The role of capsid maturation on adenovirus priming for sequential uncoating

The Role of Capsid Maturation on Adenovirus Priming for Sequential Uncoating * □ S Received for publication, June 8, 2012, and in revised form, July 11, 2012 Published, JBC Papers in Press, July 12, 2012, DOI 10.1074/jbc.M112.389957 Ana J. Pe´rez-Berna´ ‡1 , Alvaro Ortega-Esteban §2 , Rosa Mene´ndez-Conejero ‡3 , Dennis C. Winkler ¶ , Margarita Mene´ndez 储 , Alasdair C. Steven ¶ , S. Jane Flint**, Pedro J. de Pablo § , and Carmen San Martín ‡4 From the ‡ Department of Macromolecular Structure, Centro Nacional de Biotecnología, and § Department of Physics of Condensed Matter, Universidad Auto´noma de Madrid, 28049 Madrid, Spain, the 储 Instituto de Química Física Rocasolano and CIBER de Enfermedades Respiratorias, 28006-Madrid, Spain, the ¶ Laboratory of Structural Biology, NIAMS, National Institutes of Health, Bethesda, Maryland 20892, and the **Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544 Background: Adenovirus proteolytic maturation is required for correct uncoating in the cell. Results: Maturation makes the virion metastable and facilitates penton and peripheral core protein release, as well as cooperative genome ejection. Conclusion: Precursor proteins act as scaffolds favoring assembly. Maturation primes adenovirus for uncoating. Significance: Identifying the molecular determinants of virus stability and uncoating is key to understanding the infectious cycle. Adenovirus assembly concludes with proteolytic processing of several capsid and core proteins. Immature virions containing precursor proteins lack infectivity because they cannot properly uncoat, becoming trapped in early endosomes. Structural studies have shown that precursors increase the network of interactions maintaining virion integrity. Using different biophysical techniques to analyze capsid disruption in vitro, we show that immature virions are more stable than the mature ones under a variety of stress conditions and that maturation primes adenovirus for highly cooperative DNA release. Cryoelectron tomography reveals that under mildly acidic conditions mimicking the early endosome, mature virions release pentons and peripheral core contents. At higher stress levels, both mature and immature capsids crack open. The virus core is completely released from cracked capsids in mature virions, but it remains connected to shell fragments in the immature particle. The extra stability of immature adenovirus does not equate with greater rigidity, because in nanoindentation assays immature virions exhibit greater elasticity than the mature particles. Our results have implications for the role of proteolytic maturation in adenovirus assembly and uncoating. Precursor proteins favor assembly by establishing stable interactions with the appropriate curvature and preventing premature ejection of contents by tightly sealing the capsid vertices. Upon maturation, core organization is looser, particularly at the periphery, and interactions preserving capsid curvature are weakened. The capsid becomes brittle, and pentons are more easily released. Based on these results, we hypothesize that changes in core compaction during maturation may increase capsid internal pressure to trigger proper uncoating of adenovirus. Adenovirus is a mild pathogen for humans but can become clinically relevant in immunocompromised patients, and it is widely examined as a therapeutic vector (1, 2). The large (⬃950 Å) nonenveloped, pseudo T ⫽25 icosahedral capsid is assembled from at least 11 different types of proteins. The general icosahedral architecture can be described as two different systems of tiles. Nine trimers of the major coat protein, hexon, formthecentralplateofeachfacet,knownastheGroupofNine (GON). 5 Thefiveperipentonalhexontrimers,togetherwiththe penton base, form the second tile system, known as Group of Six (GOS) (3). Trimeric fibers protrude from each vertex. Minor coat proteins IIIa, VI, VIII, and IX are required for correct capsid assembly and occupy specific positions in the capsid (3). Polypeptide IX has an extended structure and forms a sort of hairnet on the outer surface of the virion, keeping together the hexon trimers in each GON and binding GONs to GONs across the icosahedral edges. Each GON is further stabilized by copies of polypeptide VIII located around the icosahedral 3-fold symmetry axis inside the capsid. Also on the inner capsid surface, the N-terminal domain of polypeptide IIIa mediates the interaction between penton base and the peripentonal hexons to keep each GOS together. Finally, IIIa and VIII cooperate to bind each GOS to its five surrounding GONs. The *Thisworkwassupported,in wholeorin part,byNational InstitutesofHealth Grants GM037705 and AI1058172 (to S. J. F.). This work was also supported by Ministry of Science and Innovation of Spain Grants BFU2010-16382/ BMC (to C. S. M.), MAT2008-02533, PIB2010US-00233, and FIS2011-29493 (to P. J. P.), FIS2010-10552-E and FIS2011-16090-E (to C. S. M. and P. J. P.), and BFU2009-10052 (to M. M.), and by Local Madrid Government Grant P2009/MAT-1467 (to P. J. P.). □ S This article contains supplemental Figs. S1–S6, Table S1, and Movies S1–S7. 1 Recipient of Juan de la Cierva Postdoctoral Contract JCI-2009-05187 from the Ministry of Science and Innovation of Spain and recipient of additional support from Spain CSIC Travel Grant PA1002892. 