Download 17 - 19 de abril, 2013 - Universitat de Barcelona

II Reunión
17 - 19 de abril, 2013
Facultad de Ciencias
Módulo C0
2
PROGRAM
Wednesday, April 17th
08:45-9:00 Opening
9:00-10:00 S. Blanc (UMR-BGPI Montpellier, France)
" Gene copy number is differentially regulated in a multipartite
virus " Page 10
10:00-10:30 Santiago F. Elena (Instituto de Biología
Molecular y Celular de Plantas (CSIC-UPV), Campus UPV
València)
" Empirical fitness landscapes reveals a limited number of
accessible adaptive pathways for an RNA virus" Page 11
10:30-11:00 Ester Lázaro (Centro de Astrobiología (CSICINTA)
" Genetic and Phenotypic Properties of Bacteriophage Qβ
Populations Evolved at Increased Error Rate" Page 12
11:00-11:30 COFFEE
11:30-12:00 José A. Cuesta (Universidad Carlos III)
" Evolving on Phenotype Landscapes" Page 14
12:00-12:30 José J. Ramasco (Instituto de Física
Interdisciplinar y de los Sistemas Complejos IFISC (CSICUIB))
"Real-time numerical forecasts of global epidemic spreading"
3
Page 15
12:30-13:00 Vicente Pallàs (Instituto de Biología Molecular y
Celular de Plantas – CSIC)
" Presentación del Grupo de Virología Molecular de Plantas
del IBMCP”Page 16
13:00-15:00 LUNCH
15:00-15:30 Carlos Briones (Centro de Astrobiología (CSICINTA))
" Magnesium-Dependent RNA Folding of the Internal
Ribosome Entry Site of Hepatitis C Virus Genome Monitored
by Atomic Force Microscopy " Page 20
15:30-16:00 Mauricio G. Mateu (Centro de Biología
Molecular “Severo Ochoa”)
" Manipulation and Biological Implications of the Thermal
Stability and Mechanical Properties of Viruses”Page 22
16:00-16:30 Carmen San Martin (Centro Nacional de
Biotecnología, CSIC)
" Structural Determinants of Adenovirus Assembly” Page 23
16:30-17:00 COFFEE
17:00-17:30 P. J. de Pablo (Unversidad Autónoma de Madrid)
"Physical virology with Atomic Force Microscopy” Page 24
17:30-18:00 D.M.A. Guérin (Unidad de Biofísica (CSICUPV/EHU) - Fundación Biofísica Bizkaia)
" Triatoma Virus (TrV) capsid disassembly and genome
4
release" Page 25
18:00-18:30 Teresa Ruiz-Herrero (Universidad Autónoma de
Madrid)
" Dynamic simulations of virus budding” Page 27
Thursday, April 18th
09:00-10:00 Félix Rey (Institut Pasteur)
" Class II viral membrane fusion proteins: virus/host gene
exchanges and cell-cell fusion events in multicellular
organisms" Page 28
10:00-10:30 Núria Verdaguer (Institut de Biologia Molecular
de Barcelona CSIC)
" Structural Characterization of RNA Viruses" Page 29
10:30-11:00 Nicola Abrescia (Structural Biology Unit, CIC
bioGUNE, CIBERehd)
" Three-dimensional Visualization of Forming Hepatitis C
Virus-like Particles by Electron-Tomography" Page 30
11:00-11:30 COFFEE
11:30-12:00 J.R. Castón (Centro Nacional de Biotecnología
CSIC)
"Cryo-EM structure of Penicillium chrysogenum virus at 4 Å
resolution" Page 32
12:00-12:30 Daniel Luque (Instituto de Salud Carlos III)
5
"Structural Analysis of Rotavirus Infection Associated
Macromolecular Complexes " Page 33
12:30-13:00 Carmela Garcia-Doval (Centro Nacional de
Biotecnología CSIC)
" Structural biology of viral fibres " Page 34
13:00-15:00 LUNCH
15:00-15:30 José L. Carrascosa (Centro Nacional de
Biotecnología CSIC)
"Studies on the double stranded DNA packaging machinery of
viruses” Page 36
15:30-16:30 Roundtable
16:30-17:00 COFFEE
17:00-18:30 Posters session
21:00 Gala dinner. Restaurant “Gasset 75”
Friday, April 19th
09:00-10:00 Antonio Šiber (Institute of Physics, Croatia)
"Are electrostatic and elastic properties of viruses tuned by
evolution and how?" Page 38
6
10:00-10:30 J. Hernández-Rojas (Universidad de la Laguna)
"A minimalist potential energy model for the self-assembly of
virus capsids" Page 39
10:30-11:00 David Reguera (Universitat de Barcelona)
“Physical Modeling of the Self-Assembly and Mechanical
Properties of Viruses” Page 40
11:00-11:30 COFFEE
11:30-12:00 A.M. Bittner (CIC nanoGUNE)
"The Physics of Tobacco Mosaic Virus" Page 41
12:00-12:30 Andres de la Escosura (Universidad Autónoma
de Madrid)
" Self-Assembly Triggered by Self-Assembly: Virus-Like
Particles Loaded with Supramo-lecular Nanomaterials" Page
43
12:30-13:00 A. Velazquez-Campoy (Universidad de
Zaragoza)
" NS3 Protease from Hepatitis C Virus: Biophysical
Characterization of a Partially Disordered Protein Domain"
Page 45
13:00-13:15 Closure
13:15 LUNCH
7
8
ORAL PRESENTATIONS
9
Gene copy number is differentially regulated in
a multipartite virus
Stéphane Blanc
UMR-BGPI Montpellier, France
Multipartite viruses are enigmatic entities with a
genome divided into several nucleic acid segments, each
encapsidated separately. An evident cost for these viral
systems, highly enhanced if some segments are rare, is the
difficulty to gather at least one copy of each segment to ensure
infection. We tackle the question of the segment frequencyrelated cost by monitoring the relative copy number of the 8
single-gene segments composing the genome of a plant
nanovirus during host infection. We show that some viral genes
accumulate at very low frequency, whereas others dominate.
We further show that the relative frequency of viral genes
impacts on both viral accumulation and symptom expression,
and specifically changes in different hosts. All earlier proposed
benefits of viral genome segmentation do not depend on the
frequency of the segments and cannot explain the situation
described here. We propose that the differential control of gene
(or segment) copy number may provide a major unforeseen
benefit for multipartite viruses, which may compensate for the
extra-costs related to the existence of low-frequency segments.
10
Empirical fitness landscapes reveals a limited
number of accessible adaptive pathways for an
RNA virus
Santiago F. Elenaa,b, Francisca de la Iglesiaa, and Jasna
Lalića
a
Instituto de Biología Molecular y Celular de Plantas (CSIC-UPV),
Campus UPV CPI 8E, Ingeniero Fausto Elio s/n, 46022 València,
Spain.
b
The Santa Fe Institute, 1399 Hyde Road Park, Santa Fe, NM 87501,
USA
RNA viruses are the main source of emerging
infectious diseases owed to the evolutionary potential bestow
by their fast replication, large population sizes and high
mutation and recombination rates. However, an equally
important parameter, which is usually neglected, is the
topography of the fitness landscape, that is, how many fitness
maxima exist and how well connected they are, which
determines the number of accessible evolutionary pathways.
To address this question, we have reconstructed a fitness
landscape describing the adaptation of Tobacco etch potyvirus
(TEV) to a new host, Arabidopsis thaliana. Two fitness traits
were measured for most of the genotypes in the landscape,
infectivity and virus accumulation. We found prevailing
epistatic effects between mutations in the early steps of
adaptation, while independent effects became more common at
latter stages. Results suggest that the landscape was highly
rugged, with a reduce number of potential neutral paths and a
alternative fitness peaks, being the one reached by the evolving
TEV population not the global optima.
11
Genetic and Phenotypic Properties of
Bacteriophage Qβ Populations Evolved at
Increased Error Rate
Ester Lázaroa, Laura Cabanillasa, and María Arribasa
a
Centro de Astrobiología (CSIC-INTA), Ctra de Ajalvir Km 4,
Torrejón de Ardoz, 28850 Madrid (Spain)
RNA virus replication takes place at a very high error
rate. When the number of mutations per genome increases
about a certain value, mutation can outpace selection, causing
fitness decreases, and sometimes population extinction.
Most mutations decrease the thermodynamic and kinetic
folding stability of proteins, reducing their capability to
perform optimally. Since virus capsids are the result of the
correct assembly of multiple copies of one or several proteins,
it would be expected that mutations leading to incorrect
foldings also reduced capsid stability, which could result in a
higher sensitivity to adverse environmental conditions.
One of the projects carried out in our laboratory is focused on
the study of the genetic and phenotypic characteristics of virus
populations evolved at increased error rate. Our experimental
model is the bacteriophage Qβ propagated in the presence of a
mutagenic nucleoside analogue (5-azacytidine or AZC). The
phenotypic traits we have evaluated include the replicative
ability of individual viruses and their thermal stability at
temperatures above 50º C. Our results show that hypermutated
12
viruses are more prone to lose their infectivity upon exposition
at high temperatures. However, the repetition of a few cycles
of exposition to this adverse condition, followed of replication
of the surviving viruses, leads to the selection of virus
populations with increased stability in hot environments.
The genetic analysis of the populations evolved at increased
error rate has allowed us to investigate how beneficial
mutations that reduce the sensitivity to AZC spread at
increased error rate. We have found that the process is affected
by interference between different mutations and also by
antagonistic epistasis, resulting in the prolonged permanence of
polymorphisms [1,2]. We have also found that the main
mutation conferring AZC resistance is located in a protein
which is present at low amount in the capsid. The result
suggests that the correct assembly of the virus capsid is one of
the main targets of selection in the presence of AZC.
1. M. Arribas, L. Cabanillas and E. Lázaro, Virology 417, 343-352
(2011).
2. L. Cabanillas, M. Arribas, and E. Lázaro, BMC Evolutionary
Biology 13:11 (2013).
