VIZIER / SPINE2 Workshop on Structural
Virology
14th - 16th July, 2008
Vienna
Organizing committee
Martino Bolognesi
Dept. of Biomolecular Sciences & Biotechnology
University of Milano
Via Celoria, 26
20131 Milano – Italy
martino.bolognesi@unimi.it
Kristina Djinovic
Department for Biomolecular Structural Chemistry
Max F. Perutz Laboratories
University of Vienna, Campus Vienna Biocenter 5
A-1030 Vienna, Austria
kristina.djinovic@univie.ac.at
Stephen Graham
Division of Structural Biology
Wellcome Trust Centre for Human Genetics
Roosevelt Drive
Oxford OX3 7BN, United Kingdom
stepheng@strubi.ox.ac.uk
David Stuart
Division of Structural Biology
Wellcome Trust Centre for Human Genetics
Roosevelt Drive
Oxford OX3 7BN, United Kingdom
dave@strubi.ox.ac.uk
Paul Tucker
EMBL Hamburg Outstation
c/o DESY, Notkestrasse 85 ,D22603 Hamburg, Germany
tucker@embl-hamburg.de
Nicola Wiskocil
Max F. Perutz Laboratories
University of Vienna & Medical University Vienna
Dr.-Bohrgasse 9/3
A-1030 Wien
nicola.wiskocil@univie.ac.at
Ursula Thalhammer
Department for Biomolecular Structural Chemistry
Max F. Perutz Laboratories
University of Vienna, Campus Vienna Biocenter 5
A-1030 Vienna, Austria
ursula.thalhammer@univie.ac.at
Werner Koenig
Department for Biomolecular Structural Chemistry
Max F. Perutz Laboratories
University of Vienna, Campus Vienna Biocenter 5
A-1030 Vienna, Austria
werner.koenig@univie.ac.at
2|Page
VIZIER / SPINE2 Workshop on Structural Virology
PROGRAMME
Monday July 14th 2008
11:30 – 13:30
13.30 – 13:40
13:40 - 17:55
Registration, snacks
Welcome
Session: Entry and Host Interactions
Chairs: Dave Stuart, Felix Rey
13:40 – 14:15
Felix Rey
Crystal structure of the Rubella virus membrane fusion
glycoprotein E1 in its post fusion conformation
14:15 – 14:50
Winfried Weissenhorn
Assembly of ESCRT-III and its role in enveloped virus
budding
14:50 – 15:20
Eric Huizinga
Structures of corona- and torovirus hemagglutininesterases offer insight in receptor specificity and
evolution
15:20 – 16:00
Coffee Break
16:00 – 16:35
Katsumi Maenaka
Crystal structure of measles virus hemagglutinin provides
the molecular basis for effective vaccination
16:35– 17:10
Dieter Blaas
Structural Basis of Rhinovirus-Receptor Interaction and
Early Processes in Infection
17:10– 17:40
Jan Kadlec
Crystal structure of the postfusion form of the baculovirus
GP64 protein
17:40 – 18:00
Ulrike Maurer
Native 3D analysis of membrane fusion in Herpes
simplex virus 1 entry: from a complex virus-host system
to single players
Free evening
3|Page
VIZIER / SPINE2 Workshop on Structural Virology
Tuesday July 15th 2008
9:00 - 12:00
Session: Entry and Host Interactions (cont)
Chairs: Winfried Weissenhorn
9:00 – 9:35
Jon Grimes
TBA
9:35– 10:00
Mark Van-Raaij
Structure of avian reovirus fibre Sigma-C and of its
double-stranded RNA-binding protein Sigma-A
10:00– 10:25
Eike Schultz
Host Recognition of Bacteriohphage K1F: The product
complex of EndoNF
10:25 – 11:00
Coffee Break
11:00 - 12:20
Session: Replication
Chairs: Bruno Canard
11:00 – 11:35
Stephen Cusack
Structure-function relationships of three domains of
influenza virus polymerase subunit PB2
11:35 – 12:10
Hongmin Li
Structure and function of flavivirus NS5 methyltransferase
12:10 – 12:45
Martino Bolognesi
Recognition of RNA cap analogues by Wesselsbron virus
NS5 methyl-transferase domain. Implications for flaviviral
RNA capping mechanism
12:45 - 15:00
Lunch break and Poster session
15:00 – 15:55
Session: Replication (cont)
Chairs: Paul Tucker
15:00 – 15:35
Rolf Hilgenfeld
Proteases of RNA viruses and retroviruses: Structures,
inhibitors, and resistance
15:35 – 16:10
Bruno Canard
The Structural Enzymologist's Gold Mine: The RNA
Replication Machinery of Nidovirales
16:10 – 16:30
Mario Milani
Structural based inhibition of flavivirus replication