2 Recipient of FPU predoctoral fellowship from the Ministry of Education of Spain. 3 Recipient of Predoctoral Fellowship FI08/00035 from the Instituto de Salud Carlos III of Spain. 4 To whom correspondence should be addressed: Centro Nacional de Biotecnología. Darwin 3, 28049-Madrid, Spain. Tel.: 34-91-5855450; Fax: 3491-5854506; E-mail: [email protected]. 5 The abbreviations used are: GON, Group of Nine; DSC, differential scanning calorimetry; BIR, breakage/indentation ratio; GOS, Group of Six; AVP, adenovirus protease; vp, virus particles; PI, propidium iodide; AFM, atomic force microscopy; EF, extrinsic fluorescence; N, newton. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 37, pp. 31582–31595, September 7, 2012 Published in the U.S.A. 31582 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287•NUMBER 37•SEPTEMBER 7, 2012 This is an Open Access article under the CC BY license. remaining minor capsid protein, polypeptide VI, could not be traced in this study, but it has been assigned to density within the internal cavity of each hexon trimer (4, 5). The adenovirus core contains a single copy of the dsDNA genome and over 25 MDa of protein, including four DNA-binding viral proteins: polypeptidesV,VII, ␮ ,andtheterminal protein. A few copies of the adenovirus protease (AVP) are also packaged (6). The disposition of DNA and proteins in the core is unclear. Adenovirus cell entry has been extensively studied using group C human adenoviruses; these are most commonly used for vector development, in particular the highly homologous types 5 (HAdV-5) and 2 (HAdV-2) (7, 8). Adenovirus uncoating in the cell proceeds in a stepwise manner (9). The virus starts to disassemble at the plasma membrane, where upon binding to its receptor some fibers are released (10), and the penton base undergoes a conformational change that might result in weakening its interactions with the rest of the capsid (11). The virus is then internalized, and disassembly continues in the early endosome with release of some internal components, such as minor coat proteins IIIa, VI, VIII, and core polypeptide V (9, 12). This is a crucial step for infection, as polypeptide VI plays the key role of altering the endosomal membrane to facilitate virus escape to the cytosol (13). The partially disassembled virion does not proceed to later stages of the endocytic pathway butescapes the earlyendosome(14).Atthis point theremustbe a certain degree of dsDNA exposure to initiate inflammatory responses (15), but the virion is still stable enough to survive transport along microtubules to the nuclear pore (16, 17). There, the final phase of uncoating takes place. Upon complete dismantling of the weakened capsid, the virus DNA, together with major core protein VII, enters the nucleus (18–21). Correct adenovirus uncoating is tightly linked to maturation. Cleavage of several structural proteins by AVP is required to yield the mature infectious virion (22). Immature virions contain the precursor versions of coat proteins IIIa, VI, and VIII and of core proteins VII, ␮ , and terminal protein. An experimental model for immature adenovirus is the HAdV-2 ts1 mutant (23). When grown at the nonpermissive temperature of 39 °C, HAdV-2 ts1 does not package AVP (24) and produces capsids containing the unprocessed protein precursors. Viral genomepackagingisunimpaired,butthevirusisnotinfectious. It has been shown that the defect in infectivity is linked to a defect in uncoating. Immature ts1 attaches to the host cell and follows the same internalization process as the wild-type (WT) virus but fails to escape the endosome and is recycled to the membrane or degraded in lysosomes (14, 25). Cryo-EM structural studies showed that in the immature virus, the presence of uncleaved precursors reinforces the network of interactions holding the virion together at three levels. First, the virus core is more compact and stable due to the condensing action of unprocessed polypeptides pVII and pre- ␮ ; second, a “molecular stitch” formed by pIIIa and pVIII underpins the boundary between GOS and GONs (5, 26); and third, there is a more ordered interaction between pVI and the inner cavity of each hexon in the capsid (4, 5). To further investigate how the presence of uncleaved precursors translates into a deficient uncoating phenotype, we have compared the in vitro stability and disassembly of ts1 and WT virions under different types of stress: thermal, chemical, or mechanical. We report the differences between mature and immature virions and discuss their implications for assembly and uncoating. EXPERIMENTAL PROCEDURES Virus Production and Purification—We used as wild-type control for mature virions the E1-deleted HAdV-5 variant Ad5GL (27), propagated in HEK293 cells. Ad5GL is completely WT for all structural polypeptides. Immature virus was obtained by propagating the HAdV-2 ts1 mutant in HeLa cells at 39.5 °C, as described (5). Particles were purified by equilibrium centrifugation in CsCl gradients, desalted on a Bio-Rad 10 DC column, and stored in 20 mMHepes, pH 7.8, 150 mMNaCl plus 10% glycerol at ⫺70 °C. Virus titers were 5 ⫻10 12 virus particles (vp)/ml for Ad5GL, and 1 ⫻10 13 vp/ml for ts1. Differential Scanning Calorimetry—DSC measurements were performed using a Microcal