cracked capsid shell. Unpacked DNA was observed in only (Supplementary Figure S5, Materials and Methods)) con- 3% of Ad5-wt particles after a single indentation, in con- firmed this aspect, with average filament heights of ∼2 nm trast to 42% of the Ad5-VII– particles (Supplementary Ta- for Ad5-wt and 1.5 nm for Ad5-VII– (Figure 6C). Also, fila- ble S1). All these results are compatible with a less con- ment heights presented a broader dispersion (∼1 to ∼3 nm) 9238 Nucleic Acids Research, 2019, Vol. 47, No. 17 Figure 5. Organization of the core inside the virus particles. (A) Central section of 3D cryo-EM maps of Ad5-VII– and Ad5-wt. Higher density is shown in white. White arrows highlight the three concentric shells in Ad5-VII. (B) Radial average profiles of the Ad5-VII– and Ad5-wt maps. in Ad5-wt. We interpret the larger thickness in Ad5-wt fila- small blob would be 324 19 × [ rrLS ]3 ∼9. However, several con- ments as being caused by the association of different regions siderations from the previous literature would be reluctant in the dsDNA genome. The thickness dispersion indicates with the formation of these VII nonamers. First, since in that this association would include at most three dsDNA one Ad5 virion there are 252 capsomers (240 hexon trimers chains. These results suggest that the presence of protein VII + 12 pentons) and between 500 and 800 copies of protein promotes condensation of dsDNA by formation of bundles, VII (13,14), a fully disrupted particle should present at most perhaps via bridging. (800/9)/(252 + (800/9)) ∼26% of small objects. In our Ad5- Topographical profiles (Figure 6D, inset) also pointed out wt images we observe ∼38% of small objects, above the to different heights for the blobs in the Ad5-wt and Ad5- highest expected value if the protein formed nonamers. This VII– images (Figure 6A and B). These particles likely corre- excess could be understood if a considerable number of cap- spond to coat and core proteins coming from the collapsed somers remained bound forming capsid debris located at virus structures after fatigue. We carried out a statistical the center of the AFM images (Figure 6A and Supplemen- analysis of the blob heights for 11 Ad5-wt and 10 Ad5- tary Figure S3). Second, assuming that the small blobs con- VII– disrupted particles analyzed in ∼10 different exper- tain 9 protein VII monomers, the estimated number of VII imental sessions. A histogram of normalized blob heights oligomers per viral particle (between 55 and 88) would be was calculated for each virus particle (Supplementary Fig- 2–4 times smaller than the number of beads previously ob- ure S5). Then, histograms for all Ad5-wt or all Ad5-VII– served for pyridine cores (60). Third, a high order multi- particles were averaged (Figure 6D). These charts reveal a mer of VII would also be incompatible with previous cross- major population of objects between 12 and 13 nm for both linking results on disrupted virions and extracted cores, Ad5-wt and Ad5-VII-, compatible with the size of aden- where only dimers were detected (61). ovirus capsomers (15,59). An additional population at ∼10 It is also possible that we are overestimating the amount nm was observed in Ad5-wt but not in Ad5-VII-. This size of VII monomers per blob, if part of the mass considered is close to the 9 nm beads previously observed in metal corresponds not only to protein VII, but also to associ- shadowed cores after high ionic strength treatments that ated DNA. In fact, an interesting model can be proposed removed most core proteins except VII (16). We classified if we consider each blob as a unit formed by one or sev- both the small and large blobs in Ad5-wt particles in two eral molecules of protein VII, wrapped or otherwise asym- categories, depending on whether they were in contact with metrically associated with a certain region of DNA. Protein the DNA or isolated on the mica surface (Figure 6A). Ob- VII appears to interact with DNA to a large extent elec- jects found isolated on the substrate corresponded mainly trostatically, as indicated by the experiments with spermi- to the larger blobs (Figure 6E, empty red), while most of dine (Figure 2). In each of these VII-DNA units, the re- the smaller blobs appeared associated with filaments (Fig- pulsive monopole electrostatic interactions would be effec- ure 6E, solid red), pointing to their interaction with ds- tively screened. Remaining higher order (multipole) inter- DNA. That is, Ad5-wt collapsed particles release objects actions in the protein-DNA complex combined with short smaller than capsomers and located preferentially in asso- range (steric) repulsion may act as effective sticky ‘patches’ ciation with DNA strands, that do not appear when Ad5- and induce the formation of particularly stable clusters with VII– particles are disrupted. Therefore, these objects must conformations determined by the structural and interaction correspond to the presence of core protein VII. anisotropy of the individual complexes, such as cubes (8 ef- We can estimate the number of protein VII monomers in fective particles), octahedra (6 effective particles) or tetra- a small blob by comparison with the known mass of the hedra (4 effective particles) (62). Depending on the inter- hexon trimer forming the larger blobs. The molecular mass action details, clusters may be more or less polydisperse, of a hexon trimer is 324 kDa, while the molecular mass of but it is worth noting that we observe a reasonably sharp mature protein VII is 19 kDa (19). If we consider the large peak in the AFM small blob size distribution (Figure 6D) and small blobs as spheres with radii rL ∼6.5 and rS ∼ 5 which suggests a largely monodisperse cluster population. nm, respectively, the number of protein VII monomers per The fact that clusters do not form in Ad5-VII– cores sug- Nucleic Acids Research, 2019, Vol. 47, No. 17 9239 Figure 6. Analysis of unpacked core contents. (A) Example images