enzymes: Helicase and Methyltransferase
16:30 – 17:00
Coffee Break
4|Page
VIZIER / SPINE2 Workshop on Structural Virology
17:00 – 18:10
Session: Assembly and Future Perspectives
Chairs: Martino Bolognesi
17:00 – 17:35
Rob Ruigrok
Structure of negative strand RNA virus nucleocapsids
17:35 – 18:10
Stephen Graham
The structures of two rhabovirus matrix (m) proteins
reveal a novel mode of self association
20:00
Workshop dinner: Wiener Rathauskeller
5|Page
VIZIER / SPINE2 Workshop on Structural Virology
Wednesday July 16th 2008
9:30: 12:50
Session: Assembly and Future Perspectives
Chairs: Stephen Graham, Rolf Hilgenfeld
09:30 – 10:05
Dennis Bamford
Virion architecture and packaging system as probes for
virus evolution and classification
10:05 – 11:40
Alasdair Steven
DNA Packaging and Capsid-based Signaling as
Regulators of Virus Maturation
10:40 – 11:20
Coffee break
11:20 – 11:55
Herman van Tillbeurgh
VIRAR : a structural genomics project on archaeal virus
proteins
11:55 – 12:30
Gerard Bricogne
Some recent developments in instrumentation, protocols
and software aimed at challenging structure determination
projects
12:30 – 12:40
Concluding remarks
12:40 – 14:00
Lunch
6|Page
VIZIER / SPINE2 Workshop on Structural Virology
ABSTRACTS
OF
INVITED SPEAKERS
7|Page
VIZIER / SPINE2 Workshop on Structural Virology
CRYSTAL STRUCTURE OF THE RUBELLA VIRUS MEMBRANE FUSION GLYCOPROTEIN
E1 IN ITS POST-FUSION CONFORMATION.
Rebecca Phillips, Marie-Christine Vaney, Rana Al-Kurdi,
Félix A. REY
Unité de Virologie Structurale, CNRS URA 3015
Department de Virologie, Institut Pasteur
25 rue du Dr. Roux, 75015 Paris
France
We will present the very recently determined crystal structure of the whole
ectodomain of the Rubella virus (RV) glycoprotein E1 at 1.8Å resolution. We
produced the recombinant protein in Drosophila Schneider 2 (S2) cells, and we
observed that a substantial fraction spontaneously adopts a trimeric, post-fusion
conformation. In size exclusion chromatography experiments, about 80% of E1
elutes as trimer, and 20% as monomer. The purified trimers bind to liposomes of
various compositions, resulting in a closed-packed hexagonal lattice of trimers
coating the surface of the liposomes, independent of the pH. The 3D structure of the
RVE1 trimer shows that, as expected, it is a class II membrane fusion protein
homologous to alphavirus E1 and flavivirus E. The latter, however, in spite of
belonging to two different virus families, appear to be more similar to each other than
they are to RVE1. In particular, RVE1 domain II is more elaborate, and several
insertions contribute to the formation of two apposed 4-stranded beta sheets. An
insertion in the cd loop (which is the fusion loop in class II proteins) in RVE1 results
in two distinct loops - separated by about 40 intervening amino acids - located at one
end of the trimer. This end is therefore predicted to interact with the target
membrane. These two putative fusion loops are rich in glycine and aromatic
residues. One of them corresponds to the previously identified “hydrophobic domain”
postulated to be the RVE1 fusion loop (Qiu et al, 2000, J Virol 74:6637-6642).
Domains I and III do not have any insertions to the standard class II fold. In the
trimer, domain III is in a similar orientation as in the post-fusion form of the alphaand flavivirus counterparts, except that its swaps protomers such that domain III from
one subunit interacts tightly with domain II of a neighboring subinit in the trimer. The
C-terminal stem region – which connects to the TM region - extends all the way
toward the membrane to reach the fusion-loop end, making an additional strand
enlarging domain II from the neighboring subunit in the trimer. The structure and the
comparison to other class II proteins will be presented in this talk.