13
Evolving on Phenotype Landscapes
José A. Cuestaa,b and Susanna C. Manrubiaa,c
a
Grupo Interdisciplinar de Sistemas Complejos (GISC),
Departamento de Matemáticas, Universidad Carlos III de Madrid,
Avda. de la Universidad 30, 28911 Leganés, Madrid, Spain
b
Instituto de Biocomputación y Física de Sistemas Complejos (BIFI),
Universidad de Zaragoza, 50009 Zaragoza, Spain
c
Centro de Astrobiología, CSIC-INTA, Carretera de Ajalvir km 4,
28850 Torrejón de Ardoz, Madrid, Spain
Despite the usefulness of genotype landscapes they are
a source of potential misunderstandings. The reason is that the
genotype-to-phenotype map is highly degenerated. Huge
patches of the genotype landscape (genotype networks, GN)
correspond to just a single phenotype. Natural selection is blind
to genotypic differences within the same GN. As genotype
landscapes are patchworks of GN, populations traversing them
by accumulating mutations will exhibit quite an unusual
dynamic behavior. We develop a simplified model of
phenotype landscape inspired by quantitative studies of GN of
RNA. As a first approximation, this landscape can be regarded
as a network of interconnected phenotypes. Individuals with
the same phenotype reproduce at the same rate. Populations
jump from a given phenotype to a neighboring one, but the rate
at which they do is determined by topological properties of the
GN, in particular its size and the time already spent within it.
This renders the evolutionary process non-Markovian. We
explore the implications of this phenotype-based evolutionary
model for the adaptability of quasi-species as well as for
14
phylogeny.
Real-time numerical forecasts of global
epidemic spreading
José J. Ramascoa and the GLEaM team
a
Instituto de Física Interdisciplinar y de los Sistemas Complejos
IFISC (CSIC-UIB), Campus UIB, 07122 Palma, Spain .
Mathematical and computational models for infectious
diseases are increasingly used to support public-health
decisions. Their capacity to forecast disease arrival times,
number of cases or even the quantities of drugs or beds needed
to treat patients could suppose a major leap forward for doctors
and health-system managers. However, the reliability of these
methods to offer good quality predictions must be proven. Data
gathered for the 2009 H1N1 influenza crisis represent an
unprecedented opportunity to validate real-time model
predictions and define the main success criteria for different
approaches. We used the Global Epidemic and Mobility Model
to generate stochastic simulations of epidemic spread
worldwide, yielding (among other measures) the incidence and
seeding events at a daily resolution for 3,362 subpopulations in
220 countries. Using a Monte Carlo Maximum Likelihood
analysis, the model provided an estimate of the seasonal
transmission potential through the Monte Carlo likelihood
analysis and generated ensemble forecasts for the activity
peaks in the northern hemisphere in the fall/winter wave. These
15
results were validated against the real-life surveillance data
collected in 48 countries, and their robustness assessed by
focusing on 1) the peak timing of the pandemic; 2) the level of
spatial resolution allowed by the model; and 3) the clinical
attack rate and the effectiveness of the vaccine.
M. Tizzoni et al., BMC Medicine 10, 165 (2012).
Presentación del Grupo de Virología Molecular
de Plantas del IBMCP
Vicente Pallàs
Instituto de Biología Molecular y Celular de Plantas (IBMCP) (UPVCSIC); Av. De los Naranjos S/N; Ed. 8E, 46022 Valencia.
The main objectives that the Plant Molecular Virology
Group of the IBMCP addresses are the following: (1) Intraand inter-cellular movement of viruses and viroids in their
susceptible host plants; (2). Protein and RNA trafficking trough
vascular tissues and (3). Characterization of host factors
interacting with viral genes that are responsible of the viral
susceptibility and/or resistance. To address these objectives we
use three different RNA pathogens: Carmovirus,
Alfamo/Ilarvirus, and viroids.
Carmoviruses are one group of viruses with the almost
simplest genome organization known. Melon necrotic spot
carmovirus (MNSV) is a small (~30 nm), isometric plant virus
that has an icosahedral symmetry with a triangulation number
of T=3. Virions are composed of 180 identical CP subunits,
16
which consist of three subunits (A, B and C). The CP is
divided into three domains, designated as the RNA-binding
domain (R), the shell domain (S), and the protruding domain
(P). The arm region is located between the R and S domains,
and the hinge region is between the P and S domains. The P
domain projects outward from the virus particle and has a
characteristic anti-parallel β-sheet called a jellyroll
conformation, which has been found in a variety of proteins
having ligand binding functions. The MNSV genome consists
of a 4.3-kb, positive-sense, ssRNA containing five ORFs,
including p29, p89, p7A, p7B and p42. The coat protein (CP)
is encoded on p42. We have demonstrated that p7A and 7B are
involved in virus movement. P7A has RNA-binding properties
being this activity essential for the cell to cell movement
(Navarro et al., 2006; Genovés et al., 2009). P7B is an integral
membrane protein harbouring a unique transmembrane domain
which is also essential for intracellular movement (MartinezGil et al., 2007: Genovés et al., 2011). We have demonstrated
that an active COPII-dependent early secretory pathway is
required for the intra- and intercellular cell-to-cell movement
of MNSV, revealing the involvement of the Golgi apparatus in
this process (Genovés et al., 2010).
Alfalfa mosaic virus (AMV), a member of the
Bromoviridae family of plant viruses, occurs predominantly as
bacilliform particles with a diameter of 19 nm and a length
varying from 30 to 56 nm composed of one of the four
genomic RNAs and a surrounding shell built from a single
gene product of 220 residues. AMV particles are labile
structures held together predominantly by RNA-protein
interactions. The RNA is exposed at the outside and the CP
assembles into T = 1 spheres of 60 subunits. In addition to
virion formation, the coat protein (CP) of AMV is involved in
the regulation of replication and translation of viral RNAs, and
17
in cell-to-cell and systemic movement of the virus. An
intriguing feature of the AMV CP is its nuclear and nucleolar
accumulation. We have recently identified an N-terminal
lysine-rich nucleolar localization signal (NoLS) in the AMV
CP required to both enter the nucleus and accumulate in the
nucleolus of infected cells, and a C-terminal leucine-rich
domain which might function as a nuclear export signal (NES)
(Herranz et al., 2012). Moreover, we demonstrated that AMV
CP interacts with importin-α, a component of the classical
nuclear import pathway. A mutant AMV RNA 3 unable to
target the nucleolus exhibited reduced plus-strand RNA
synthesis and cell-to-cell spread. Moreover, virion formation
and systemic movement were completely abolished in plants
infected with this mutant. In vitro analysis demonstrated that
specific lysine residues within the NoLS are also involved in
modulating CP-RNA binding and CP dimerization, suggesting
that the NoLS represents a multifunctional domain within the
AMV CP. The observation that nuclear and nucleolar import
signals mask RNA-binding properties of AMV CP, essential
for viral replication and translation, supports a model in which
viral expression is carefully modulated by a cytoplasmic/
nuclear balance of CP accumulation.
Navarro JA, Genoves A, J. Climent, A. Sauri, L. Martinez-Gil, I.
Mingarro and Pallas V. (2006) RNA-binding properties and
membrane insertion of Melon necrotic spot virus (MNSV)
double gene block movement proteins. Virology 356: 57-67.
Martinez-Gil, L., Sauri, A., Vilar, M., Pallas, V. and Mingarro, I.
(2007). Membrane insertion and topology of the p7B
movement protein of Melon necrotic spot virus (MNSV).
Virology 367 (2): 348-357.
Martínez-Gil, L., Sanchez-Navarro, J.A., Cruz, A., Pallas, V., PerezGil, J., Mingarro, I. (2009). Plant Virus Cell-to-Cell
Movement Is Not Dependent on the Transmembrane
18
Disposition of Its Movement Protein. J. Virol. 83(11): 55355543.
Genovés A, Navarro JA and Pallas V. (2009). A self-interacting
carmovirus movement protein plays a role in binding of viral
RNA during the cell-to-cell movement and shows an actin
cytoskeleton dependent location in cell periphery. Virology
395: 133-142.
Genovés A, Navarro JA and Pallas V. (2010). The Intra- and
Intercellular movement of Melon necrotic spot virus
(MNSV) depends on an active secretory pathway. Molecular
Plant Microbe Interactions 23(3): 263-272.
Genovés, A., Pallás, V. and Navarro, J.A. (2011).Contribution of
topology determinants of a viral movement protein on its
membrane association, intracellular traffic and viral cell-tocell movement. J. Virol. 85(15): 7797-7809.
Herranz, M.C., Pallas V, Aparicio F. (2012). Multifunctional roles for
the N-terminal basic motif of Alfalfa mosaic virus coat
protein: nucleolar/cytoplasmic shuttling, modulation of
RNA-binding activity and virion formation. Mol. Plant
Microbe Interact. 25(8): 1093-1103.
19
Magnesium-Dependent RNA Folding of the
Internal Ribosome Entry Site of Hepatitis C
Virus Genome
Monitored by Atomic Force Microscopy.
Ana García-Sacristána,b, Elena López-Camachoa,c, Ascensión
Ariza-Mateosb,d,
a
Miguel Moreno , Rosa M. Jáudenesa, Jordi Gómezb,d, José
Ángel Martín-Gagoa,c and
Carlos Brionesa,b.
a
Department of Molecular Evolution, Centro de Astrobiología
(CSIC-INTA), Torrejón de Ardoz, Madrid.
b
Centro de Investigación Biomédica en Red de enfermedades
hepáticas y digestivas. (CIBERehd), Spain.
c
Instituto de Ciencia de Materiales de Madrid (CSIC), Cantoblanco,
Madrid.
d
Instituto de Parasitología y Biomedicina “López-Neyra” (CSIC),
Granada.
The 5’ untranslatable region (5’UTR) of the hepatitis C
virus (HCV) genomic RNA is highly structured and contains
an internal ribosome entry site (IRES) element responsible to
drive cap-independent translation initiation (1). The iondependent tertiary fold of the minimal HCV IRES element
(containing domains II to IV) has been investigated (2), and
significant progress has been made in determining the threedimensional structure of individual IRES domains and
subdomains at high resolution (3). Nevertheless, little
information is still available (4) on the tertiary structure of the
HCV IRES element.
20
Atomic Force Microscopy (AFM) is a useful
nanotechnology-based tool for the analysis of a wide range of
biological entities, including nucleic acids and their complexes
(5). We have optimized AFM technology for analysing HCV
IRES structure in native conditions as well as for monitoring
its conformational rearrangements in diverse physicochemical
environments, in particular at magnesium ion concentrations
ranging from 0 to 10 mM. Here we report the Mg2+-dependent
folding of the HCV IRES in a sequence context that includes
its structured, functionally relevant flanking regions (domains
I, V and VI). In the 571 nt-long HCV genomic RNA molecule
analyzed, a structural switch has been monitored when Mg 2+
concentration increases from 2 to 4 mM. This effect has been
confirmed by classical molecular biology techniques for RNA
structural characterization, such as gel-shift analysis and partial
RNase T1 cleavage. Our results suggest a magnesium-driven
transition from an ‘open’ to a relatively ‘closed’ conformation
of the HCV IRES.