VP-DSC instrument (Microcal, Inc., Northampton, MA) at a heating rate of 60 °C/h and under an extra constant pressure of 2 atm to prevent degassing during the scan. The VP-viewer package was used for data acquisition. Samples of WT and ts1 at ⬃5–7 ⫻10 12 vp/ml were dialyzed against 8 mMNa 2 HPO 4 ,2mMKH 2 PO 4 , 150 mMNaCl, and 0.1 mMEDTA, pH 7.4. The reference cell was loaded with buffer from the last dialysis, which was also used to run the buffer-buffer baseline. Apparent excess heat capacity curves were obtained after subtraction of the buffer-buffer baseline from the experimental heat capacity profiles and division by hexon molar concentration. The Origin-DSC software package (7.0 version) was used for data analysis. The excess heat capacity profiles were theoretically analyzed in terms of independent, irreversible two-state transitions (N i 3D i ) according to Equation 1 (28), ⌬Cpex共T兲⫽ 冘 i⫽1 6⌬Hi appEi app RTmi 2⫻exp 冉 Ei app共T⫺Tmi兲 RTmi 2 冊 ⫻exp 冉 ⫺exp 冉 Ei app共T⫺Tmi兲 RTmi 2 冊冊 (Eq. 1) where ⌬Cp ex (T) is the excess heat capacity value at temperature T;T mi is the temperature of the maximum for transition i; ⌬H i app and⌬E i app aretheapparent calorimetric enthalpy change and the activation energy for the same transition, and Ris the gas constant. Fluorescence Spectroscopy—Extrinsic fluorescence spectra were obtained from virus preparations subject to different kinds of stress: high temperature, pyridine, or acidic pH. Mature and immature virus samples (5 ⫻10 10 vp/ml) were incubated in different buffers, depending on the experiment. Thermal disruption experiments were carried out in 8 mM Na 2 HPO 4 ,2mMKH 2 PO 4 ,150 mMNaCl, and 0.1 mMEDTA, pH 7.4. For pH disruption experiments, the sample was diluted and subsequently dialyzed overnight at 4 °C against buffers at the appropriate values of pH: HCl/KCl in the 1–2.5 pH range, citric acid/sodium citrate at pH 3–6, and Na 2 HPO 4 /KH 2 PO 4 at pH range 6.5–7.5, always with 150 mMNaCl and 0.1 mMEDTA. Fluorescence emission spectra were obtained employing a Hitachi Model F-2500 FL spectrophotometer equipped with a How Maturation Primes Adenovirus for Uncoating SEPTEMBER 7, 2012•VOLUME 287•NUMBER 37 JOURNAL OF BIOLOGICAL CHEMISTRY 31583 Peltier temperature control device. 1 mMpropidium iodide (PI) (Molecular Probes) was added to the sample that was then allowed to equilibrate for 5 min before data acquisition. Sample volumes of 0.150 ml were used in sealed quartz cuvettes. The sample was excited at 535 nm, and fluorescence emission was monitored from 580 to 700 nm, using excitation and emission slit widths of 8 nm. Raw spectra were corrected by subtraction of the PI spectrum at each tested condition. The ratio of PI fluorescence to the initial emission (I/I 0 ) at the maximum position ( ␭ max ⫽607 nm) ⫾S.E. for three independent experiments was plotted as a function of stress condition values. Fluorescence intensity changes were fitted to a sum of sigmoids using the Origin software package, according to Equation 2, I/I0⫽A1⫹B1⫺A1 1⫹exp 冉 C1⫺x C1D1 冊 ⫹ 冘 i⫽2 n 冢 Bi⫺1⫹Bi⫺Bi⫺1 1⫹exp 冉 Ci⫺1⫺x CiDi 冊 冣 (Eq. 2) where A i and B i are the lower and upper platform values for each sigmoid (notice that starting from the second sigmoid, the lower platform A i is forced to coincide with the previous upper platform B i⫺1 ); C i is the transition midpoint; and D i is the slope. The number of sigmoid curves in the summatory (up to n⫽3) was the minimum necessary to fit the experimental curves, based on best R 2 values. The fluorescence thermal-disruption curve was also fitted in terms of the model used to analyze the calorimetric curves using Equation 3, I/I0共T兲⫽IF⫺共IF⫺1兲⫻ 冘 i⫽1 3 fi ⫻exp 冉 ⫺exp 冉 Ei app共T⫺Tmi兲 RTmi 2 冊冊 (Eq. 3) where I F is the final value of I/I 0 , and f i is the relative contribution of transition ito the total variation of PI fluorescence (29). Negative Staining Electron Microscopy—For imaging of disassembly products, samples treated as described for fluorescence spectroscopy were adsorbed, at a concentration of 5 ⫻ 10 11 vp/ml, onto glow-discharged, collodion/carbon-coated EM grids, negatively stained with 2% uranyl acetate, and observedinaJeol 1200EX-II transmission electron microscope. Percentages of intact, damaged, and collapsed capsids were quantified by counting the number of particles of each type per micrograph. Error bars refer to differences between different micrographs. Cryoelectron Tomography—Viral samples subject to different kinds of stress were mixed at a concentration of 1 ⫻10 12 vp/ml with 10-nm colloidal gold particles (AURION, Wageningen, The Netherlands) and vitrified as described (30). A Tecnai-12 electron microscope (FEI, Hillsboro, OR) operating at 120 kV with a LaB6 source and equipped with an energy filter (GIF 2002; Gatan, Pleasanton, CA) was used to collect tilt series at ⬃4- ␮ m underfocus, covering the range ⫾70° in 2° increments. The data were acquired using the SerialEM package (31), for a total dose of ⬃70–80 electrons/Å 2 per series. Images were recorded on a 2048 ⫻2048-pixel CCD camera (Gatan, Pleasanton, CA) at ⫻38,500 magnification (7.8 Å/pixel). Data were pre-processed and aligned using the IMOD software package (32), with gold particles as fiducial markers. The final aligned tilt series were normalized and reconstructed using the simultaneous iterative reconstruction