8|Page
VIZIER / SPINE2 Workshop on Structural Virology
ASSEMBLY OF ESCRT-III AND ITS ROLE IN ENVELOPED VIRUS BUDDING
Suman Lata1, Guy Schoehn1,2, Ankur Jain3, Ricardo Pires1, Jacob Piehler3, Heinrich
G. Gőttlinger4 and Winfried WEISSENHORN1
1Unit
for Virus Host Cell Interaction, UMR 5233 UJF-EMBL-CNRS, 6 rue Jules
Horowitz 38042 Grenoble cedex 9, France
2Institut
de Biologie Structurale Jean-Pierre UMR 5075 CEA-CNRS-UJF, 41, rue
Jules Horowitz, 38027 Grenole cedex 01, France
3Institute
of Biochemistry, Johann Wolfgang Goethe-University, Frankfurt/Main,
Germany
4Program
in Gene Function and Expression, Program in Molecular Medicine,
University of Massachusetts Medical School, Worcester, MA 01605, USA
The ESCRT-III complex, formed by CHMP family member proteins, is recruited to
membranes and functions at budding steps during multivesicular body biogenesis,
cytokinesis and enveloped virus release. Notably enveloped viruses hijack the
ESCRT machinery to obtain access to ESCRT-III and the AAA-type ATPase VPS4
to catalyze their release from host cell membranes. We show that ESCRT-III CHMP3
folds into an elongated helical structure that exposes polymerization and membrane
targeting surfaces, which are important for HIV-1 budding. At least CHMP3 exists in
a soluble closed conformation and an open conformation that probably represents
the membrane active form. Co-incubation of C-terminally truncated CHMP2A and
CHMP3 results in the assembly of helical tubular structures in vitro. We applied the
iterative helical real space reconstruction algorithm to reconstruct volumes from the
helical tubular structures. This revealed a structure with a 32 Å helical pitch,
containing 16.57 repeating units per turn and inner and outer diameters of 43 nm
and 52 nm, respectively; this low resolution structure fits the model of a
CHMP2A/CHMP3 heterodimer. We show further that VPS4 binds on the inside of the
tubes via the C-terminal region of the CHMP3 and disassembles the tubular
structures upon ATP hydrolysis in vitro. In contrast the membrane binding surface of
the polymer is positioned on the outside. Our data suggests a model where an
ESCRT-III helical structure assembles on the inside of an evolving bud; VPS4
interaction may then control CHMP composition and the diameter of ESCRT-III ring
assemblies. Thus the combined function of ESCRT-III and VPS4 might lead to
membrane constriction similarly to the mode of action of dynamin in endocytotic
membrane fission steps.
9|Page
VIZIER / SPINE2 Workshop on Structural Virology
CRYSTAL STRUCTURE OF MEASLES VIRUS HEMAGGLUTININ PROVIDES THE
MOLECULAR BASIS FOR EFFECTIVE VACCINATION
Takao Hashiguchi, Mizuho Kajikawa, Makoto Takeda, Kimiko Kuroki, Yusuke
Yanagi, Katsumi MAENAKA
Department of Virology, Faculty of Medicine
Medical Institute of Bioregulation, Kyushu University
3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka 812-8582
Japan
Worldwide, measles virus (MV) still causes 4% of all deaths in children under 5 years
of age despite the availability of efficacious vaccines. Unlike many other
paramyxoviruses, MV uses signalling lymphocyte activation molecule (SLAM), rather
than sialic acid, as a cellular receptor. CD46 also acts as an additional receptor for
vaccine strains of MV. A better understanding of MV’s interaction with receptors will
facilitate the design of novel strategies for the prevention and treatment of measles
infection. Here we report the crystal structure of the MV receptor-binding attachment
glycoprotein hemagglutinin (MV-H), which directly recognizes SLAM. Our study
reveals an unexpected sugar-shielded structure for MV-H. This conformation renders
large areas of the protein inaccessible to both receptor and neutralizing antibodies.
Limited surface areas of MV-H are able to act as neutralizing antibody epitopes, one
of which corresponds to the SLAM-binding site crucial for MV entry. N-linked sugars
also appear to tilt the relative orientation of the dimeric MV-H head domains such
that the receptor-binding sites are readily accessible to the cellular receptors.
MV vaccines are highly successful; the progenies of the first MV isolate obtained a
half century ago are still in use as live vaccines, and no escape mutant strains have
been reported. Our structural study clearly revealed that sugar shields of MV-H
appear to account for one serotype property of MV, ensuring an effective immune
response, in contrast to the sugars of HIV gp120 which allow for immune evasion.