1. P. J. Lukavsky. Virus Res. 139: 166 (2009)
2. J. S. Kieft et al. J. Mol. Biol. 292: 513 (1999)
3. K. E. Berry et al. Structure 19: 1456 (2011)
4. J. Pérard et al. Nat Commun. 4: 1612 (2013)
5. H. G. Hansma et al. Curr. Op. Struct. Biol. 14: 380 (2004)
21
Manipulation and Biological Implications of the
Thermal Stability and Mechanical Properties of
Viruses
Mauricio G. Mateu
Centro de Biología Molecular “Severo Ochoa” (CSIC-UAM),
Universidad Autónoma de Madrid, Cantoblanco 28049 Madrid
We are using protein engineering to manipulate the
thermal stability and mechanical properties of small viruses.
We aim at understanding the molecular determinants that
underlie the physical properties of viruses, and also at the
design of viral particles with improved thermal and/or
mechanical resistance for bio/nanotechnological applications.
We have recently discovered that electrostatic repulsions
between subunits in the capsid of foot-and-mouth disease virus
(FMDV) underlie its low thermostability, and we have
engineered thermostable FMDV variants for improved
vaccines. In collaboration with Dr. P.J.de Pablo´s and
J.Gómez´s groups (Dept. of Physics of the Condensed Matter,
UAM) we found that, in the minute virus of mice (MVM),
segments of the viral DNA bound to specific sites in the capsid
act like molecular buttresses that decrease the mechanical
elasticity of most regions in the viral particle. However, the
regions around channels involved in biologically relevant
molecular translocation events are kept free from bound DNA,
and remain as elastic as in the empty capsid. Our recent studies
using atomic force microscopy indicate that this anisotropic
distribution of mechanical stiffness may be a biological
22
adaptation to prevent MVM inactivation without impairing
infection. We have also mechanically disassembled single
MVM particles using AFM, and experimentally identified
theoretically predicted assembly/disassembly intermediates.
Finally, these studies have led to the engineering of the
mechanically stiffer viral capsids known to date.
Structural Determinants of Adenovirus Assembly
Carmen San Martín
Department of Macromolecular Structure. Centro Nacional de
Biotecnología (CNB-CSIC). Darwin 3, 28049 Madrid (Spain)
We focus on the principles governing assembly and
stabilization of complex viruses, using adenovirus (AdV) as a
model system. The dsDNA AdV genome is bound to large
amounts of positively charged proteins that help condense it
forming the core, which is confined inside a T=25 icosahedral
capsid composed by multiple copies of seven different viral
proteins. The final stage of AdV morphogenesis consists in
proteolytic processing of several capsid and core proteins. The
immature virus, containing all precursor proteins, is not
infectious due to an uncoating defect. To determine why the
presence of precursor proteins impairs uncoating, we have
carried out in vitro disruption analyses of mature and immature
capsids. The results show how maturation primes the virus for
stepwise uncoating in the cell, and reveal the structural changes
undergone by the virion in conditions similar to those
23
encountered during entry(1). We have also contributed to define
the role of the viral genome as a cofactor of the AdV protease
during maturation(2,3). Current research interests include the
mechanism of genome packaging and the organization of the
non-icosahedral components in the virion.
1. A. J. Pérez-Berná et al., J Biol Chem 287, 31582 (2012).
2. V. Graziano et al., J Biol Chem 288, 2068 (2013).
3. P. C. Blainey et al., J Biol Chem 288, 2092 (2013).
Physical virology with Atomic Force Microscopy
Pedro J. de Pablo Gómez
Universidad Autónoma de Madrid, 28049 Madrid, Spain.
Viruses are striking examples of macromolecular
assembly of proteins, nucleic acids, and sometimes lipid
envelopes that form symmetric objects with sizes ranging from
10s to 100s of nanometers. The basic common architecture of a
virus consists of the capsid a protein shell made up of repeating
protein subunits, which packs within it the viral genome which
can be single or double stranded DNA or RNA depending on
the type of the virus. Virtually every aspect of the virus cycle
from DNA packing to maturation to interaction with the host
modifies and, in turn, is influenced by the material properties
of the virus. In this talk I will show how Atomic Force
Microscopy has emerged as a unique technique to unveil some
24
physical properties of viruses, such as stiffness and elasticity,
which can be directly related to their structure and function (1).
In addition, AFM enables monitoring the dynamics of virus
disassembly in real time to unveil the ultimate physical
changes to trigger virus infectivity (2).
1. Hernando-Pérez, M., Miranda, R., Aznar, M., Carrascosa, J. L.,
Schaap, I. A. T., Reguera, D., and de Pablo, P. J. Small 8, 2365
(2012).
2. Ortega-Esteban, A., Pérez-Berná, A. J., Menéndez-Conejero, R.,
Flint, S. J., San Martín, C., and de Pablo, P. J. Scientific Reports 3,
1434 (2013).
Triatoma Virus (TrV) capsid disassembly and
genome release
Rubén Sánchez-Eugenia and Diego M.A. Guérin
Unidad de Biofísica (CSIC-UPV/EHU), and §Fundación Biofísica
Bizkaia.
Bº Sarriena S/N, 48940 Leioa, Bizkaia, Spain. Email:
[email protected]
TrV is a small spherical, non-enveloped, +ssRNA virus
that infects triatomines (Hemiptera: Reduviidae), and belongs
to the Dicistroviridae family (1). Dynamic Light Scattering and
intrinsic fluorescence experiments at low pH (<5.0) indicate
that acidification does not affect capsid integrity(2). Cryo-EM
3D reconstructions show that, after genome release, the
25
resulting empty capsid do not displays striking conformational
changes in reference to the native viral capsid (3). Atomic
Force Microscopy nanoindentation and Native Mass
Spectrometry experiments show that the encapsidated RNA
plays an important role in stabilizing the viral integrity, and
that the interplay between protein shell and genome is highly
dependent on the pH (4). These and other experiments,
demonstrate that in TrV, the genome release displays features
that are in contrast with the current model of genome delivery
based on the mammalian viruses poliovirus and rhinovirus.
1) "Characterization of Triatoma virus, a Picorna-like virus
isolated from the Triatomine bug Triatoma infestans". O. A.
Muscio, J. L. La Torre, E. A. Scodeller (1988). J. Gen. Virol. 69
:2929–2934.
2) "Capsid protein identification and analysis of Triatoma Virus
(TrV) mature virions and naturally occurring empty particles".
Agirre, J., Aloria, K., Arizmendi, J.M., Iloro, I., Elortza, F., Marti,
G.A., Neumann, E., Rey, F.A., and Guérin, D.M.A. (2011) Virology
409:91-101.
3) “Cryo-TEM reconstruction of Triatoma virus particles: a clue
to unravel genome delivery and capsid disassembly”. Agirre, J.,
Goret, G., LeGoff, M., Sánchez-Eugenia, R., Marti, G.A., Navaza, J.,
Guérin D.M.A., and Neumann, E. (2013) J. Gen. Virol. In press
(DOI: 10.1099/vir.0.048553-0).
4) "Probing the biophysical interplay between a viral genome
and its capsid" J. Snijder, C. Uetrecht, R.J. Rose, R. SanchezEugenia, G.A. Marti, J. Agirre, D.M.A. Guérin, G.J.L. Wuite, A.J.R.
Heck, W.H. Roos (2013). Nature Chemistry. In press.
26
Dynamic simulations of virus budding
Teresa Ruiz-Herreroa and Michael Haganb
a
Departamento de Física Teórica de la Materia Condensada,
Universidad Autónoma de Madrid.
b
Department of Physics, Brandeis University, Waltham, MA, USA
For many viruses assembly and budding occur
simultaneously during the last stage of the replication cycle.
Understanding the basic mechanisms of this process could
promote biomedical efforts to block viral replication and
enable use of capsids in nanomaterials applications. To this
end, we have performed molecular dynamics simulations on a
coarse-grained model to elucidate the special characteristics for
virus assembly on a fluctuating surface. Our simulations show
that the membrane promotes assembly through dimensional
reduction of adsorbed subunits, but also introduces barriers that
inhibit complete assembly. We find that a domain within the
membrane (i.e. lipid raft) can enhance assembly by reducing
these barriers. Furthermore, the simulations demonstrate that
assembly and budding depend crucially on the system
dynamics via multiple timescales related to membrane
deformation, protein diffusion, association, and adsorption
onto the membrane.
27
Class II viral membrane fusion proteins:
virus/host gene exchanges and cell-cell fusion
events in multicellular organisms
Félix Rey
Institut Pasteur / CNRS
Paris, France
Class II proteins are viral membrane fusogenic molecules
folded essentially as β-sheet and having an internal fusion
peptide. In particular, they lack the characteristic central alphahelical coiled coil present in the post-fusion conformation of
all other viral fusion proteins. The regular, icosahedrally
symmetric enveloped viruses that have been studied so far,
such as flaviviruses, alphaviruses and phleboviruses have been
shown to have class II fusion proteins, which in their prefusion conformation make an icosahedral shell surrounding the
viral membrane. Yet despite having very similar envelope
proteins, these viruses belong to three different viral families
with totally different genome replication machineries. We have
recently identified the rubella virus fusion a belonging to class
II, although the virus particles appear pleomorphic and lack
icosahedral symmetry. In spite of the lack of any detectable
sequence conservation, the available structures indicate that
class II proteins have undergone divergent evolution from a
distal, ancestral gene. We have now discovered that the cellular
fusion protein EFF-1, involved in syncytium formation during
the genesis of the skin in nematodes (C. elegans) and in other
multicellular organisms, is also folded as a class II viral fusion
protein, thereby indicating common ancestry, highlighting an
unprecedented amount of exchange of genetic information
28
between viruses and cells. My talk will discuss the
implications of this finding, which highlights the intricate
exchange of genetic information that has taken place between
viruses and cells during evolution. This analysis also suggests a
mechanism for the homotypic cell-cell fusion process, which
has not been studied so far.
Structural Characterization of RNA Viruses
Núria Verdaguer
Institut de Biologia Molecular de Barcelona CSIC, Parc
Científic de Barcelona Baldiri i Reixac10, 08028, Spain.
The replicative cycle in RNA viruses relies on: i) the
attachment to the appropriate cellular receptors, efficient entry
into the host cell and delivery of the viral RNA into the
cytoplasmon, ii) the activity of unique virus-encoded enzymes,
leading to viral RNA and protein synthesis and, iii) the
assembly of infectious virions that are released from the cell to
continue the infectious process. The structural and nonstructural viral proteins that orchestrate these steps are
potentially vulnerable targets for “attack” by appropriate
ligands that interfere with their functionality.