showing the final stages of Ad5-VII– (left) and Ad5-wt (right) particles after mechanical fatigue disruption. (B) Close-up details of the released contents. (C) Statistics of fibered material heights calculated as explained in Supplementary Figure S4. The inset shows two example topographical profiles across the fibered material (blue and red solid lines in B). (D) Histograms of the blob height distribution obtained for 11 Ad5-wt (red) and 9 Ad5-VII– (blue) disrupted particles. Solid lines present the average of the dotted lines coming from individual particles (Supplementary Figure S5). Inset: example topographical profiles across the blobs (dashed lines in B). (E) Histograms showing the height of the Ad5-wt blobs in contact (red) and not in contact (white) with DNA. The equivalent histogram for Ad5-VII– in shown in Supplementary Figure S5C. (F) Cartoon showing a hypothetical model for the condensing role of protein VII. Green, DNA; blue, protein VII; red, protein ␮. The inset illustrates the situation where the bridging action of ␮ is hindered by the absence of clusters formed by the VII-DNA complexes. 9240 Nucleic Acids Research, 2019, Vol. 47, No. 17 gests that protein VII is essential for their formation. The disrupted capsid while trafficking across the cytosol, before clustering model should be taken with some care, as it does reaching the nuclear pore. not take explicitly into account the role of other core pro- teins and the fact that the Ad5 DNA is a single topological entity. If this model is correct, it would mean that the inter- DATA AVAILABILITY action between the protein-DNA complexes is more com- The Ad5-wt and Ad5-VII– maps have been deposited at the plicated than previously proposed in the full confinement Electron Microscopy Databank with ID EMD-4448 (Ad5- (isotropic, weakly repulsive potential) (17). In any case, our wt) and EMD-4424 (Ad5-VII-). data show that objects compatible with multimers of protein VII bound to DNA, and bundles of dsDNA, are present in Ad5-wt unpacked cores, but not in Ad5-VII-. SUPPLEMENTARY DATA Supplementary Data are available at NAR Online. CONCLUSIONS ACKNOWLEDGEMENTS We have used AFM and cryo-EM to explore how the ab- We acknowledge the CNB-CIB cryo-EM facility for data sence of core protein VII influences the physical properties acquisition, and Mara I. Laguna for expert technical help. and core organization of human adenovirus particles. In the Author contributions: N.M.G. performed AFM experi- absence of protein VII, the Ad5 particle becomes stiffer and ments. M.H.P., M.P.I. and C.S.M. carried out cryo-EM the core becomes softer. Incubation with spermidine softens studies. P.O., P.H., M.P.I. and G.N.C. produced virus the Ad5-VII– particle. Thus, our results support the role of samples. D.R. performed theoretical calculations. N.M.G., protein VII in screening the DNA negative charges, thereby M.H.P., C.S.M., A.S. and P.J.dP. analyzed data. C.S.M. and modulating capsid pressure. A decrease in the core conden- P.J.dP. coordinated the study and wrote the manuscript, sation when VII is not present accelerates genome diffusion with input from all other authors. through fractures of the capsid shell. Therefore, the defect in virus entry observed in Ad5-VII– particles (24) is not due to either lack of internal pressure or trapping of the genome FUNDING inside the particle. Our images of the Ad5 core as it is pro- gressively extracted from the capsid in a physiological envi- Spanish Ministry of Economy and Competitivity [FIS2017- ronment do not show beads-on-a-string structures as pre- 89549-R; “Marı́a de Maeztu” Program for Units of Ex- viously shown in released cores dried on a surface for EM cellence in R&D MDM-2014-0377; and FIS2017-90701- analysis where most proteins but VII had been chemically REDT] and Human Frontiers Science Program [HFSPO removed (16). Rather, we find beads that can be attributed RGP0012/2018] to P.J.P. Spanish State Research Agency to clusters of protein VII-DNA complexes, adjacent to bun- and European Regional Development Fund [BFU2016- dles consisting of a few dsDNA chains that suggest con- 74868-P] and Spanish Ministry of Economy, Industry and densing by a bridging interaction (Figure 6A and B, right). Competitivity [BIO2015-68990-REDT, the Spanish Aden- Since the bridging seems to happen away from the actual ovirus Network, AdenoNet] to C.S.M. MINECO/FEDER, position of the VII-DNA cluster, we propose that protein UE [FIS2015-67837-P] to D.R. National Institutes of VII promotes bundling of DNA by facilitating the bridging Health [CA122677 and AI102577] to P.H. M.H.-P. is a re- action of the small core protein ␮ (18). The 9 kDa precursor cipient of a Juan de la Cierva postdoctoral contract funded form of protein ␮ is cleaved in three 20–30 residue long frag- by the Spanish Ministry of Economy and Competitiv- ments by the Ad5 protease (19), which would correspond to ity. M.P.-I. holds a predoctoral fellowship funded by La objects of radii ∼1.3 nm as estimated in a similar way as for Caixa Foundation. Funding for open access charge: Secre- the red blobs above. We suggest that protein VII could as- tarı́a de Estado de Investigación, Desarrollo e Innovación sist the action of ␮ by bending the DNA molecule so that [FIS2017-89549-R]. two branches of it become closer, within the bridging range Conflict of interest statement. None declared. of ␮ (Figure 6F). Although the condensing action of protein VII is not REFERENCES needed for DNA packaging during adenovirus assembly, it 1. Luijsterburg,M.S., White,M.F., Driel,R. and Dame,R.T. (2008) The might play a role in other steps of the infectious cycle. For major architects of chromatin: architectural proteins in bacteria, instance, it has been proposed that protein VII protects the Archaea and Eukaryotes. Crit. Rev. Biochem. Mol. Biol., 43, 393–418. Ad5 genome from the cellular DNA damage response once 2. 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