Thus, our results suggest the potential ability of sugar shields of microbial
glycoproteins to modulate the immune responses by directing them to restricted
regions, which may be exploited as a useful tool for vaccine design.
10 | P a g e
VIZIER / SPINE2 Workshop on Structural Virology
STRUCTURAL BASIS OF RHINOVIRUS-RECEPTOR INTERACTION
AND EARLY PROCESSES IN INFECTION
Dieter BLAAS
Department of Medical Biochemistry
Max F. Perutz Laboratories (MFPL)
Medical University of Vienna
Dr.-Bohr-Gasse 9/3, 1030 Wien
Austria
Human rhinoviruses (HRVs) are a main cause of upper respiratory infections that
usually take a mild course. However, HRVs can also attain the lungs and become
life-threatening when other conditions such as asthma, chronic obstructive
pulmonary disease and otitis media are pre-existing. The more than 120 HRV types
currently known are divided into subgenera A, B, and (tentatively) C. Independent
from this classification and despite high sequence and structure similarity HRVs use
intercellular adhesion molecule 1 (ICAM-1), low-density lipoprotein receptors (LDLR,
VLDLR, and LRP), heparan sulphate, and other, so far unknown receptors for cell
entry. This distinction is only partly understood on the molecular basis but
comparisons of HRV 3-D structures and sequences of the capsid proteins yield hints
as to what might determine receptor selectivity. Furthermore, the receptors also
control the entry pathway and mode of uncoating (i.e. release of the genomic RNA
into the cytosol). I shall give a summary on the current knowledge on the early
stages of HRV infection i.e. virus-receptor interaction, cell surface attachment, entry,
and RNA transport through cellular membranes.
11 | P a g e
VIZIER / SPINE2 Workshop on Structural Virology
CRYSTAL STRUCTURE OF THE POSTFUSION FORM OF THE
BACULOVIRUS GP64 PROTEIN
Jan KADLEC
WTCHG
University of Oxford
Roosevelt Drive
OX3 7BN Oxford
United Kingdom
12 | P a g e
VIZIER / SPINE2 Workshop on Structural Virology
To Be Announced
Jonathan GRIMES
WTCHG
University of Oxford
Roosevelt Drive
OX3 7BN Oxford
United Kingdom
13 | P a g e
VIZIER / SPINE2 Workshop on Structural Virology
STRUCTURE-FUNCTION RELATIONSHIPS OF THREE DOMAINS OF I
NFLUENZA VIRUS POLYMERASE SUBUNIT PB2
Stephen CUSACK
EMBL Grenoble Outstation
UJF-EMBL-CNRS Unit of Virus Host Cell Interactions
6 rue Jules Horowitz, 38042 Grenoble cedex 9
France
Using a novel high-throughput screening technique, called ESPRIT, to
experimentally find soluble domains in multi-domain proteins we have identified and
determined the crystal structures of three independently folded domains from the
polymerase PB2 subunit of influenza A virus. These include a small domain involved
in nuclear import of PB2 (1), a cap-binding domain involved in the cap-snatching
mechanism of viral transcription (2) and a third domain containing important hostspecific determinants. The structures and their functional implications will be
presented.
(1)
(2)
Structure and nuclear import function of the C-terminal domain of influenza virus polymerase
PB2 subunit. Tarendeau F, Boudet J, Guilligay D, Mas PJ, Bougault CM, Boulo S, Baudin F,
Ruigrok RW, Daigle N, Ellenberg J, Cusack S, Simorre JP, Hart DJ. Nat Struct Mol Biol. 2007,
14(3):229-33.
The structural basis for mRNA cap-binding by influenza virus polymerase subunit PB2.
Delphine Guilligay, Franck Tarendeau, Patricia Resa-Infante, Rocío Coloma, Thibaut Crepin,
Rob W. H. Ruigrok, Juan Ortin, Darren J. Hart and Stephen Cusack. Nat. Struct. Mol. Biol.
2008, 15(5):500-506.
The work has been partly financed by the EU FLUPOL contract (SP5B-CT-2007-044263).