Results of our recent research, aimed at the elucidation of
the X-ray structures of different viral proteins and protein-RNA
complex assemblies involved in RNA uncoating and RNAdependent RNA replication, will be presented.
29
Three-dimensional Visualization of Forming
Hepatitis C Virus-like Particles by ElectronTomography
Daniel Badia-Martineza, Bibiana Peraltaa, German Andrésb,
Milagros Guerrab, David Gil-Cartóna and Nicola Abresciaa,c
a
Structural Biology Unit, CIC bioGUNE, CIBERehd, 48160 Derio,
Spain.
b
Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Campus
Cantoblanco,
28049 Madrid, Spain.
c
IKERBASQUE, Basque Foundation for Science, 48011 Bilbao,
Spain.
Hepatitis C virus (HCV) infects almost 170 million people per
year being one of the major causes for chronic liver disease. As
other flaviviruses, HCV is thought to replicate in the cytoplasm
acquiring the viral envelope by budding through the endoplasmatic reticulum (ER) but its assembly pathway with the involvement of lipiddroplets, architecture and structures of its
envelope proteins are poorly understood. With this paucity of
three-dimensional (3D) structural information, applying a reductionist and mechanistic approach we embarked in studying
HC virus-like particles produced in insect cells. Using electron
tomography of plastic-embedded sections of Sf9 cells, we have
provided a 3D morphological description of these HCV-LPs at
the ER site as surrogate of wt-HCV allowing to view the particles one-by-one and each in its budding stage (differently to
the previously used 2D imaging technique that displays the
HCV-LPs as projection and whose shape doesn’t necessarily
reflect the budding stage). Tomographic data were collected on
our JEOL JEM2200-FS microscope on a 4Kx4K CCD camera.
30
Tomograms were processed with IMOD, denoised using Tomobflow and analysed with Chimera and Amira softwares. Our
data provide a 3D sketch of viral assembly at the ER site with
different budding stages identified as three main classes: (i)
membrane areas of protein concentration, (ii) cup-shaped particles and (iii) particles on the verge of scission. Furthermore we
could detect proximity of buds from which we hypothesize a
mechanism of large particles formation.
Acknowledgments
We are extremely grateful to Genentech and S. Foung for providing
respectively the AP33 antibody and the
antibodies CBH-2, -5 and -7 against glycoprotein E2.
31
Cryo-EM structure of Penicillium chrysogenum
virus at 4 Å resolution
J.R. Castóna, J. Gómez-Blancoa, D. Luquea, D. Garrigab, José
M. Gonzáleza, A. Brilotc, W.H. Havensd, J.L. Carrascosaa, B.L.
Truse, N. Verdaguerb, N. Grigorieffc and S.A. Ghabriald
a
Centro Nacional de Biotecnología/CSIC, 28049 Madrid, Spain;
b
IBMB/CSIC, 08028 Barcelona, Spain; cBrandeis University,
Waltham MA, USA; dUniversity of Kentucky, Lexington KY, USA;
e
CIT-NIH, Bethesda MD, USA
Penicillium chrysogenum virus (PcV) is a fungal
dsRNA virus with a genome comprised of four segments. The
PcV capsid is based on a T=1 lattice formed by 60 subunits.
Whereas the PcV capsid protein (CP) has two motifs with a
similar fold, most dsRNA virus capsid subunits consist of
dimers of a single protein (with a 120-subunit capsid). This
ubiquitous stoichiometry provides an optimal framework for
genome replication and organization.
We report the 3D structure by single-particle cryo-EM analysis
of PcV at ~4 Å resolution. The full-atom model of the 982amino-acid CP showed the critical contacts among subunits
that mediate capsid assembly, and specific RNA-protein
interactions. Despite the lack of sequence similarity between
the two halves, the CP is an almost perfect structural
duplication of a single domain in which most -helices and chains matched very well. Superimposition of secondary
structure elements showed a single “hot spot” into which
structural and functional variations can be introduced by
insertion of distinct segments.
32
The near-atomic structure of the PcV capsid protein derived
from cryo-EM data has allowed us to determine that its
conserved core is a hallmark fold preserved in the dsRNA virus
lineage.
Structural Analysis of Rotavirus Infection
Associated Macromolecular Complexes
Daniel Luquea, Esther Martín-Foreroa, Fernando GonzálezCamachoa, María del Carmen Terróna, José L. Carrascosab,
José R. Castónb, Javier M. Rodrígueza
a
CNM-ISCIII. Carretera de Majadahonda - Pozuelo, Km. 2.200.
28220 - Majadahonda (Madrid).
b
CNB-CSIC. C/ Darwin nº 3, Cantoblanco. 28049 Madrid.
Our laboratory combines electron microscopy and image
processing methods with molecular biology techniques in order
to determine structure-function relationships in medically
important human pathogens. One of our main research topics is
Rotavirus, the most relevant member of the family Reoviridae
due to its public health significance and its role as a model in
the research of this complex family of dsRNA viruses.
To become fully infectious, the rotavirus virion spike
protein must be proteolytically cleaved by trypsin-like
proteases in the intestinal lumen. To investigate the mechanism
underlying this step we have analyzed cleaved and intact
rotavirus particles by cryo-electron microscopy. These studies
have revealed a new trypsin-independent reorganization of the
33
virus spike and its importance for virus infectivity.
Unlike the rest of the Reoviridae, whose morphogenesis is
purely cytoplasmic, the immature rotavirus particles enter the
reticulum, are surrounded by a membrane, lose that membrane
and are released into the cytoplasm as mature. We have
addressed the production and structural characterization of this
rotavirus Membrane Enveloped Particles (MEPs) to understand
the rotavirus morphogenesis and as a model for the study of the
transport of protein complexes across the endoplasmic
reticulum.
Structural biology of viral fibres
Carmela Garcia-Dovala, Laura Córdoba Garcíaa, Meritxell
Granell Puiga, Abhimanyu K. Singha, Thanh H. Nguyena,
Marta Sanz Gaiteroa, Mark J. van Raaija
a
Departamento de Estructura de Macromoléculas, Centro Nacional
de Biotecnología (CNB-CSIC) c/Darwin, 3 28049 Madrid
Our research focuses on the fibres some viruses use to
attach to their host cells. These fibres have a common
structure: a N-terminal virus attachment domain, a shaft
domain and a C-terminal receptor-binding domain involved in
host recognition. Our goal is to determine the structure of these
fibres and determine their role in host recognition.
During the last two years we solved the structures of the Cterminal head domains of two different adenovirus fibres from
34
hitherto unknown genera: one from the Turkey type 3
Siadenovirus and one from the Snake type 1 Atadenovirus.
In 2010, we published the structure of the C-terminal
domain of gp37 (1), the distal half of bacteriophage T4 fibre.
Now we have also solved the structure of part of the proximal
fibre protein gp34 and in collaboration with the group of Pedro
de Pablo (UAM) we are performing AFM experiments on fulllength gp37.
We also solved the structure of the C-terminal domain of the
bacteriophage T7 fibre (2) and we are doing a mutational
analysis of the residues that may be involved in receptor
recognition.
1. Bartual, S. G., Otero, J. M., Garcia-Doval, C., Llamas-Saiz, A. L.,
Kahn, R., Fox, G. C. and van Raaij, M. J. Proc Natl Acad Sci U S A
107, 20287-20292 (2010).
2. Garcia-Doval, C. and van Raaij, M. J. Proc Natl Acad Sci U S A
109, 9390-9395 (2012).
35
Studies on the double stranded DNA packaging
machinery of viruses.
José L. Carrascosaa,c Rebeca Bocanegraa, A. Cuervoa and M.
Ibarraa
a
c
Centro Nacional de Biotecnología, CSIC, c/Darwin 3, Cantoblanco,
28049 Madrid, Spain
Instituto Madrileño de Estudios Avanzados en Nanociencia (IMDEA
Nanociencia), Cantoblanco, 28049 Madrid, Spain
Double stranded DNA bacteriophages (dsDNA)
package actively their genetic material inside the capsid using
a protein motor which requires the hydrolysis of ATP as energy
source. DNA enters inside the viral head through the channel
formed by the connector that sits at a unique five-fold vertex of
the icosahedral capsid, a vertex which is also involved in the
delivery of the genome during DNA ejection.
We are studying the different components of this
machinery, namely the connector (which forms the
dodecameric portal at the viral vertex), the terminase (which is
a powerful motor that converts ATP hydrolysis into mechanical
movement of the DNA) and the tail proteins (which prevent
DNA exit and, upon a signal, promote the ejection of the DNA
from the viral head).
We have determined that the terminase assembled into
the portal complex shows a different conformation when
compared to the isolated terminase pentamer. The function
of the portal vertex is studied by efficient orthogonal
integration of the connector into lipid bilayers that allows to
36
perform DNA packaging in liposomes with integrated
connectors. We use a new experimental set up based in the
combination of patch clamp and optical tweezers. We are also
working in the structure of the DNA ejection machinery. We
have determined the precise topology of the tail structural
proteins by comparing the structure of the T7 tail extracted
from viruses and a recombinant complex formed by gp8, gp11
and gp12, and our high resolution model reveals the existence
of a common architecture with other Podoviridae tail
complexes.
37
Are electrostatic and elastic properties of viruses
tuned by evolution and how?
Antonio Šiber
Institute of physics, Bijenička c. 46, 10000 Zagreb, Croatia
Viruses have been studied as electrostatic and
elastic entities, yet, not much is really known regarding
the possible evolutionary convergence of their
electrostatic and elastic properties. Are there physical
constraints and reasons for these properties to converge at
all? Can we possibly say something regarding the
evolutionary relatedness of different viruses on the basis
of their physical properties? I will present some results
from my studies of elastic [1-3] and electrostatic [4-8]
properties of viruses and discuss the constraints that these
properties impose on a functional, evolutionary viable
virus.
[1] A. Šiber, "Buckling transition in icosahedral shells subjected to
volume conservation constraint and pressure: Relations to virus
maturation", Phys. Rev. E 73, 061915 (2006).
[2] A. Šiber and R. Podgornik, "Stability of elastic icosadeltahedral
shells under uniform external pressure: Application to viruses under
osmotic pressure“, Phys. Rev. E 79, 011919 (2009).
[3] A. Lošdorfer Božič, A. Šiber and R. Podgornik, “Statistical
analysis of sizes and shapes of virus capsids and their resulting
elastic properties”, in press in J. Biol. Phys., DOI 10.1007/s10867013-9302-3
[4] A. Šiber and R. Podgornik, "Role of electrostatic interactions in
38
the assembly of empty spherical viral capsids", Phys. Rev. E 76,
061906 (2007).
