Supplementary Information
Monitoring dynamics of human adenovirus disassembly induced by mechanical fatigue
A. Ortega-Esteban1, A. J. Pérez-Berná2, R. Menéndez-Conejero2, S. J. Flint3, C. San Martín2, P. J. de
Pablo1*
* p.j.depablo@uam.es
1
Departamento de Física de la Materia Condensada, Universidad Autónoma de Madrid, 28049 Madrid
(Spain)
2
Department of Macromolecular Structure, Centro Nacional de Biotecnología (CNB-CSIC). Darwin 3,
28049 Madrid (Spain)
3
Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA
Rupture patterns obtained by single nanoindentation assays
Classical nanoindentation assays consisted of exerting a force exceeding that of breakage
1
at the
top of the particle (figure S1b). Afterwards an image of the virus was taken to confirm its disruption
(figure S1c). For each virus analyzed, a breakage map corresponding to the force-distance curve
(figure S1d) was computed by subtracting the final image after the nanoindentation from the intact
virion image. The difference image was binarized to a gray level of 1 within the cracking area
(figure S1e). After aligning all the virus images in the dataset using cross-correlation procedures in
Xmipp2, breakage patterns were overlaid to obtain the average breakage pattern due to the
nanoindentations (figure S1f and S1g). These breakage patterns do not provide evident differences
between WT and immature virions.
-1-
Supplementary figures
a
b
c
force (nN)
3
2
1
0
0
d
20
40
indentation (nm)
46 nm
e
f
WT
g
ts1
Figure S1
Standard single nanoindentation experiments using large forces. (a) AFM image showing an intact
adenovirus virion. (b) Example of a nanoindentation force-distance curve. Black and red are
forward and backward cycles, respectively. (c) Final state of the virion showing the crack produced
by the nanoindentation performed close to the center of the facet. (d) Difference map resulting from
the subtraction of image (c) from (a). (e) Binarization of (d). (f and g) are average breakage maps of
9 WT and 12 ts1 virions, respectively. Darker gray levels indicate higher frequency of breakage at
these regions. These analyses show that viruses are mainly broken at the center, because the tip is
meant to perform the indentation at the center of the triangular facet.
-2-
c
a
b
0
5
7
18
22
27
28
31
34
Figure S2
Virus disruption is due to mechanical fatigue, and not to the prolonged experimental time. (a) Two
WT virions attached to the surface before the disassembly experiment. (b) A set of different stages
throughout the disassembly of the lower viral particle are shown. The typical crumbling pattern for
WT virus is observed. Frame numbers are indicated in each image. (c) The same area as in (a)
imaged after the end of the disassembly experiment. The neighboring particle at the top remains
intact.
-3-
a
b
Dilation model from EM
1
2
3
3
Intact capsid
Pentonless capsid
80
60
40
30
C
5
4
40
50
nm
60
Intact capsid
Pentonless capsid
100
height (nm)
100
height (nm)
5
4
Experimental AFM data
1
2
80
60
40
40
70
60
nm
80
100
EM dilation
Experimental data
100
height (nm)
vacancy
80
60
40
60
nm
80
100
Figure S3
(a) Adenovirus dilation
3
simulations performed on EM data before (a1) and after (a2) penton
removal (a3 and a4 respectively) by using a 12 nm diameter tip. (a5) Comparison of profiles at
the penton region (dotted lines). Figures (b3) and (b4) show the experimental AFM data, which
are high-pass filtered in (b1) and (b2), respectively, to enhance penton vacancies. The profiles of
the dotted lines are depicted at (b5), showing the same topography predicted in simulations. (c)
Profiles demonstrate an excellent agreement between the theoretical and the experimental data of
the penton vacancies.
-4-
In vivo
Mechanical
fatigue
0
9
66 8
15 min
61
45-60 min
Figure S4
Mechanical disruption recapitulates the temporal pathway of adenovirus uncoating in the cell.
Virions escape the endosome 15 min p.i., having released some pentons. They reach the nuclear
envelope at 45 min p.i. and are completely dismantled by 60 min p.i .In mature virions, the
partially disrupted structure persists for 50-75% of the disassembly elapsed time which can be
compared to the 66±8% due to mechanical fatigue disassembly.
-5-
c
WT
ts1
3.0
2.0
2.0
E3
1.0
1.0
E (10-16cal)
E (105KBT)
3.0
E2
E1
0.0
vertex1
vertex2
0.0
vertex3
Penton release
Figure S5
Procedure to estimate the energy supplied for each penton release event. (a) Schematics showing
the calculation of supplied energy for the scanning force in one point for WT (black) and ts1 (red)
viruses. (b) Area within the virus perimeter and number of pixels, i.e. number of force-distance
curves in one image. (c) Supplied energies for each penton release event averaged to all ts1 and WT
virions vacancies occurrences, indicated as E1, E2 and E3 for WT.