14 | P a g e
VIZIER / SPINE2 Workshop on Structural Virology
STRUCTURE AND FUNCTION OF FLAVIVIRUS NS5 METHYLTRANSFERASE
Hongmin LI
Wadsworth Center
New York State Department of Health
120 New Scotland Ave, Albany, NY 12208
USA
The plus-strand RNA genome of flavivirus contains a 5' terminal cap 1 structure
7
(m GpppAmG). The flaviviruses encode one methyltransferase (MTase), located at
the N-terminal portion of the NS5 RNA-dependent RNA polymerase (RdRp). The
NS5 MTase catalyzes both guanine N-7 and ribose 2'-OH methylations during viral
cap formation (1, 2). Representative flavivirus MTases from dengue, yellow fever,
7
7
and West Nile virus (WNV) sequentially generate GpppA-->m GpppA-->m GpppAm
(3, 4). Despite exhibiting two distinct methylation activities, the crystal structures of
flavivirus MTases showed a single binding site for S-adenosyl-L-methionine (SAM),
the methyl donor (1, 3, 5). This indicates that substrate GpppA-RNA should be
repositioned to accept the N-7 and 2'-O methyl groups from SAM during the
sequential reactions. Further studies demonstrated that distinct RNA elements are
required for the methylations at guanine N-7 on the cap and ribose 2'-OH on the first
transcribed nucleotide (4). In a WNV model, N-7 cap methylation requires specific
nucleotides at the second and third positions and a 5' stem-loop structure; in
contrast, 2'-OH ribose methylation requires specific nucleotides at the first and
second positions, with a minimum 5' viral RNA of 20 nucleotides. Mutant enzymes
with different methylation defects can trans complement one another in vitro,
demonstrating that separate molecules of the enzyme can independently catalyze
the two cap methylations in vitro (6, 7). Mutation analysis further demonstrated that
defects in the MTase domain of NS5 can be compensated by mutations in the RdRp
domain, indicating a direct correlation between the MTase and RdRp functions of the
NS5 protein to complete virus replication (8-10). In the context of complete virus,
defects in both methylations are lethal to WNV; however, viruses defective solely in
2'-O methylation are attenuated and can protect mice from later wild-type WNV
challenge (2, 3). The results demonstrate that the N-7 methylation activity is
essential for the WNV life cycle and, thus, methyltransferase represents a novel
target for flavivirus therapy.
1) Egloff, M. P., D. Benarroch, B. Selisko, J. L. Romette, and B. Canard. 2002. An RNA cap
(nucleoside-2'-O-)-methyltransferase in the flavivirus RNA polymerase NS5: crystal structure and
functional characterization. Embo J 21:2757.
2) Ray, D., A. Shah, M. Tilgner, Y. Guo, Y. Zhao, H. Dong, T. Deas, Y. Zhou, H. Li, and P. Shi.
2006. West nile virus 5'-cap structure is formed by sequential guanine N-7 and ribose 2'-O
methylations by nonstructural protein 5. J. Virol. 80:8362.
3) Zhou, Y., D. Ray, Y. Zhao, H. Dong, S. Ren, Z. Li, Y. Guo, K. A. Bernard, P. Y. Shi, and H. Li.
2007. Structure and function of flavivirus NS5 methyltransferase. J Virol 81:3891.
4) Dong, H., D. Ray, S. Ren, B. Zhang, F. Puig-Basagoiti, Y. Takagi, C. K. Ho, H. Li, and P. Y. Shi.
2007. Distinct RNA elements confer specificity to flavivirus RNA cap methylation events. J Virol
81:4412.
5) Egloff, M. P., E. Decroly, H. Malet, B. Selisko, D. Benarroch, F. Ferron, and B. Canard. 2007.
Structural and Functional Analysis of Methylation and 5'-RNA Sequence Requirements of Short
Capped RNAs by the Methyltransferase Domain of Dengue Virus NS5. J Mol Biol 372:723.
15 | P a g e
VIZIER / SPINE2 Workshop on Structural Virology
6) Dong, H., S. Ren, H. Li, and P. Y. Shi. 2008. Separate molecules of West Nile virus
methyltransferase can independently catalyze the N7 and 2'-O methylations of viral RNA cap.
Virology 377:1.
7) Dong, H., S. Ren, B. Zhang, Y. Zhou, F. Puig-Basagoiti, H. Li, and P. Y. Shi. 2008. West Nile
virus methyltransferase catalyzes two methylations of the viral RNA cap through a substraterepositioning mechanism. J Virol 82:4295.
8) Zhang, B., H. Dong, Y. Zhou, and P. Y. Shi. 2008. Genetic interactions among the West Nile virus
methyltransferase, the RNA-dependent RNA polymerase, and the 5' stem-loop of genomic RNA. J
Virol.