[5] A. Šiber and R. Podgornik, "Nonspecific interactions in
spontaneous assembly of empty versus functional single-stranded
RNA viruses“, Phys. Rev. E 78, 051915 (2008).
[6] A. Šiber and A. Majdandžić, "Spontaneous curvature as a
regulator of the size of virus capsids“, Phys. Rev. E 80, 021910
(2009).
[7] A. Lošdorfer Božič, A. Šiber, and R. Podgornik, "How simple can
a model of an empty viral capsid be? Charge distributions in viral
capsids“, J. Biol. Phys. 38, 657 (2012).
[8] A. Šiber, A. Lošdorfer Božič, and R. Podgornik, "Energies and
pressures in viruses: contribution of nonspecific electrostatic
interactions“, Phys.Chem.Chem.Phys. 14, 3746 (2012).
A minimalist potential energy model for the selfassembly of virus capsids
J. Hernández-Rojas, J. Bretón, and J.M. Gomez Llorente
Departamento de Física Fundamental II and IUdEA,
Universidad de La Laguna, 38205, Tenerife, Spain
We present a simple potential energy model built as a sum
of pair-wise anisotropic interactions for viruses with two types
of capsomers: pentamers and hexamers. While the pentamerhexamer interaction parameters depend on the number of
capsomers, the hexamer-hexamer potential parameters are the
same for all capsids. “Basin-hopping” global optimization
method is used to find the lowest energy structures for virus
39
capsids with up to N=176 (Bacteriophage T4 head). Among
these structures we find those of the icosahedral viruses. We
also find prolate and oblate capsids based on Moody’s rules
and other new structures derived from the hexagonal lattice.
Physical Modeling of the Self-Assembly and
Mechanical Properties of Viruses
David Reguera
Departament de Física Fonamental, Universitat de Barcelona, C/
Martí i Franquès 1, 08028 Barcelona, SPAIN
Viruses are fascinating biological entities, in the fuzzy
frontier between life and inert matter. Contrary to most
biological organisms, viral particles are made of a minimal
number of relatively simple components that are not capable of
any metabolic activity, except when their genome sequesters
the metabolism of the infected host to achieve the replication
of new particles. Despite the lack of sophisticated biological
machinery, viruses have found the way to efficiently infect the
host, assemble, and egress the cell following, in many cases, a
coordinated sequence of passive and spontaneous processes.
This strongly suggests that, during their life cycle, viruses must
rely on general physical and chemical mechanisms to succeed
in their different tasks and to achieve the required resistance
against possible extreme environmental conditions.
In this talk, I will summarize some of our recent efforts
40
to understand the basic physical principles behind the
virus life-cycle. In particular, I will focus on the kinetics
and thermodynamics of assembly of empty capsids using
computer simulations of simplified coarse-grained
models. We will also discuss how these models can be
very useful to understand the remarkable mechanical
properties of the resulting capsid. The results of these
studies provide new insights into the microscopic
mechanisms of the assembly process and the physical
ingredients controlling the selection of a particular
structure that can be potentially very useful to develop
biomedical and nanotechnological applications.
The Physics of Tobacco Mosaic Virus
A.M. Bittnera,b
a
CIC nanoGUNE, San Sebastián, Spain.
b
Ikerbasuqe, Bilbao, Spain.
How do nanoscale fibres (and tubes!) interact with water
(and with other liquids)? The answer has to rely on a very good
choice of the fibre (or tube). The use of plant viruses is
motivated by their simple structure, well-defined diameter, and
well-characterised chemical behaviour.
The Self-Assembly group employs scanning probe and
environmental electron microscopy techniques to Tobacco
41
mosaic virus. We observe wetting scenarios below 50 nm.
However, the big challenge is the molecular scale below 5 nm.
We were able to address it indirectly with TEM: We try to
clarify how solutions of metal complexes interact with the
virus, with a special focus on effusion from the 4 nm channel
inside the virion. Potential uses of plant viruses include acting
as templates for nanoscale materials, and as drug delivery
vehicle.
1. J.M. Alonso et al., review Trends in Biotechnol., subm. (2013).
2. A.A. Khan et al. Langmuir 29 (2013) 2094-2098.
3. J.M. Alonso et al. Nanotechnol. 24 103405 (2013).
4. S. Balci et al., Nanotechnol., 23 045603 (2012).
5. A. Mueller et al. ACS Nano, 5 (2011) 4512-4520.
6. A. Kadri et al. Virus Res., 157 (2011) 35-46.
42
Self-Assembly Triggered by Self-Assembly:
Virus-Like Particles Loaded with Supramolecular Nanomaterials
Andres de la Escosura,a Melanie Brasch,b Jealemy
Galindo,b Eduardo Anaya,a Francesca Setaro,a Daniel
Luque,c Jose L. Carrascosa,c Jose R. Caston,c Jeroen J. L.
M. Cornelissenb and Tomas Torresa,d
a
Universidad Autónoma de Madrid, Organic Chemistry Department,
Cantoblanco, 28049 Madrid (Spain)
f
Laboratory for Biomolecular Nanotechnology, MESA+ Institute,
University of Twente, PO Box 207, 7500 AE Enschede (The
Netherlands)
c
Department of Structure of Macromolecules, Centro Nacional de
Biotecnología/CSIC, Cantoblanco, 28049 Madrid (Spain)
d
IMDEA-Nanociencia, Facultad de Ciencias, Ciudad Universitaria
de Cantoblanco, 28049 Madrid (Spain)
The self-assembly of biomolecules such as the coat proteins
(CP) of virus capsids offer great opportunities in
nanotechnology and nanomedicine, leading to monodisperse
platforms where different chemical species can be organized
through covalent or non-covalent bonding. Yet, because the
covalent approach for the modification of virus capsids is still a
demanding task, efficient and straightforward supramolecular
strategies are highly desirable. The Cowpea Chlorotic Mottle
Virus (CCMV), in particular, is a plant virus of 28 nm in
diameter with an interesting sensitivity to pH and ionic
strength. Depending on these factors, CCMV capsids can
rapidly be disassembled in vitro into CP dimers and then reassembled again. In this presentation, we will show several
43
examples of hierarchical and cooperative processes in which
self-assembled organic chromophores serve as templates for
the assembly of the CCMV CP around them. In such processes,
the structure of the self-assembled templates determines the
size and geometry of the resulting virus-like particles (VLP),
while confinement within the VLP also determines the
structure of the chromophore self-assemblies. The precise
structure and assembly properties of these particles have been
studied in detail by microscopy techniques, and sophisticated
VLP have been designed and prepared for multimodal
photodynamic therapy (PDT) and imaging.
“Viruses and Protein Cages as Nanocontainers and Nanoreactors”,
A. de la Escosura, R. Nolte and J. Cornelissen, J. Mat. Chem. 2009,
19, 2274-2278.
“Encapsulation of DNA-Templated Chromophore Assemblies within
Virus Protein Nanotubes”, A. de la Escosura, P. Janssen, A.
Schenning, R. Nolte and J. Cornelissen, Angew. Chem. Int. Ed. 2010,
49, 5463-5466.
“Encapsulation of Phthalocyanine Supramolecular Stacks into VirusLike Particles”, M. Brasch, A. de la Escosura, Y. Ma, A. Heck, T.
Torres and J. Cornelissen, J. Am. Chem. Soc. 2011, 133, 6881.
“Self-Assembly Triggered by Self-Assembly: Protein Cage
Encapsulated Micelles as MRI Contrast Agents ”, J. Galindo, M.
Brasch, E. Anaya, A. de la Escosura, et al. Submitted.
“Structure and Assembly Properties of Phthalocyanine-Loaded VirusLike Particles”, D. Luque, A. de la Escosura, et al. Manuscript in
preparation.
44
NS3 Protease from Hepatitis C Virus:
Biophysical Characterization of a Partially
Disordered Protein Domain
O. Abiana,b, S. Vegaa, C. Marcuelloc, A. Lostaoc,d, J.L.
Neiraa,e, A. Velazquez-Campoya,d,f
a
Institute for Biocomputation and Physics of Complex Systems
(BIFI), Joint Unit BIFI-IQFR (CSIC), Universidad de Zaragoza,
Zaragoza, Spain
b
Centro de Investigación Biomédica en Red en el Área Temática de
Enfermedades Hepáticas y Digestivas (CIBERehd), ISCIII; Aragon
Health Sciences Institute (I+CS) – IIS Aragon, Zaragoza, Spain
c
Laboratorio de Microscopías Avanzadas (LMA), Instituto de
Nanociencia de Aragón (INA), Universidad de Zaragoza, Spain
d
ARAID Foundation, Government of Aragon, Spain
e
Instituto de Biología Molecular y Celular, Universidad Miguel
Hernández, Elche (Alicante), Spain
f
Department of Biochemistry and Cellular and Molecular Biology,
Faculty of Sciences, Universidad de Zaragoza, Zaragoza, Spain
The NS3 protease from the hepatitis C virus is located at the
N-terminal domain of the non-structural protein 3. It has been
considered as a drug target since its identification as a key
enzyme in the viral life cycle. A biophysical characterization
performed on this protein has unraveled a quite complex
conformational landscape for this allosteric enzyme, with a
substantial interplay between its intrinsic plasticity and the
interactions with cofactors (zinc and viral protein NS4A) and
substrates (1-3).
45
1. X. Arias-Moreno, O. Abian, S. Vega, J. Sancho and A.
2.
3.
Velazquez-Campoy. Curr. Protein Pept. Sci. 12, 325-338 (2011).
O. Abian, S. Vega, J. L. Neira and A. Velazquez-Campoy.
Biophys. J. 99, 3811-3820 (2010).
O. Abian, J. L. Neira and A. Velazquez-Campoy. Proteins 77,
624-636 (2009).
46
POSTERS
Aznar P1 .......................................................................................................... 48
Bocanegra P2 .................................................................................................. 50
Carrillo, P.J.P P3.............................................................................................. 51
Condezo P4 ..................................................................................................... 53
Correia P5 ........................................................................................................ 54
Cuervo A P6 .................................................................................................... 56
Ferrero D P7 .................................................................................................... 57
Guérin P8......................................................................................................... 59
Hernando-Pérez P9 ......................................................................................... 60
Iranzo P10 ....................................................................................................... 62
Llauró P11 ....................................................................................................... 64
Mertens P12 .................................................................................................... 65
Ortega-Esteban P13 ........................................................................................ 66
Pérez-Berná AJ P14 ....................................................................................... 67
Rincón V P15 .................................................................................................. 69
Rodrigo P16 .................................................................................................... 71
47
P1. Physical ingredients controlling the viral
capsid
María Aznar and David Reguera
Universidad de Barcelona,
Dpto. Física Fundamental, Martí i Franqués, 1,
08028 Barcelona
Spain.
