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 biology Atomic Force Microscopy Binnig, Quate, Gerber, PRL 1986 Physiol siologi ogical al condi diti tions ons Functional protein shells Mechan anics Manip Ma ipula lati tion on Re Real al time e ex experi erimen ments ts

Atomic Force microscopy in liquids

Cantilever/virus size

Atomic Force Microscopy scanning probe

AFM imaging of viruses De Pablo o et al, APL 1998 Ortega-Est Esteb eban n et al. Ultram amicr crosc oscopy py 2012 2012

High resolution AFM of adenovirus hexons

Single indentation assay

Single indentation assay breaking force 1 2 3 spring constant 3 k v 2 1 after before

Self-recovery of vault particles 3 2 1 Llauró et al Biophysical Journal 2014

Human adenovirus Penton loss and disassembly Capsid id 240 hexons, 12 pentons, proteins IIIa, VI, VIII, IX Fiber flexible, specific host recognition Core re 35kbp dsDNA, proteins TP, VII, μ 50% DNA + 50% histone-like proteins Greber et al. Cell 1993

Maturation changes the core protease cleaved protein protease protein Immature Mature non infectious infectious Pérez-Berná et al. JMB 2009

DNA • Does DNA modulate the mechanical properties of adenovirus particles? • Interplay between physical properties and virus function? disassembly DNA diffusion

Adsorption geometries

Mechanical evolution

Interpretation k virus =k shell +k DNA k DNA (mature) > k DNA (immature)

Crack-open the shell

Crack-opening the shell mature inmature

Mechanics of cores 1.4 mature 60 immature 1.2 50 Young's Modulus (MPa) 1.0 Deflection (nm) 40 0.8 30 0.6 20 Dimitriadis 0.4 Biophys J. 2002 10 0.2 0 0.0 0 20 40 60 80 mature immature Indentation (nm) E < E Pressurization? mature immature k k > immature mature

DNA condensate Adding counterions to DNA induce toroidal condensates (3+) Gronbech-Jensen et al. PRL 1997

Core mechanics < E E E < mature mature immature spermidine

Pressure estimation Unbranched polymer Irrespective of the physical origin Vella et al. The Indentation of Pressurized Elastic Shells: From Polymeric Capsules to Yeast Cells. 2011, Journal of The Royal Society Interface . 1 τ 2 − 1 k 1 = π 2 2 k 0 . 1 arctanh 1 − τ − 2 2 p=3 ± 1 MPa

DNA-DNA repulsion pressurizes the shell after maturation

Biological implications Pentons are the weakest capsomers W. Klug et al, PRL 10/2012; 109(16):168104. Ortega-Esteban Sci. Rep. 2013, 3, 14434 Ortega-Esteban et al ACS Nano 2015 We propose that pressure helps to pop-off pentons at the early endosoome

Biological implications diffusion of DNA?

Fatigue

Multiple indentation assay below the breaking force (fatigue) Force  100 pN 30 times less than breaking force! Ortega-Esteban et al. Ultramicroscopy 2012

Uncoating dynamics Mat ature Immat ature

Core exposure # image # image 0 10 20 30 40 50 60 0 10 20 30 40 50 60 90 90 80 75 average height (nm) 70 60 60 height (nm) 50 70 nm 70 45 40 nm 30 30 20 15 10 0 0 0 20 40 60 80 100 120 140 160 0 20 40 60 80 100 120 140 160 time (m) time (m) Immat ature Mat ature Can an we we vi visual alize genome uncoat ating?

Core exposure 86.6 nm nm m 0.0 nm Mat ature Immat ature nm Can we we vi visual alize th the genome uncoat ating?

Fluorescence YOYO-1 absorption YOYO-1 emission 1.0 0.8 Intensity (a.u.) 0.6 0.4 0.2 0.0 350 400 450 500 550 600 650 700 750 Wavelength (nm) YOYO-1

AFM – fluorescence combination

AFM – fluorescence combination

AFM induced unpacking of adenovirus

AFM induced unpacking of adenovirus

AFM forced unpacking of Adenovirus 15 600 photons above background 12 400 9 Force (nN) 6 200 3 0 0 0 1 2 3 4 time (s) simultaneous single particle fluorescence with AFM observe DNA release with YOYO-1

Quantifying DNA release mature average count wt 60 immature average count ts1 50 Emission (counts) 40 30 20 10 0 0 20 40 60 80 time (s)

Quantifying DNA release Ortega ga-Es Esteba teban, , de Pablo, , Schaa aap et al. ACS Nano 2015 45

Controlled capsid disassembly

Quantifying DNA release Ma Matu ture Immat ature - Mature core spreads more the genome - Immature emits less photons 47 Ortega ga-Es Esteba teban, , de Pablo, , Schaa aap et al. ACS Nano 2015

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

Cementing/decorative proteins An alternative strategy to strengthen virus capsids during maturation 49

Lambda phage  60 nm in diamter  420 gpE + 415 gpD. 72 capsomers  DNA ~ 48.5 kbp ~ 14.5 µm . C.G Lander

Single indentation assay undecorated 32nm 30nm decorated Nature Communications 5, 4520 (2014)

Mechanical fatigue undecorated 5 2,5x10 # load cycles 5 2,0x10 5 1,5x10 5 1,0x10 decorated 4 5,0x10 52 Nature Communications 5, 4520 (2014)

Decorated particles are mechanically more robust that undecorated Can we use cementing proteins to recover weaken protein shells? 53

Tuning viral capsid nanoparticle stability with simmetrycal morphogenesis 20 m 65 0 C P22 phage (EX)panded P22 phage “ Wiffle Ball” WB EX-Dec Lian Tang et al Structure 2006 WB-Dec Parent et. Al 54 Structure 2010, Biomaterials 2012 P22 binds phage L Dec proteins at quasi-three fold locations

P22 particles 20 m 65 0 C WB EX EX-Dec WB-Dec 55

Collapse of p22 particles after adsorption on the surface 20 nm 56

Collapse of p22 particles after adsorption on the surface 20 nm 57

Collapse of p22 particles after adsorption on the surface 57 0,92 56 0,90 55 0,88 54 20 nm h (nm) 53 0,86 h/d 52 0,84 51 0,82 50 49 0,80 EX EX+ Dec WB WB+ Dec 58

Which structure is more stable? Llauró et al. ACS Nano 2016 59

Which structure is more stable? Llauró et al. ACS Nano 2016 60

WB EX+Dec EX WB-Dec 0,24 EX+Dec EX 0,22 WB+Dec WB 0,20 K (N/m) 0,18 0,16 0,14 0,12 0,7 0,8 0,9 1,0 1,1 1,2 1,3 1,4 Breaking Force (nN)

How much work is used to crack the particles? Llauró et al. ACS Nano 2016 62

Cementing proteins improve capsid performance

Summary Core mechanics indicates adenovirus pressurization that helps for disassembly and genome delivery Immatur ure Matur ure Genome condensation influences on diffusion Cementing proteins recovers weak particles

Natalia González Francisco J. Moreno-Madrid Manuel Jiménez Former members Marina López Mercedes Hernando Carolina Carrasco Alvaro Ortega Aida Llauró

Collaborators Funding Arvind Raman Carlos E. Catalano Carmen San Martín Daniel Luque Dave Evans David Reguera Iwan Schaap José Ruiz Castón Mark van Raaij Mauricio García Mateu Nuria Verdaguer Rudi Podgornik Salvatore Cannistraro Trevor Douglas Urs Greber Thank you!