technique implemented in Tomo3D (33). Individual virus particles were extracted from tomograms using IMOD. Tomograms were denoised by 100 iterations of anisotropic nonlinear diffusion (34). The in-plane resolution of the full tomograms was in the 43–55-Å range, as estimated using the Bsoft program TOMORES (35). Dihedral angles were measured from central slices of single virion tomograms using the angle measurement tool in Adobe Photoshop. A virion was considered to conserve fibers if at least one fiber was clearly visible in the denoised tomogram. The presence or absence of pentons was determined manually by observation of single virion tomogram slices after alignment with an icosahedral three-dimensional map as a reference, and comparison with model maps of full and pentonless virions. Radial profiles were calculated from average virus tomograms calculated after aligning each individual map with an icosahedral reference. Icosahedral symmetry was considered for alignment but not imposed in the final averaged map or in the individual aligned maps, except for supplemental Fig. S6. Alignment was carried out using maximum likelihood procedures for tomography as implemented in XMIPP (36, 37). Nanoindentation Assays—Stocks of WT and ts1 virus in HBS buffer (20 mMHepes, 150 mMNaCl, pH 7.8) were diluted in a solution of NiCl 2 in HBS to obtain a final solution with 5 mMof Ni 2⫹ , and adsorbed onto freshly cleaved Muscovite mica (V-1 quality). A drop of 20 ␮ l of the final solution, containing virus particlesat concentrations between 1.5and 2 ⫻10 12 vp/ml,was deposited on the substrate and incubated for 30 min at 4 °C before washing with 5 mMNiCl 2 in HBS. The AFM tip was prewetted with 30 ␮ l of the same buffer. The mica was placed on the AFM sample holder and immersed in 500 ␮ l of buffer. The AFM (Nanotec Electro´nica S.L., Madrid, Spain) was operated in Jumping Mode Plus (38) in liquid using rectangular cantilevers RC800PSA and Biolevers (BL-AC40TS) (Olympus, Tokyo, Japan) with nominal spring constants of 0.05 and 0.03 N/m, respectively. Cantilever spring constants were routinely calibrated by using the Sader method (39). AFM exploration at forces of ⬃100 pNindicated a random population of virions on the mica surface. Single virus particles were deformed by the AFM tip by performing individual indentation (force versus z-piezo displacement (FZ)) experiments. To ensure that the indentation is performed at the top of the virus, the virus shell is zoomed in continuously by reducing the x-y scanning size until the bump of the very top is under the whole piezo scan. Then a large force indentation was executed at the top of the particle. Finally, an image of the virus was taken to confirm its disruption. The maximum force applied during each indentation was high enough to How Maturation Primes Adenovirus for Uncoating 31584 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287•NUMBER 37•SEPTEMBER 7, 2012 ensurethat the virusparticlesaredestroyed.The FZ speed is about 60 nm/s (40). Images were processed using the WSxM software (41). RESULTS Thermal Capsid Disruption—To directly detect the energy requiredto disrupt thecapsid and to monitorthe stability ofthe intermediates formed along the heat-induced disassembly process, DSC was used. DSC scans of mature (WT) and immature (ts1) adenovirus at physiological ionic strength and neutral pH are shown in Fig. 1A. As expected for a large, complex macromolecular specimen, thermal denaturation of both WT and ts1 adenovirus was irreversible upon heating above 90 °C. Both thermograms show three well defined transitions (denoted as T2, T3, andT4inTable1)precededbyabroadendothermbetween36and 65 °Cinthets1 mutantandthreediscretepeaksinWTthatextend over a similar range of temperature (Fig. 1A,inset). Indeed, deconvolutionof the heat capacity profiles showed thattheoverallendotherm of both WT and ts1 adenovirus can be described in terms of six irreversible two-state transitions whose apparent transition enthalpies, activation energies, and T m values are summarized in Table 1. Thetwolast peaks of the thermograms(T m valuesat71.7 and 78.4 °C for WT and 72.8 and 81.0 °C for ts1) have previously been assigned to denaturation of the purified hexon protein (42), which is the most abundant component (⬎60%) of the protein shell. The minor transition (T 2 ) appearing at 67.3 °C for WT and 68.9 °C for ts1 seems to be related to hexon stabilization upon GON formation, as it is absent in polypeptide IXdeleted recombinant viruses (42). The three low temperature transitions (denoted as T 1A ,T 1B , and T 1C in Table 1) may correspond to capsid disassembly and reflect changes undergone by adenovirus during uncoating in the cell. However, whether they are directly due to disruption of hexon interactions or reflect denaturation of other virion components cannot at present be ascertained.Alltransitionsoccurredathighertemperaturesforts1 than for WT, with differences in the 1–2 °C range for all T m values except T 1A and T 1B , where differences were as high as 5 °C. These differences reveal an increased thermal stability of hexon and other virion structures in the immature capsid context. FIGURE 1. Thermal capsid disruption. A, thermograms showing WT and ts1 thermally induced events upon virus heating at a constant rate of 60 °C/h (10 mM phosphatebuffer, 150mMNaCl,0.1 