Email: [email protected].
One of the crucial steps in the viral life cycle is precisely the
self-assembly of its protein shell. Typically, each native virus
self-assembles into a unique T- number structure, with some
exceptions like Hepatitis B Virus, which makes T=3 and T=4
capsids. But many viruses have the capability to self-assemble
into different T-number and shape structures in vitro by
changing the assembly conditions (i.e. typically the pH, salt
and protein concentrations). For example, Polyoma [1] or
Simian Virus 40 [2] self-assemble in vitro into T=1, snub
cubes, T=7 and different size tubes.
A proper understanding of the ingredients that control the in
vitro assembly of viruses is essential to get capsids with welldefined size and structure that could be used for promising
applications in medicine or bionanotechnology. However, the
mechanisms that determine which of the possible capsid shapes
and structures is selected by a virus and that avoid its
polymorphism are still not well known.
We present a coarse-grained model to analyze and understand
the physical mechanisms controlling the size and structure
48
selection in viral self-assembly [3]. We have characterized the
phase diagram and the stability of T = 1, 3, 4, 7 and snub cube
structures using Monte Carlo simulations. In addition, we have
studied the tolerance of the different shells to changes in
physical parameters related to ambient conditions. Finally, we
will discuss the factors that select the shape of the capsid as
spherical, faceted, elongated and decapsidated, in the range of
parameters
(directly related to measurable biophysical
parameters: bending constant and spontaneous curvature )
where a structure is stable.
[1] Howatson A.F. and Almeida J.D. 1960. Observations on the fine
structure of polyoma virus. Journal of Biophysical and Biochemical
Cytology, 8, 828-834.
[2] Kanesashi SN.et al 2003. Simian virus 40 VP1 capsid protein
forms polymorphic assemblies in vitro. Journal of General Virology,
84, 1899–1905.
[3] M.Aznar and D. Reguera. Physical ingredients controlling the
polymorphism and stability of viral capsid. In preparation.
49
P2. Optimizing a Combined Optical TweezersPatch Clamp Set Up to Study ϕ-29 Connector
Rebeca Bocanegraa, Lara H. Moleirob, Francisco
Monroyb, José L. Carrascosaa,c
a
Centro Nacional de Biotecnología, CSIC, c/Darwin 3, Cantoblanco,
28049 Madrid, Spain
b
Departamento de Química Física I, Universidad Complutense,
28040 Madrid, Spain
c
Instituto Madrileño de Estudios Avanzados en Nanociencia (IMDEA
Nanociencia), Cantoblanco, 28049 Madrid, Spain
Bacteriophage ϕ-29 encapsidates its DNA in a preformed
prehead using its packaging motor, located in one unique
vertex of the prehead. This packaging motor consists of three
macromolecular components: the connector protein, pRNA (an
RNA pentamer with structural function) and terminase (the
ATPase wich provides the energy for packaging from ATP
hydrolysis).
We have developed an optimized reconstitution
method for efficient orthogonal integration of native viral
connector into lipid bilayers, particularly of giant unilamelar
vesicles (1). We have optimized the bilayer in order to afford
the assembly of the complete ϕ-29 motor and we are currently
optimizing the DNA packaging in liposomes with integrated
connectors.
We also propose a new experimental set up based in
the combination of two powerful techniques: patch clamp and
optical tweezers. With this set-up we will be able to study the
forces implied in the DNA translocation through the channel,
by isolating a membrane patch with inserted connectors from
the GUVs previously formed.
50
1. L. H. Moleiro, I. López-Montero, I. Márquez, S. Moreno,
M. Vélez, J. L. Carrascosa and F. Monroy. ACS Synth. Biol 1(9),
414-424 (2012).
P3. Mechanical Disassembly of Single Virus
Particles Reveals Kinetic
Intermediates Predicted by Theory
Castellanos, M.a, Carrillo, P.J.P. a, Pérez, R. a, de Pablo
P.J. b, Mateu, M.G. a
a
Centro de Biología Molecular Severo Ochoa (Consejo Superior de
Investigaciones Científicas-Universidad Autónoma de Madrid) and
b
Departamento de Física de la Materia Condensada C-III,
Universidad Autónoma de Madrid, Madrid, Spain.
New experimental approaches are required to detect the
conformational dynamics of viruses [1, 2] and elusive transient
intermediates predicted by simulations of virus assembly or
disassembly. We have used an atomic force microscope (AFM)
to mechanically induce partial disassembly of single
icosahedral T = 1 capsids and virions of the minute virus of
mice (MVM) [3]. The kinetic intermediates formed were
imaged by AFM. The results revealed that induced disassembly
of single MVM particles is frequently initiated by loss of one
of the 20 equivalent capsomers (trimers of capsid protein
subunits) leading to a stable, nearly complete particle that does
not readily lose further capsomers. With lower frequency, a
fairly stable, three-fourths-complete capsid lacking one
pentamer of capsomers and a free, stable pentamer were
51
obtained. The intermediates most frequently identified (capsids
missing one capsomer, capsids missing one pentamer of
capsomers, and free pentamers of capsomers) had been
predicted in theoretical studies of reversible capsid assembly
based on thermodynamic-kinetic models [4], molecular
dynamics [5], or oligomerization energies [6,7]. We conclude
that mechanical manipulation and imaging of simple virus
particles by AFM can be used to experimentally identify
transient, kinetic intermediates predicted by simulations of
assembly or disassembly.
1. M. Castellanos et al., PNAS 109, 12028-33 (2012).
2. M. G. Mateu, Virus Res. 168, 1-22 (2012).
3. M. Castellanos et al., Biophys J. 102, 2615-24 (2012).
4. S. Singh and A. Zlotnick, J. Biol. Chem. 278, 18249–55 (2003).
5. D. C. Rapaport, Phys. Rev. Lett. 101, 186101-4 (2008).
6. V. S. Reddy and Johnson, Adv. Virus Res. 64, 45–68 (2005).
7. V. S. Reddy et al., Biophys J. 74, 546–558 (1998).
52
P4. Comparative Study of Cellular
Modifications Induces by Adenovirus: Wild
Type, Packaging and Maturation Mutants
Gabriela Condezoa, Marta del Alamoa, S. Jane Flintb,
Miguel Chillónc, Carmen San Martína
a
Centro Nacional de Biotecnología CNB-CSIC. Madrid (Spain)
b
Princeton University, Princeton, New Jersey (USA)
c
Centro de Biotecnología Animal y Terapia Génica CBATEG-UAB.
Barcelona (Spain)
The maximum viral titer of human adenovirus type 5 (Ad5) is
obtained at 36hpi (hours post-infection). At this time of
infection, Ad5 has induced several well-characterized cellular
modifications. Ad5/FC31, an Ad5 mutant with two insertions
(attB/attP-ΦC31) flanking the packaging domain, has a
delayed viral cycle, 20 hours longer than wt (wild type);
however, its replication and protein expression is normal.
Studies showed that the delay is mainly affecting packaging of
the viral genome. We are taking advantage of this alteration in
the viral cycle to study adenovirus assembly within the cell.
Using electron microscopy (EM), we have compared changes
in the nuclear structure of cells infected with wt Ad5 or
Ad5/FC31. Apart from the changes previously described in the
bibliography, we observed a new structure specific for
Ad5/FC31 that we called “speckled bodies” (SBs) due to their
aspect at the electron microscope. SBs seem to contain subviral
particles trapped in DNA-rich regions, and their size varies in
range between 0.5 and 3 µm. Interestingly, SBs also appear in
cells infected with ts1, a mutant defective not in packaging but
in maturation. This observation suggests that packaging and
53
maturation could be coupled during adenovirus assembly. To
determine the composition of SBs, we have followed viral
DNA and DNA-packaging proteins in immune-fluorescence
assays. We are currently expanding the fluorescence study to
EM.
P5. Procapsids of Infectious Pancreatic
Necrosis Virus
Ana R. Correiaa, Daniel Luquea, Natalia Ballesterosb,
Sylvia R. Saint-Jeanb, Sara Pérez Prietob, JL Carrascosaa
and JR Castóna
a
Centro Nacional de Biotecnologia/CSIC, Department of Structures
of Macromolecules, Campus de Cantoblanco, c/ Darwin 328049
Madrid, Spain
b
ICentro de Investigaciones Biologícas/CSIC, Department of
Molecular Microbiology, Ramiro de Maeztu 9, 28040 Madrid, Spain
Birnaviruses are nonenveloped dsRNA viruses with an
icosahedral T=13l capsid built of a single protein, VP2. Most
of our understanding of birnaviruses is based on studies of
infectious bursal disease virus (IBDV) and of infectious
pancreatic necrosis virus (IPNV). VP2 polymorphism is
controlled by an inherent switch, a transient C-terminal a-helix
in the precursor pVP21,2. This switch is processed by viral and
cellular proteases3; cleavage takes place in a in a procapsid-like
structure stabilized by many copies of the VP3 scaffold
protein4,5. IPNV is a model for studying the coordination of
54
molecular factors involved in this multistep process. IPNV
procapsids, termed A particles, can be purified from IPNVinfected BF-2 cells after 72 h in TNE buffer (10 mM Tris, 200
mM NaCl, 1 mM EDTA, pH 7.4). A mixture of immature and
mature particles (A and B, respectively) is purified if PES
buffer (25 mM PIPES, 150 mM NaCl, 20 mM CaCl 2, p H 6.2)
is used throughout the purification process. A particles have
lower mobility than B particles in native agarose gels, although
they show the same protein composition. Both particle types
have a similar appearance by cryo-EM. B particles purified
from BF-2 cells have a similar electrophoretic mobility in
agarose gels to virions purified from CHSE cells using PES or
TNE buffers. Furthermore, A particles can be converted into B
particles after dialysis in PES buffer. Data suggest that
differences in pH and/or Ca2+ concentration are involved in
conformational changes in the capsid and might be associated
with distinct infectivities. This maturation mechanism, together
with other shared features, is reminiscent of the maturation
process triggered by acidic pH of nodavirus. 3D cryo-EM
analysis with A and B particles and IPNV virions are in
progress.
1. Saugar et al, Structure, 13, 1007 (2005)
2. Luque D. et al, J. Virol., 81 (13), 6869 (2007).
3. Irigoyen et al, JBC, 287(27), 24773 (2012)
4. Irigoyen et al, JBC, 284 (12), 8064 (2009)
5. Saugar et al, J. Biol. Chem. 285 (6), 3643 (2010);
55
P6. Structural Characterization Of The T7 Tail
Complex
Cuervo A., Pulido M., Martín-Benito J., Chagoyen M.,
Arranz R., Castón J.R., González-García V., García-Doval
C., Valpuesta J.M., van Raaij MJ. and Carrascosa J.L.
