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Viruses X-ray, EM structure function structure function - - PowerPoint PPT Presentation
Viruses X-ray, EM structure function structure function - - PowerPoint PPT Presentation
Viruses X-ray, EM structure function structure function properties thermal stability mechanics electrostatics vibrations Etc PHYSICAL VIROLOGY Single molecule techniques provide complementary information to structural
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Viruses
structure function X-ray, EM
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structure function properties
- thermal stability
- mechanics
- electrostatics
- vibrations
- Etc
PHYSICAL VIROLOGY
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Single molecule techniques provide complementary information to structural biology
Physiol siologi
- gical
al condi diti tions
- ns
Functional protein shells Mechan anics Ma Manip ipula lati tion
- n
Re Real al time e ex experi erimen ments ts
Binnig, Quate, Gerber, PRL 1986
Atomic Force Microscopy
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Atomic Force microscopy in liquids
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Cantilever/virus size
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Atomic Force Microscopy scanning probe
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De Pablo
- et al, APL 1998
Ortega-Est Esteb eban n et al. Ultram amicr crosc
- scopy
py 2012 2012
AFM imaging of viruses
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High resolution AFM of adenovirus
hexons
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Single indentation assay
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1 2 3
Single indentation assay
before after 2 1 3 spring constant
kv
breaking force
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Self-recovery of vault particles
1 2 3
Llauró et al Biophysical Journal 2014
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Capsid id 240 hexons, 12 pentons, proteins IIIa, VI, VIII, IX Fiber flexible, specific host recognition Core re 35kbp dsDNA, proteins TP, VII, μ
Human adenovirus
Greber et al. Cell 1993
Penton loss and disassembly 50% DNA + 50% histone-like proteins
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Immature non infectious Mature infectious cleaved protein
Maturation changes the core
protease protein protease
Pérez-Berná et al. JMB 2009
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DNA
- Does DNA modulate the mechanical properties of adenovirus particles?
- Interplay between physical properties and virus function?
disassembly DNA diffusion
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Adsorption geometries
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Mechanical evolution
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Interpretation
kvirus=kshell+kDNA kDNA (mature) > kDNA(immature)
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Crack-open the shell
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Crack-opening the shell
mature inmature
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Mechanics of cores
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 immature Young's Modulus (MPa) mature 20 40 60 80 10 20 30 40 50 60 mature immature Deflection (nm) Indentation (nm)
mature
E <
immature
E
Dimitriadis Biophys J. 2002
mature
k >
immature
k
Pressurization?
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DNA condensate
Adding counterions to DNA induce toroidal condensates (3+) Gronbech-Jensen et al. PRL 1997
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Core mechanics
mature
E <
mature spermidine
E
immature
E <
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Pressure estimation
k1 = π 2 k0 τ2 − 1
1 2
arctanh 1 − τ−2
1 2
.
Vella et al. The Indentation of Pressurized Elastic Shells: From Polymeric Capsules to Yeast
- Cells. 2011, Journal of The Royal Society
Interface. p=3±1 MPa
Irrespective of the physical origin Unbranched polymer
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DNA-DNA repulsion pressurizes the shell after maturation
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We propose that pressure helps to pop-off pentons at the early endosoome
Biological implications
- W. Klug et al, PRL 10/2012; 109(16):168104.
Ortega-Esteban Sci. Rep. 2013, 3, 14434 Pentons are the weakest capsomers Ortega-Esteban et al ACS Nano 2015
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Biological implications diffusion of DNA?
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Fatigue
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Multiple indentation assay below the breaking force (fatigue)
Force 100 pN
Ortega-Esteban et al. Ultramicroscopy 2012
30 times less than breaking force!
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Uncoating dynamics
Mat ature Immat ature
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Immat ature
20 40 60 80 100 120 140 160 15 30 45 60 75 90
# image height (nm) time (m)
10 20 30 40 50 60
70 70 nm nm
Mat ature
20 40 60 80 100 120 140 160 10 20 30 40 50 60 70 80 90
average height (nm) time (m)
10 20 30 40 50 60
# image
Core exposure
Can an we we vi visual alize genome uncoat ating?