mMEDTA,pH 7.4). Thesolid line is the theoreticalfit of theexperimental envelopes toirreversible two-state transitionsusing parameters given in Table 1. The inset shows the deconvolution of peaks likely corresponding to capsid disassembly. B, extrinsic fluorescence measurements showingDNA exposureto solventuponheating. Averagevalues anderrorbars correspondingtothe standarddeviation ofthreeindependent experimentsare plotted. Long dashed lines correspond to the fit in terms of the model used for the calorimetric curves. Short dashed lines correspond to the fit according to a multiple sigmoid function. C, histogram showing structural effects of heating on the virus particles, as analyzed by negative staining. Categories are as follows: I, intact virions; D, damaged particles, including various degrees of disruption but retaining some degree of curvature; F, flattened capsids. Error bars indicate standard deviation in the particle counts between different micrographs. The total number of particles in the dataset, N, is indicated at the top right corner. A–C,blacksymbols andlines refertoWTvirus;gray symbols andlines refertots1.D, galleryshowing examplesofintact,damaged,andflattenedcapsids. Thescale bar corresponds to 100 nm. Arrows indicate missing vertex pentons; arrowheads indicate missing GOSs. How Maturation Primes Adenovirus for Uncoating SEPTEMBER 7, 2012•VOLUME 287•NUMBER 37 JOURNAL OF BIOLOGICAL CHEMISTRY 31585 To define the conditions in which the particle undergoes partial disassembly and the DNA is exposed, we measured the DNA accessibility to solvent by using the DNA intercalating dye PI. PI fluorescence is quenched until the virus capsid is compromised and enhanced upon binding to DNA when it becomes accessible (43). As we have shown previously (5), similar degrees of DNA exposure occurred at higher temperatures for ts1 than for WT (Fig. 1B). EF measurements were fitted to multiple sigmoidal curves with parameters shown in Table 2. WT presents a fluorescence burst at 47.5 °C. The sharp slope of the curve at this temperature indicates a highly cooperative process, also observed for WT forms of HAdV-2 and HAdV-5 at ⬃45 °C (5, 44, 45). In contrast, all transitions showed low cooperativity in ts1. EF data were also analyzed using the irreversible transition model used for the DSC curves (Fig. 1B), and the best fitting parameters are compared with those derived from the heat capacity profiles in Table 3. The T m values obtained from EF experiments for WT closely agreed with those deduced from DSC (Table 3), indicating that capsid protein denaturation (DSC) and DNA exposure (EF) events are correlated. Transition overlapping in the immature virus impaired a reliable analysis of the EF curve, as around 90% of the total emission variationcanbeapparentlydescribedbya single transition with a T m value of 57.0 °C. Introduction of a third transition approached the T m values to the DSC estimates but increased significantly the errors of the thermal parameters without improving the R 2 value. Structuralchanges due tothermaldisassemblywerefollowed by EM. We had previously observed that WT capsids were completely disrupted and their contents released at 47 °C, whereas the ts1 virion only lost vertex structures (pentons and peripentonal hexons, or GOS) and extruded part of its contents as a single nucleoprotein filament (5). Here, we report a quantitative description of capsid disruption patterns at different temperatures. WT and ts1 preparations were imaged by negative staining EM after heating at the temperatures corresponding to T m values derived for WT. To interpret the EM observation, we grouped the observed particles into three categories (Fig. 1C): intact virions (I); damaged capsids, still retaining a spherical arrangement (D); and collapsed, flattened capsids (F). Fig. 1D shows examples of particles in the three categories (see also supplemental Fig. S1). At the T 1A transition temperature (40 °C), most ts1 virions were intact, whereas a considerable proportion (⬃30%) of WT capsids showed small defects consistent with loss of pentons (Fig. 1D). The fact that the interior of these capsids does not appear dark indicates that they still contain stain-excluding material (protein/DNA), in agreement withthemodestincrease inDNAaccessibilitytoPI indicatedby EF. At the T 1B transition temperature (47 °C), only 10% of WT virions retained their structural integrity, with the rest showing different degrees of damage. In particular, about 40% of the WT virions appear as collapsed flat structures, with the icosaheTABLE 1 Thermal capsid disruption parameters from deconvolution of DSC endotherms 1 ⌬Hunits are kilocalories/mol of hexon monomer (720 hexon monomers/virion). TABLE 2 EF analysis of thermal capsid disruption Fitting of thermal disruption EF data to multiple sigmoid curves. Boldface indicates transitions with high cooperativity. WT ts1 Midpoint Slope Midpoint Slope °C °C Sigmoid 1 38 ⫾2 2.8 ⫾0.5 41 ⫾1 1.1 ⫾0.1 Sigmoid 2 48 ⴞ1 170 ⴞ10 54 ⫾18⫾4 Sigmoid 3 58 ⫾34⫾1 65.0 ⫾0.4 4.6 ⫾0.6 R 2 0.99482 0.99483 How Maturation Primes Adenovirus for Uncoating 31586 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287•NUMBER 37•SEPTEMBER 7, 2012 drons split open, and large defects at the vertices consistent with loss of complete GOS. Immature ts1 virions, however, are not