Department of Macromolecular Structure. Centro Nacional de
Biotecnología, CSIC. Darwin 3, Cantoblanco, 28049 Madrid, Spain.
Most of bacterial viruses need an specialised machinery named
the tail to deliver its genome inside the bacterial cytoplasm
without disrupting cellular integrity. T7 bacteriophage is a
well- characterized member of the Podoviridae bacteriophage
family infecting E. coli, and it presents a short non-contractile
tail that assembles sequentially in the viral head after DNA
packaging. T7 tail is a complex of around 2.5 MDa composed
by at least four proteins: connector (gp8), fibres (gp17) and the
tail tubular proteins (gp11 and gp12). Using cryo-electron
microscopy (Cryo-EM) and single particle image
reconstruction techniques we have determined the precise
topology of the tail structural proteins by comparing the
structure of the T7 tail extracted from viruses and a
recombinant complex formed by gp8, gp11 and gp12 proteins.
Furthermore, cloning and purification of the different tail
proteins allowed performing interaction assays to define the
location and the order of assembly of the proteins within the
complex. The existence of common folds among similar tail
proteins allowed to obtain pseudo-atomic threaded models of
the gp8 (connector) and gp11 (tubular) proteins, which were
mapped into the corresponding cryo-EM volumes of the tail
complex, generating a high resolution model of the connector-
56
gatekeeper interaction, and revealing the existence of a
common architecture with other Podoviridae tail complexes.
P7. Structural and functional characterization
of a non-cannonical replicase of ssRNA virus
(Thosea asigna virus): understanding
regulatory elements
Ferrero Dsa, Buxaderas Ma, Rodriguez JFb, Verdaguer Na
a
Instituto de Biología Molecular de Barcelona-CSIC, c/Baldiri
Reixac 10, 08028, Barcelona .
B
Centro Nacional de Biotecnología, c/Darwin 3, 28049, Madrid.
During infection most viruses employ a viral polymerase to
replicate and transcribe the viral genome. Do to their crucial
role, polymerases are broadly conserved in viruses following
the right hand architecture, with fingers, palm and thumb
subdomains. They also conserved six ordered sequence motifs
(A-B-C-D-E-F), four located into the palm subdomain (A to D)
and two (E-F) only present in RNA-dependent RNA
polymerases (RdRps). However a small group of ssRNA
viruses (Permutotetraviridae family) and some dsRNA viruses
(members of Birnaviridae family) not follow the canonical
organization, having a RdRp with a permuted palm motifs
organization (C-A-B-D). Given the shortage of this atypical
polymerases, there is scarce structural and biochemical
information about them.
Thosea asigna virus (TaV) is an insect restricted (+)ssRNA
57
virus that belong to the Permutotetravirus genus within the
Permutetetraviridae family. The paricular TaV RdRp, have a
non-canonical connectivity yielding a permuted palm
organization. In this work, we resolve the structure of TaV
RdRp and performed a biochemical characterization in order to
better understand this replicases.
The exhaustive analysis of the RdRp structure allow us to
identify several structural elements that potentially regulate the
polymerase activity. The amino terminus (30 aa) and an
extensive loop blocking the active site cavity may inhibit it.
They may undergo in a structural rearrengement, allowing the
polimerase to be active as we confirm by biochemical analysis.
The mutagenical analysis could gain insight into how RdRp
generally work and are regulated by their own structural
elements. In addition, we provide structural information to
support the existence of a common ancestor between ssRNA
and dsRNA viruses.
58
P8. The cryoEM reconstruction of Drosophila
C Virus (DCV) at 5.4 Å
Leandro Estrozia,, Jon Agirreb, Jean-Luc Imlerc, Estelle
Santiagoc, Jorge Navazaa, Guy Schoehna, Diego M.A.
u rinb
a
Institut de Biologie Structurale Jean-Pierre Ebel. 41, rue Jules Horowitz F-38027 Grenoble Cedex 1, France.
B
Unidad de Biof sica and Fundaci n Biof sica Bizkaia. PO Box 644,
E-48080, Bilbao, Spain.
C
Institut de Biologie Molculaire et Cellulaire. 15 rue Rene Descartes, F-67084 Strasbourg Cedex, France.
The Dicistroviridae family, which is currently classified under
the Picornavirales order, groups a pool of arthropod-infecting
viruses with bicistronic genomes. The interest in this family of
viruses has been fueled due to the economical implications of
their hosts, which range from beneficial arthropods (bees and
shrimps) to insect pests (crickets, ants and triatomines). Two
crystallographic structures of dicistroviruses have been reported to date: Cricket Paralysis Virus (CrPV, type species of the
Cripavirus genus) and Triatoma Virus (TrV). Their structures
revealed that dicistroviruses share a core archetypal organization, which is complemented by external and internal capsidwide differences that likely have arisen from unique host adaptation. In this work we report the cryoEM reconstruction at 5.4
Å resolution, and C-alpha trace of Drosophila C Virus (DCV),
a viral pathogen that infects Drosophila melanogaster, among
other Drosophila species. This virus holds a 65.8% sequence
identity with CrPV and, given the ability of the latter to repli-
59
cate in Drosophila hosts, a detailed comparison can give insight into the infective cycle of dicistroviruses.
Keywords: cryoEM, reconstruction, dicistroviridae, DCV
P9. Mapping in vitro physical properties of
intact and disrupted virions at high resolution
using multi-harmonic atomic force microscopy
Mercedes Hernando-Pérez1 Alexander Cartagena2, José
L. Carrascosa3, Pedro J. de Pablo1, and Arvind Raman2
1
Departamento de Física de la Materia Condensada, Universidad
Autónoma de Madrid,
Madrid, Spain
2
School of Mechanical Engineering and the Birck Nanotechnology
Center, Purdue University,
West Lafayette, IN, USA
3
Centro Nacional de Biotecnología. CSIC, 28049 Madrid, Spain
Viruses are striking examples of macromolecular nanomachines which carry out complex functions with minimalistic
structure. Understanding the relationships between viral material properties (stiffness, charge density, adhesion, viscosity),
structure (protein sub-units, genome, receptors, appendages),
and functions (self-assembly, stability, disassembly, infection)
60
is of significant importance in physical virology and nanomedicine application (1-2).
We present quantitative maps at nanometer resolution of local
electro-mechanical force gradient, adhesion, and hydration
layer viscosity within individual Bacertiophage ɸ29 using the
multi-harmonic atomic force microscopy technique under
physiological condition. The technique significantly generalizes recent multi-harmonic theory and enables high-resolution in
vitro quantitative mapping of multiple material (3).
High-resolution quantitative maps of bacteriophage ɸ29 show
that the material properties changes over the entire virion
provoked by the local disruption of its shell, providing
evidence of bacteriophage despressurization (4).
(1) Carrasco C, et al Proc. Natl. Acad. Sci. U. S. A. , (2006),
103:13706-13711
(2) T. Douglas and M. Young, Nature, 1998, 393, 152-155
(3) Raman A, et al. (2011) Nature Nanotech 6: 809-814
(4). Hernando-Pérez, M et al., Small, 2012, 8, 2365
61
P10. Evolutionary Dynamics of Genome
Segmentation in Multipartite Viruses
Jaime Iranzo and Susanna C. Manrubia
Centro de Astrobiología, INTA-CSIC, Ctra. de Ajalvir km. 4, 28850
Torrejón de Ardoz, Madrid, Spain
The origin and evolutionary history of viral genomes is a
classical problem that has inspired a long series of questions
and hypotheses in evolutionary biology. An especially
intriguing case concerns multipartite viruses, which are formed
by a variable number of genomic fragments packed in
independent viral capsids. This fact poses stringent conditions
on their transmission mode, demanding in particular a high
multiplicity of infection (MOI) for successful propagation.
Because the actual advantages of the multipartite viral strategy
are as yet unclear, the origin of multipartite viruses represents
an evolutionary puzzle. While classical theories suggested that
a faster replication rate or higher replication fidelity would
favour shorter segments, recent experimental results seem to
point to an increased stability of virions with incomplete
genomes as a factor able to compensate for the disadvantage of
mandatory complementation [1]. Using as main parameters
differential stability as a function of genome length and MOI,
we calculate the conditions under which a set of
complementary segments of a viral genome would outcompete
the non-segmented variant. Further, we examine the likeliness
that multipartite viral forms could be the evolutionary outcome
of the competition among the defective genomes of different
lengths that spontaneously arise under replication of a
62
complete, wild type genome [2]. We conclude that only
multipartite viruses with a small number of segments could be
produced in our scenario, and discuss alternative hypotheses
for the origin of multipartite viruses with more than four
segments [3].
1. S. Ojosnegros, J. García-Arriaza, C. Escarmís, S. C. Manrubia, C.
Perales, A. Arias, M. García Mateu and E. Domingo, PLoS Genet. 7,
e1001344 (2011).
2. J. García-Arriaza, S. C. Manrubia, M. Toja, E. Domingo and C.
Escarmís, J. Virol. 78, 11678-11685 (2004).
3. J. Iranzo and S. C. Manrubia, Proc. R. Soc. Lond. B. 279, 38123819 (2012).
63
P11. Mechanical stability and reversible failure
of vault particles
A. Llauró1, P. Guerra2, N. Irigoyen3, J. F. Rodríguez4, N. Verdaguer2, P. J. de Pablo1
1
Departamento de Física de la Materia Condensada, UAM,
Francisco Tomás y Valiente 7,28049-Madrid, Spain.
2
Institut de Biologia Molecular de Barcelona, CSIC. Baldiri i Reixac
10, 08028-Barcelona, Spain.
3
Division of Virology, Department of Pathology, University of
Cambridge, Tennis Court, Cambridge CB2 1QP, United Kingdom.
4
Centro Nacional de Biotecnología, CSIC, Calle Darwin nº 3, 28049Madrid, Spain.
Vaults are the largest ribonucleoprotein particles found in
eukaryotic cells, with an unclear cellular function and
promising applications as drug delivery containers. In this
paper we study the local stiffness of individual vaults and
probe their structural stability with Atomic Force Microscopy
(AFM) under physiological conditions. Our data show that the
barrel, the central part of the vault, governs both the stiffness
and mechanical strength of these particles. In addition, we
provoke single protein fractures in the barrel shell and monitor
their temporal evolution. Our high-resolution AFM
topographies show that these fractures occur along the contacts
between two major vault proteins and disappear over time, thus
removing any mark of the previous rupture. This
unprecedented systematic self-healing mechanism, which may
enable these particles to reversibly adapt to certain geometric
64
constraints, might help vaults safely pass through the nuclear
pore complex.