9) Yap, T. L., T. Xu, Y. L. Chen, H. Malet, M. P. Egloff, B. Canard, S. G. Vasudevan, and J. Lescar.
2007. Crystal structure of the dengue virus RNA-dependent RNA polymerase catalytic domain at
1.85-angstrom resolution. J Virol 81:4753.
10) Malet, H., M. P. Egloff, B. Selisko, R. E. Butcher, P. J. Wright, M. Roberts, A. Gruez, G.
Sulzenbacher, C. Vonrhein, G. Bricogne, J. M. Mackenzie, A. A. Khromykh, A. D. Davidson, and B.
Canard. 2007. Crystal structure of the RNA polymerase domain of the West Nile virus non-structural
protein 5. J Biol Chem 282:10678.
16 | P a g e
VIZIER / SPINE2 Workshop on Structural Virology
RECOGNITION OF RNA CAP ANALOGUES BY WESSELSBRON VIRUS NS5 METHYLTRANSFERASE DOMAIN. IMPLICATIONS FOR FLAVIVIRAL RNA CAPPING MECHANISM
Eloise Mastrangelo, Mario Milani, Michela Bollati, Graziella Sorrentino and Martino
BOLOGNESI
Department of Biomolecular Sciences and Biotechnology
University of Milan
Via Celoria 26, 20133 Milan
Italy
Viral methyltransferases are involved in completion of the mRNA capping process,
transferring a methyl group from S-adenosyl-L-methionine to capped RNA. The
Flavivirus genome is capped at the 5’ terminus with the “cap I” structure, that is
essential for mRNA stability and proper replication. N7- and 2’O-methyltransferase
activities have been recently associated to the N-terminal domain of the viral NS5
protein. In order to further characterize the series of enzymatic reaction supporting
capping, our lab analyzed several crystal structures of Wesselsbron virus MTase, in
complexes with the AdoMet cofactor, with AdoHcy (the product of the methylation
reaction), with Sinefungin (a molecular analogue of AdoMet), and with three different
cap-analogues (GpppG, N7MeGpppG, N7MeGpppA; Bollati et al. in preparation).
The different crystal structures show that the 5’ Guanine is constantly bound to a
“high affinity” site region located next to the N-terminal region of the enzyme.
Conformational disorder affects systematically the 3’ end of the cap analogues,
following the second or the third phosphate of the ppp bridge. The N7-methyl group
does not display any contact with the protein, being solvent accessible; on the other
hand, electrostatic effects related to N7-methylation may play a recognition role.
Following incubation with GTP, Wesselsbron MTase displays a GMP molecule
covalently bound to residue Lys28, although to a fraction of the whole molecular
population; such an observation may suggest implications for transfer of a guanine
group to the ppRNA. The structures of the MTase complexes obtained and the
binding assays are discussed in the context of a model for N7-MTase and 2’OMTase activities.
This work has been developed in close collaboration with Laboratoire Architecture et
Fonction des Macromolécules Biologiques (Marseille, France)
17 | P a g e
VIZIER / SPINE2 Workshop on Structural Virology
PROTEASES OF RNA VIRUSES AND RETROVIRUSES:
STRUCTURES, INHIBITORS, AND RESISTANCE
Rolf HILGENFELD
Institute of Biochemistry
Center for Structural and Cell Biology in Medicine (CSCM)
University of Lübeck
Ratzeburger Allee 160, 23538 Lübeck
Germany
Viral proteases are important targets for structure-based drug discovery. Prime
examples are the proteinases of coronaviruses and picorna- as well as
enteroviruses, and the retroviral proteases, Progress over the past few years in
developing inhibitors for these targets will be described.
The development of viral resistance against drugs seems to be inevitable. The
structural evolution of resistance mutants of the HIV-1 proteinase in a patient treated
with ritonavir will be discussed and efforts to overcome such resistance will be
presented. It will be suggested to abandon the dogma that the best inhibitors are
those offering maximum interaction between their side chains and the target
enzyme.
References:
1) J. Weber, J.R. Mesters, M. Lepsik, J. Prejdova, M. Svec, J. Sponarova, P. Mlcochova, K.
Skalicka, K. Strisovsky, T. Uhlikova, M. Soucek, L. Machala, M. Stankova, J. Vondrasek, T.