flattened; they retain the capsid spherical arrangement although they have lost complete GOS. At 57 °C, the vast majority (⬃80%) of WT capsids are flattened, while most ts1 virions still retain curvature, even when extensively damaged. Although capsid flattening is most likely an artifact induced by the negative staining preparation, the differences found between WT and ts1 virions suggest that capsid precursors strengthen the seams at the icosahedral edges, preserving capsid curvature. At temperatures beyond 57 °C, only isolated capsomers or small capsid fragments are observed for both WT and ts1. Chemical Capsid Disruption—Next, we subjected the adenovirus mature and immature capsids to different sources of chemical stress. Immature ts1 virions showed higher stability than WT both in the presence of denaturant compounds and, most interestingly, upon acidification. A critical point in adenovirus uncoating in vivo is acidification within the early endosome, where the luminal pH ranges between 6.5 and 6 (46). Upon acidification, the increase of PI fluorescence for WT, indicative of DNA accessibility, proceeds in a biphasic way (Fig. 2A). The first highly cooperative transition is centered at pH 6.1 and starts as the medium acidity approaches the endosomal range of pH (Table 4). In contrast, the immature virus withstood more severe values of pH (Fig. TABLE 3 Comparison between capsid thermal disruption parameters obtained from DSC and calculated from EF spectroscopic curves using the same irreversible transition model T 1A T 1B T 1C T m1A E 1A app f 1A T m1B E 1B app f 1B T m1C E 1C app f 1C °C kcal/mol °C kcal/mol °C kcal/mol WT DSC 40.0 ⫾0.3 130 ⫾30 47.4 ⫾0.2 61 ⫾6 57.2 ⫾0.3 55 ⫾6 Spectroscopy 40 ⫾625⫾10 0.23 47.8 ⫾0.1 ⬎10 3 0.30 57 ⫾238⫾10 0.47 ts1 DSC 45.1 ⫾0.4 57 ⫾5 52.3 ⫾0.7 55 ⫾10 60 ⫾140⫾10 Spectroscopy R 2 ⫽0.998 (42 ⫾40) (21 ⫾10) (0.15) (55.9 ⫾0.4) (90 ⫾40) (0.44) (60 ⫾10) (40 ⫾70) (0.41) R 2 ⫽0.997 39 ⫾339⫾20 0.1 57.0 ⫾0.2 48 ⫾4 0.9 R 2 ⫽0.996 56.8 ⫾0.2 40 ⫾1 1.0 FIGURE 2. Chemical capsid disruption: effect of acidification. A, extrinsic fluorescence measurements showing DNA exposure to solvent upon acidification. Average values and error bars corresponding to the standard deviation of three experiments are plotted. Dashed lines correspond to the fit according to a multiple sigmoid function. The shadowed area indicates the pH range in the early endosome. B, histogram showing structural effects of acidification on the virus particles, as analyzed by negative staining. Categories are as follows: I, intact virions; D, damaged particles, including various degrees of disruption but retaining some degree of curvature; F, flattened capsids. Error bars indicate standard deviation in the particle counts between different micrographs. The total number of particles in the dataset, N, is indicated at the top right corner.Aand B,black symbols and lines refer to WT virus; gray symbols and lines refer to ts1. C, gallery showing examples of intact, damaged, and flattened capsids. Particles where the staining agent penetrates are indicated with a star. Particles lacking large capsid fragments are indicated with arrows. The scale bar corresponds to 100 nm. How Maturation Primes Adenovirus for Uncoating SEPTEMBER 7, 2012•VOLUME 287•NUMBER 37 JOURNAL OF BIOLOGICAL CHEMISTRY 31587 2A) and the ts1 curve showed, apparently, only a broad transition centered at pH 4.1, which is near the midpoint of the second WT transition. At pH values below 3, both viruses present the same behavior. A highly cooperative transition leading to almost complete DNA exposure was also observed upon WT capsid disruption by the denaturant agent pyridine (supplemental Fig. S3 and supplemental Table S1). EM analysis (Fig. 2Band supplemental Fig. S2) indicated that at pH 6 over 80% of the ts1 particles were intact but more than 70% WT virions already showed damage. Again, complete collapse and flattening were rarely observed for ts1 and WT. The same tendency regarding flattening was observed for disruption by pyridine (supplemental Fig. S3). Acidification damage produced capsids with dark centers, indicating penetration of the staining agent. However, no evident gaps indicating loss of pentons were observed, meaning that the capsid defect allowing DNA exposure to PI and uranyl acetate is small and of low occurrence in the icosahedral network. Additionally, new kinds of disassembly intermediates were observed: particles with fissures and particles that had lost capsid fragments of different sizes and shapes (“chunks”) but still retaining the polyhedral curvature (Fig. 2C). These kinds of images are consistent with the capsid releasing the stress imposed by acidification via cracks at hexameric positions, instead of by releasing vertex structures (47). Interestingly, 74% of WT damaged particles at pH 6 (55% over total number of particles) were stain-filled capsids. In ts1, however, disruption via fissures and chunk release was more abundant: 56% (46% of the total) of ts1 damaged particles corresponded to this category at pH 4.5, and 60% (58% of the total) at pH 3.5. This difference in disruption patterns suggests that the defect in the WT capsid that allows stain penetration is the physiologicalpathforpartialuncoatingintheendosome,rather thanthedisruptionvia cracks.However,fromthenegative stain images, it was