P12. Imaging and stiffness measurement of
IBDV virions by jumping mode AFM
Johann Mertensa, Santiago Casadoa, Carlos P. Matab,
Mercedes Hernando-Perézc, Pedro J. De Pabloc, José R.
Castónb and José L. Carrascosaa,b
a
IMDEA Nanociencia, Unidad asociada CNB-IMDEA
Nanociencia, Campus de Cantoblanco, 28049 Madrid, Spain.
b
Centro Nacional de Biotecnología - CSIC. Darwin 3,
Campus de Cantoblanco, 28049 Madrid, Spain.
c
Dpto. de Física de la Materia Condensada, Universidad
Autonoma de Madrid, Campus de Cantoblanco, 28049
Madrid, Spain.
We imaged surface-attached IBDV virions by using jumpingmode AFM, which allowed us to control maximal tip-sample
forces accurately. Six natural populations of the virus (E1 to
E6), which share a similar protein composition but increasing
copy number of genome segments inside the viral capsid, have
been identified and probed separately. Our results show that
one can probe nanoscale IBDV shells and quantitatively extract
their mechanical properties. This constitutes the first direct
evaluation of the mechanical properties of IBDV capsids.
Surprisingly, the stiffness of the capsids changes and seems to
increase with the amount of RNA packed inside the virus, from
65
0.4 N/m for E1 to 0.78 N/m for E6. Mechanical reinforcement
of IBDV capsids is a new feature to be explored in relation
with their biochemical properties and almost to understand the
variations in the infectivity of the virus.
P13. Monitoring Dynamics of Human
Adenovirus Disassembly Induced by
Mechanical Fatigue
A. Ortega-Estebana, A. J. Pérez-Bernáb, R. MenéndezConejerob, S. J. Flintc, C. San Martínb and P. J. de Pabloa
a
Departamento de Física de la Materia Condensada, Universidad
Autónoma de Madrid, 28049 Madrid, Spain
b
Department of Macromolecular Structure, Centro Nacional de
Biotecnología (CNB-CSIC). Darwin 3, 28049 Madrid, Spain
c
Department of Molecular Biology, Princeton University, Princeton,
NJ 08544, USA
The standard pathway for virus infection of eukaryotic cells
requires disassembly of the viral shell to facilitate release of
the viral genome into the host cell. Here we use mechanical
fatigue, well below rupture strength, to induce stepwise
disruption of individual human adenovirus particles under
physiological conditions, and simultaneously monitor
disassembly in real time. Our data show the sequence of
dismantling events in individual mature (infectious) and
immature (noninfectious) virions, starting with consecutive
release of vertex structures followed by capsid cracking and
66
core exposure. Further, our experiments demonstrate that
vertex resilience depends inextricably on maturation, and
establish the relevance of penton vacancies as seeding loci for
virus shell disruption. The mechanical fatigue disruption route
recapitulates the adenovirus disassembly pathway in vivo, as
well as the stability differences between mature and immature
virions.
2. A. Ortega-Esteban, A. J. Pérez-Berná, R. Menéndez-Conejero, S. J.
Flint, C. San Martín and P. J. de Pablo, Sci. Rep. 3, 1434 (2013).
P14. The Non-icosahedral Components in
Adenovirus Studying By Cryo-electron
Tomography
Pérez-Berná AJa, Chichón FJa,Fernández JJa, Winkler Db,
FontanaJb, Flint SJc, Carrascosa JLa, Steven ACb, San
Martín Ca
a
Centro Nacional de Biotecnología (CNB-CSIC), Madrid, Spain
b
IAMS, National Institutes of Health, Bethesda, Maryland,
c
Princeton University, Princeton, New Jersey
Adenovirus has a non-enveloped icosahedral capsid enclosing
a 35 kbp linear dsDNA genome associated with ~25 MDa of
DNA-binding proteins, making up a non-icosahedral core. We
are using cryo-electron tomography to visualize the nonicosahedral elements of adenovirus. We have extracted, aligned
and classified the vertex regions from 612 individual virus
67
tomograms
using
maximum-likelihood
subtomogram
averaging methods. This procedure revealed that the vertices in
each icosahedral virion can be categorized in three groups,
according to the relation between the shell and the internal
contents. In each viral particle, one vertex is in direct contact
with the core, while the opposed vertex presents a gap between
the icosahedral shell and the core, and the other 10 vertices
present an intermediate situation. This observation may
indicate the presence of additional proteins beneath one
singular vertex (eg the packaging machinery), or an asymmetry
in the distribution of the genome and accompanying proteins
within the virion. Additionally, our cryo-electron tomography
analysis of adenovirus shows that each particle contains 150180 discrete ellipsoidal densities with asymmetrical
distribution profile with approximate radii 14 x 6 nm. This is
the first time that the “adenosomes” have been directly
observed within the virion.
68
P15. Electrostatic repulsions at neutral pH
underlie the weak thermal stability of foot-andmouth disease virus, and guide the engineering
of modified virions of increased stability for
improved vaccines
Rincón Va, Rodríguez-Huete Aa, Harmsen MMb and
Mateu MGa
a
Centro de Biología Molecular “Severo Ochoa” (CSIC-UAM),
Universidad Autónoma de Madrid, Cantoblanco, 28049. Madrid,
Spain
b
Central Veterinary Institute of Wageningen UR, P.O. Box 65, 8200
AB Lelystad, The Netherlands
We are investigating the molecular basis of physical stability of
virus particles in order to understand virus assembly, stability
and dynamics, and also for bio-nanotechnological purposes
including thermostable vaccines. One of our model systems is
foot-and-mouth disease virus (FMDV), the causative agent of
one of the economically most important animal diseases
worldwide. In the present study we have investigated the
molecular mechanism by which mutation A2065H in capsid
protein VP2 exerts a greatly thermostabilizing effect on the
virion against dissociation into pentameric subunits. The
results have revealed the presence in the virion of coulombic
repulsions between pentamers, even at neutral pH, which
contribute to explain the low thermostability of FMDV and its
empty capsid. Several acidic residues not far from residue
A2065 contribute to this repulsion. Most likely, mutation
A2065H stabilizes the virion because the additional positive
69
charge introduced may partly neutralize some of the excess
negative charge around, thus weakening the interpentameric
repulsion. The discovery of this repulsive effect between
pentamers at neutral pH allowed us to undertake a new rational
protein engineering approach on FMDV that led to obtain four
virus variants of increased thermostability. These engineered
FMDVs constitute good candidates for development of
thermostable vaccines against FMD based on virions or empty
capsids.
70
P16. An evolutionary systemic approach to
virus-host interactions
Guillermo Rodrigoa, Javier Carreraa, and Santiago F.
Elenaa
a
Instituto de Biología Molecular y Celular de Plantas, Consejo
Superior de Investigaciones Científicas - Universidad Politécnica de
Valencia, València, Spain.
Understanding the mechanisms by which plants trigger host
defenses in response to viruses has been a challenging problem
owing to the multiplicity of factors and complexity of
interactions involved. The advent of genomic techniques,
however, has opened the possibility to grasp a global picture of
the interaction. Here, we used Arabidopsis thaliana to identify
and compare genes that are differentially regulated upon
infection with seven distinct (+)ssRNA and one ssDNA plant
viruses. In a first approach, we established lists of genes
differentially affected by each virus and compared their
involvement in biological functions and metabolic processes.
We found that phylogenetically-related viruses significantly
alter the expression of similar genes and that viruses naturally
infecting Brassicaceae display a greater overlap in the plant
response. In a second approach, virus-regulated genes were
contextualized using models of transcriptional and proteinprotein interaction networks of A. thaliana. Our results confirm
that host cells undergo significant reprogramming of their
transcriptome during infection, which is possibly a central
requirement for the mounting of host defenses. We uncovered a
general mode of action in which perturbations preferentially
affect genes that are highly connected, central and organized in
71
modules.
72
LIST OF PARTICIPANTS
Last Name
Abian
Abrescia
Arribas Hernán
Aznar Palenzuela
Bittner
Blanc
Bocanegra Rojo
Briones
Cabanillas
Carrascosa
Casado
Castrillo Briceño
Catalan
Condezo Castro
Cordoba Garcia
Cuervo Gaspar
Cuesta
de la Escosura
Navazo
First Name
Olga
Nicola G
María
María
Alexander
Stéphane
Rebeca
Carlos
Laura
Jose L.
Santiago
Mariana
Pablo
Gabriela
Laura
Ana
Jose
Email
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
Andrés
[email protected]
de Pablo Gómez
Domingo
Elena
Fernández Arias
Pedro José
Esteban
Santiago
Clemente
Diego Sebastián
Miguel Angel
Carmela
Mauricio
Meritxell
Diego M.A.
[email protected]
[email protected]
[email protected]
[email protected]
Ferrero
Fuertes
Garcia Doval
García-Mateu
Granell
Guérin
73
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
Hernández Rojas
Hernando
Iranzo Sanz
Lázaro
Llauró Portell
Luque Buzo
Manrubia
Mertens
Nguyen
Ortega Esteban
P. Mata
Pallàs Benet
Perales Viejo
Perez Berna
Pérez Carrillo
Ramasco
Reguera
Rey
Rincón Forero
Rodrigo
Rodriguez
Rodríguez
Rodriguez Martinez
Rubí
Ruiz Castón
Ruiz Herrero
San Martín
Sanz Gaitero
Siber
Singh
Valbuena Jiménez
van Raaij
Javier
Mercedes
Jaime
Ester
Aida
Daniel
Susanna
Johann
Thanh
Alvaro
Carlos
Vicente
Celia
Ana Joaquina
Pablo José
Jose J.
David
Felix
Verónica Del
Pilar
Guillermo
Alicia
Dolores
Javier Maria
J. Miguel
José
Teresa
Carmen
Marta
Antonio
Abhimanyu
Alejandro
Mark
74
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
Vega
VelazquezCampoy
Verdaguer
Viegas Correia
Sonia
[email protected]
Adrian
[email protected]
Nuria
Ana Raquel
[email protected]
[email protected]
Organizer:
P. J. de Pablo Gómez
[email protected]
Departamento de Física de la Materia Condensada
Facultad de Ciencias
Universidad Autónoma de Madrid
Coordinator:
David Reguera
[email protected]
Departament Física
Fonamental
Universitat de
Barcelona
75
76