-6-
Figure S6
Time cumulative disruption maps (TCDMs). Examples of two-dimensional rupture maps for WT (a-c) and
ts1 (d-f) virions. The elapsed time in minutes corresponding to the whole grayscale for each image is 104
(a), 81 (b), 174(c), 104 (d), 90 (e), 92 (f), respectively. The scale bar corresponds to 45 nanometers in (a), 38
in (b), 46 in (c), 45 in (d), 49 in (e), and 50 in (f). Red contour plots indicate areas removed at 73% (a), 45%
(b), 49% (c), 33% (d), 38% (e), 34% (f), of the total elapsed time, respectively. Green contour plots indicate
areas removed at 90% (a), 75% (b), 75% (c), 73% (d), 65% (e), 79% (f), of the total elapsed time,
respectively. Blue contour plots indicate areas removed at 96% (a), 94% (b), 92% (c), 80% (d), 73% (e),
93% (f), of the total elapsed time respectively. See also movies S1, S2, S3, S4 and S5.
-7-
# particle
1
2
3
4
5
6
7
average
WT
68±4
38±4
38±5
150±16
48±8
70±4
127±61
77
ts1
22±9
5±1
7±1
10±1
17±3
17±4
--
13
Table S1
Average and mean statistical error of the cooperative factors n of height decrease during
disassembly.
Penton Vacancies
Virion
WT
ts1
Time (min)  2.8 min·frame-1
Frame
Particle
Upright facet
Lateral
Upright facet
Lateral
1
4
4
4
11.2
11.2
11.2
2
5
5
5
14
14
14
3
9
13
17
25.2
36.4
47.6
4
3
4
5
3
8.4
11.2
14
8.4
5
-
2
13
13
-
5.6
36.4
36.4
6
3
6
16
8.4
16.8
44.8
7
4
5
12
11.2
14
33.6
8
4
6
9
11.2
16.8
25.2
1
18
46
50
50.4
128.8
140
2
20
20
20
56
56
56
3
-
4
6
-
8.4
14
4
5
15
17
14
42
47.6
5
-
18
21
-
50.4
58.8
6
-
10
23
-
28
64.4
7
1
13
16
2.8
36.4
44.8
8
13
32
40
36.4
89.6
112
16
44.8
Table S2
Frames in which appearance of penton vacancies was observed, used for penton release distribution
estimation shown in figure 3f. Data pertain to more particles than those included at figure 3a,b
because some viruses were detached from the surface just after losing their pentons.
-8-
# particle
1
2
3
4
5
6
7
average
SE
WT
89
91
41
67
74
43
57
66
8
ts1
87
48
16
0
53
24
--
40
10
Table S3
Pentonless time expressed in % of the total disassembly time for each virion.
Movies S1, S2 and S5
Mechanical fatigue disruption of two different Wild Type (mature) virions.
First, a three-dimensional rendered topography of the virus is shown. Afterwards, the same
sequence is shown stopping at clue points of the disassembly (7 frames per second). Lastly, a video
of time cumulative disruption maps in which the breakage dynamics can be seen is shown (5
frames per second). The time cumulative disruption maps (TCDMs) of figures S4c and S4b are
obtained at the end of the movies, respectively.
Movies S3 and S4
Mechanical fatigue of two different ts1 (immature) virions.
First, a three-dimensional rendered topography of the virus is shown. Afterwards, the same
sequence is shown stopping at clue points of the disassembly (7 frames per second). Lastly, a video
of time cumulative disruption maps is shown in wchich the breakage dynamics can be seen (5
frames per second). The time cumulative disruption maps (TCDMs) of figures 4b and S4f are
obtained at the end of the movies, respectively.
Supporting references:
1
2
3
Pérez-Berná, A. J. et al., The role of capsid maturation on adenovirus priming for
sequential uncoating. J. Biol. Chem. 287 (37), 31582 (2012).
Scheres, S. H. W. et al., Image processing for electron microscopy single-particle
analysis using XMIPP. Nat. Protoc. 3 (6), 977 (2008).
Villarrubia, J. S., Algorithms for scanned probe microscope image simulation, surface
reconstruction, and tip estimation. Journal of Research of the National Institute of
Standards and Technology 102 (4), 425 (1997).
-9-