not clear exactly what this defect was, as no clear pattern of lost capsomers (e.g. vertices) was observed. Cryoelectron Tomography of Disassembly Intermediates— DSCand EFprovidebulkinformation on changesundergoneby the specimen, but they do not reveal changes in individual virions. Imaging disassembled virions by negative stain provided single virion images; however, interaction with the carbon support and the acidic staining agent, plus drying and flattening, may result in additional changes not directly related to the stress conditions being tested. Furthermore, in negative stain images only a two-dimensional projection of the three-dimensional virion is obtained, and it is not possible to observe the internal structures of the particle. To obtain a better picture of the structural changes occurring during capsid disruption, we used cryoelectron tomography. First, we investigated the changes in the structure of WT viruses at the stressor conditions where the first transition was observed in DSC or EF assays and where ts1 virions remained mostly unaltered (Figs. 1–3 and supplemental Movies S1–S3): at 40 °C or pH 6.0. In both cases, tomograms showed that capsids conserved to a large extent their structural integrity, including attached fibers (Fig. 3Aand supplemental Fig. S5). However, the aspect of the core differed between control and stressedviruses. Incontrolconditions (25 °C,pH7.4),the virion interior presented a smooth, flat density with gray levels similar to those of the capsid shell. In viruses incubated at pH 6, the core density appeared more heterogeneous and slightly weaker than that of the capsid. This same difference, sometimes more pronounced,was observedinthe coreofvirusesheated at40 °C. A radial average profile calculated from aligned individual virus tomograms indicated that, for both types of mild stress, the density of the outer region of the core was lower than in control viruses (Fig. 3B). This change in electron density is compatible witha relaxation of the DNA-protein complex or loss of peripheral core material. No decrease in core density was observed for ts1 virions at pH 6 (supplemental Fig. S4). Next, we searched for capsid defects that would account for the increase in DNA exposure observed in EF assays. Slice-byslice analysis of individual virion tomograms indicated that both mild heating and acidification resulted in loss of pentons (Fig. 3Cand supplemental Fig. S5). However, the degree of loss was different depending on the kind of stress; acidified viruses lost only one to two pentons, whereas heating produced the release of three to four pentons (Fig. 3D). Concomitantly with a larger loss of pentons, fewer fibers were observed in heat-disrupted WT virions, although upon disruption at pH 6 more than 70% virions still conserved fibers (supplemental Fig. S5), i.e. mild acidification by itself is not causing massive fiber release. Occasionally, both at 40 °C or at pH6,ashort(25–30nmlong and 4–5nmwide)stretch of DNA was seen to protrude out of the missing penton position (supplemental Fig. S6). However, this was an extremely rare event (less than 5% of analyzed viruses under mild stress conditions). Tomograms of WT and ts1 virions subject to high stress conditions (heating at 47 °C or acidification at pH 5; also pH 4 for ts1) were also obtained. At 47 °C, most WT virions appeared as empty broken shells with missing GOS (Fig. 4A,supplemental Fig. S5 and supplemental Movies S4 and S5). Only in a few cases some loose core structures remained attached to capsid fragments. Most immature ts1 virions had also lost GOS and in some cases were cracked open; however, the core remained attached to cracked capsids and in a compacted state. We have previously reported that ts1 virions disrupted at 47 °C extrude a nucleoprotein filament (5). However, no such filaments were observed in cryoelectron tomograms, although they are common occurrences in negative staining (5) or platinum/carbon shadowing preparations (data not shown). We conclude that filament extrusion is induced by the EM preparation conditions,namelyacombination ofadsorptiontothesupport, staining with low pH uranyl salts or shadowing, and drying. TABLE 4 EF analysis of capsid disruption induced by acidification Fitting of EF data to multiple sigmoid curves. Boldface indicates transitions with high cooperativity. WT ts1 Midpoint (pH units) Slope Midpoint (pH units) Slope Sigmoid 1 6.1 ⴞ0.1 48 ⴞ54.4 ⫾0.3 7.4 ⫾0.3 Sigmoid 2 4.1 ⫾0.4 5.4 ⫾0.9 R 2 0.99951 0.99916 How Maturation Primes Adenovirus for Uncoating 31588 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287•NUMBER 37•SEPTEMBER 7, 2012 A considerable proportion of ts1 capsids remained intact at low pH values (more than 90% at pH 6 and up to 60% at pH 4; supplemental Fig. S5). Notably, fibers (which are not directly involved in proteolytic processing) remain attached to immature virions even under conditions as harsh as pH 4 (supplemental Fig. S5). Upon high acidification, WT shells had gaps consistentwithlossofGOSandwereonlyrarelyseenincontact withanycoreremains. Conversely,ts1 capsidsonly lostpentons and cracked open, whereas cores remained attached to shell fragments and in a condensed state (Fig. 4Band supplemental Movies S6 and S7). Unlike for negative staining EM, no flattened capsids were observed in cryoelectron tomography