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0.0 nm nm 86.6 nm nm
m
Mat ature Immat ature
Core exposure
Can we we vi visual alize th the genome uncoat ating?
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Fluorescence
350 400 450 500 550 600 650 700 750 0.0 0.2 0.4 0.6 0.8 1.0
Intensity (a.u.) Wavelength (nm)
YOYO-1 absorption YOYO-1 emission
YOYO-1
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AFM – fluorescence combination
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AFM – fluorescence combination
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AFM induced unpacking of adenovirus
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AFM induced unpacking of adenovirus
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simultaneous single particle fluorescence with AFM
- bserve DNA release with YOYO-1
AFM forced unpacking of Adenovirus
1 2 3 4 3 6 9 12 15
Force (nN) time (s)
200 400 600
photons above background
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20 40 60 80 10 20 30 40 50 60
Emission (counts) time (s) average count wt average count ts1
Quantifying DNA release
immature mature
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Quantifying DNA release
Ortega ga-Es Esteba teban, , de Pablo, , Schaa aap et al. ACS Nano 2015
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Controlled capsid disassembly
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- Immature emits less photons
- Mature core spreads more the genome
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Quantifying DNA release
Ortega ga-Es Esteba teban, , de Pablo, , Schaa aap et al. ACS Nano 2015
Immat ature Ma Matu ture
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Topics today
- 1. Introduction
- 2. Mechanics of human adenovirus: capsid and core
- 3. Genome release: watching a virus undress
- 4. Mechanical role of cementing proteins: tuning particles stability with symmetrical
morphogenesis
- 5. Summing up
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Cementing/decorative proteins
An alternative strategy to strengthen virus capsids during maturation
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- 60 nm in diamter
- 420 gpE + 415 gpD.
72 capsomers
- DNA ~ 48.5 kbp ~ 14.5 µm .
C.G Lander
Lambda phage
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Single indentation assay
undecorated decorated
30nm 32nm
Nature Communications 5, 4520 (2014)
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5,0x10
4
1,0x10
5
1,5x10
5
2,0x10
5
2,5x10
5
# load cycles
undecorated decorated
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Mechanical fatigue
Nature Communications 5, 4520 (2014)
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Can we use cementing proteins to recover weaken protein shells?
Decorated particles are mechanically more robust that undecorated
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Tuning viral capsid nanoparticle stability with simmetrycal morphogenesis
P22 phage (EX)panded P22 phage “Wiffle Ball” WB
Lian Tang et al Structure 2006 Parent et. Al Structure 2010, Biomaterials 2012
20 m 65 0C
P22 binds phage L Dec proteins at quasi-three fold locations
EX-Dec WB-Dec
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P22 particles
WB 20 m 65 0C EX-Dec WB-Dec EX
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Collapse of p22 particles after adsorption
- n the surface
20 nm
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Collapse of p22 particles after adsorption
- n the surface
20 nm
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Collapse of p22 particles after adsorption
- n the surface
EX EX+ Dec WB WB+ Dec 49 50 51 52 53 54 55 56 57
h (nm)
0,80 0,82 0,84 0,86 0,88 0,90 0,92
h/d
20 nm
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Which structure is more stable?
Llauró et al. ACS Nano 2016
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Which structure is more stable?
Llauró et al. ACS Nano 2016
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0,7 0,8 0,9 1,0 1,1 1,2 1,3 1,4 0,12 0,14 0,16 0,18 0,20 0,22 0,24
EX+Dec
EX WB+Dec WB
K (N/m) Breaking Force (nN)
WB EX+Dec WB-Dec EX
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How much work is used to crack the particles?
Llauró et al. ACS Nano 2016
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Cementing proteins improve capsid performance
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Summary
Core mechanics indicates adenovirus pressurization that helps for disassembly and genome delivery Genome condensation influences on diffusion
Immatur ure Matur ure
Cementing proteins recovers weak particles
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Natalia González Francisco J. Moreno-Madrid Manuel Jiménez
Former members Marina López Mercedes Hernando Carolina Carrasco Alvaro Ortega Aida Llauró
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