images, confirming that flattening was a preparation artifact. However, measurement of dihedral angles in tomograms indicated that in high stress conditions the curvature of disrupted capsids departed from the icosahedral value (Table 5 and Fig. 4C) in both WT and ts1 but with a more noticeable effect in WT. We conclude that flattened capsids observed by negative staining in WT correspond in solution to empty deformed capsids with reduced curvature reinforcements that become flat upon adsorption to carbon staining and drying. In ts1, loss of curvature is not so pronounced, and capsids are less prone to flattening during the negativestaining procedure. Interestingly,curvaturecharacteristics are preserved in ts1 even in conditions where large vertex structures (GOS) are lost (47 °C). This observation suggests that the tighter interactions between capsid and compacted core established by precursor proteins play a role in determination and maintenance of capsid curvature during assembly. For both WT and ts1, a small proportion of particles that appeared as intact capsids was found to lack some pentons after closer inspection (supplemental Fig. S5, class “P”). When these FIGURE 3. Cryoelectron tomography analysis of structural changes induced by mild stress in WT adenovirus. A, representative sections of tomograms showing WT virus in control conditions, 40 °C or pH 6, as indicated. The bar represents 100 nm. Some visible fibers are labeled with arrowheads.B, radial density profiles of average maps calculated from 22 individual virus tomograms for each case. The central section of each average map is shown at right.C, example sections of individual virus tomograms showing penton loss. Equivalent sections of a model lacking pentons, and of the full virion icosahedral map, are shown for comparison. Model maps are low pass-filtered at 50 Å resolution. Red dashed circles indicate the position of the vertex. D, histogram showing number of pentons lost per virion. No penton loss was observed in an equivalent dataset of control viruses. 33 individual virion tomograms (396 vertices) were analyzed for each condition. How Maturation Primes Adenovirus for Uncoating SEPTEMBER 7, 2012•VOLUME 287•NUMBER 37 JOURNAL OF BIOLOGICAL CHEMISTRY 31589 particles were aligned and averaged, radial profiles indicated that the WT core density had decreased not only in the periphery but also in the center of the virion, i.e. core relaxation or component loss increased with increasing stress levels. Conversely, core density in ts1 virions lacking some pentons remained unaltered, even at pH 5 or 47 °C (supplemental Fig. S4). Mechanical Capsid Disruption—AFM nanoindentation assays allow exploring virus stability under mechanical stress. After locating an individual intact virus on the surface (Fig. 5A), the cantilever pushes the virion at the very top of the particle. Cantilever bending is recorded as a function of zdisplacement, thus eliciting the virus deformation as a function of the vertical force (48). During the first stages of indentation, the virus particles undergo a linear deformation that is reflected by a sustained climbing in the force indentation curve (Fig. 5, Cand D) and provides the spring constant of the virus k v (49). At a certain point, the curve tendency sharply changes, and the force suddenly starts decreasing due to capsid failure. The point where tendency changes determines the breaking force. Finally, an image of the virus is taken to confirm its disruption (Fig. 5B). In the experimental conditions used here, viruses preferentially attached to the mica in a 3-fold orientation. We recorded data from single nanoindentations on 25 WT (Fig. 5C) and 28 ts1 (Fig. 5D) particles. From the results obtained in conditions of thermal or chemical stress, we would have expected to find the mature capsid more fragile and softer than the immature one. On the contrary, WT virions presented both higher breaking force (3.3 ⫾0.2 nN) and stiffness (0.46 ⫾ 0.02 N/m) than ts1 particles (2.3 ⫾0.2 nN and 0.38 ⫾0.04 N/m). Further analysis of indentation curves can supply extra information about the core. From the experimental curves in FIGURE 4. Cryoelectron tomography analysis of structural changes induced by high stress. A, representative sections of tomograms showing WT and ts1 virus at 47 °C; B, at pH 5. The bar represents 100 nm. Black arrows indicate missing GOSs; stars indicate core remnants in open capsids; white arrows indicate capsid-core contacts; black arrowhead indicates a tangential view of a capsid crack. C, analysis of capsid curvature. Top panel, schematics showing examples of dihedral angles on central sections of individual virus tomograms. Middle and bottom panels, histograms of angle values for WT and ts1 control, intact virions; and for disrupted capsids at 47 °C or pH 5. TABLE 5 Statistics of dihedral angles in disrupted capsids under high stress A total of 30 individual virus tomograms were examined for each condition. The number of angle measurements n for each case is indicated. Condition WT ts1 Perfect icosahedron 138° 138° Control 137 ⫾3° (n⫽180) 136 ⫾3° (n⫽180) 47 °C 125 ⫾13° (n⫽160) 133 ⫾8° (n⫽170) pH 5 132 ⫾14° (n⫽168) 134 ⫾11° (n⫽169) How Maturation Primes Adenovirus for Uncoating 31590 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287•NUMBER 37•SEPTEMBER 7, 2012