Klimkait, H.-G. Kraeusslich, R. Hilgenfeld & J. Konvalinka: Unusual binding mode of an HIV-1
protease inhibitor explains its potency against multi-drug-resistant virus strains. J. Mol. Biol. 324,
739-754 (2002).
2) K. Anand, G.J. Palm, J.R. Mesters, S.G. Siddell, J. Ziebuhr & R. Hilgenfeld: Structure of
coronavirus main proteinase reveals combination of a chymotrypsin fold with an extra alpha-helical
domain. EMBO J. 21, 3213-3224 (2002).
3) K. Anand, J. Ziebuhr, P. Wadhwani, J.R. Mesters & R. Hilgenfeld: Coronavirus main proteinase
(3CLpro) structure: Basis for design of anti-SARS drugs. Science 300, 1763-1767 (2003).
4) H. Yang, M. Yang, Y. Ding, Y. Liu, Z. Lou, Z. Zhou, L. Sun, L. Mo, S. Ye, H. Pang, G.F. Gao, K.
Anand, M. Bartlam, R. Hilgenfeld & Z. Rao: The crystal structures of severe acute respiratory
syndrome virus main protease and its complex with an inhibitor. Proc. Natl. Acad. Sci. USA 100,
13190-13195 (2003).
5) J. Tan, K.H.G. Verschueren, K. Anand, J. Shen, M. Yang, Y. Xu, Z. Rao, J. Bigalke, B. Heisen, J.
Mesters, K. Chen, X. Shen, H. Jiang & R. Hilgenfeld: pH-dependent conformational flexibility of the
SARS-CoV main proteinase (Mpro) dimer: Molecular dynamics simulations and multiple X-ray
structure analyses. J. Mol. Biol. 354, 25-40 (2005).
6) J.R. Mesters, J. Tan & R. Hilgenfeld: Viral enzymes. Curr. Opin. Struct. Biol. 16, 776-786 (2006).
7) K.H.G. Verschueren, K. Pumpor, S. Anemüller, S. Chen, J.R. Mesters & R. Hilgenfeld: A
structural view of the inactivation of the SARS coronavirus main proteinase by benzotriazole esters.
Chem. Biol. 15, 597-606 (2008).
18 | P a g e
VIZIER / SPINE2 Workshop on Structural Virology
THE STRUCTURAL ENZYMOLOGIST'S GOLD MINE: THE RNA REPLICATION
MACHINERY OF NIDOVIRALES
Bruno CANARD
Architecture and Function of Biological Molecules
CNRS-Université AIX-Marseille I & II
163, Av de Luminy, 13288 Marseille
France
The order Nidovirales is comprised of the Coronaviridae (Coronavirus and
Torovirus), the Arteriviridae, and the Roniviridae. They infect animals and man, and
the resulting pathology can range from benign to fatal. A common feature of the
order is a complex organisation of both gene products and gene expression.
Nidovirales have in common a polycistronic genome, a large replicase open reading
frame whose proteolytic product stoechiometry is controlled by a frameshift before
the RNA dependent RNA polymerase gene cluster, and the ability to synthesize a
set of nested subgenomic RNAs encoding sructural proteins.
In 2003, the emergence of a Severe Acute Respiratory Syndrome due to a
Coronavirus (SARS-CoV) has dramatically shown the scarcity of biochemical and
structural data available regarding Nidovirales. The Nidovirales replication machinery
is however an unbelievable treasure for the enzymologist. The main replicative ORF
encodes up to 16 multifunctional proteins involved in sophisticated coordinated RNA
replication/transcription. Interestingly, many putative enzyme activities are
coronavirus-unique and not directly related to "traditional" viral RNA replication.
Although some proteins remain with no putative function associated, many enzyme
activities have been predicted using bio-informatic approaches, and the
corresponding activities demonstrated.
There are however a large number of question marks regarding the role these
activities play in any of the processes occuring during a Nidovirales life-cycle. There
are a number of possibilities regarding the involvement of the different orf1a and b
partners. The presence of seemingly RNA replication-irrelevant enzyme activities,
such as the macro domain of Coronavirus nsp3, argues in favor of a role into
counteracting the host defence mechanisms, whereas the presence of endo and
exonuclease activities suggests the presence of RNA repair systems or microRNA
production machineries absent in other (+)RNA genome viruses.
We have engaged into a structural and functional study of the Nidovirales order in
order to cast light into this most interesting and sophisticated RNA replication
machinery.
We have reported, for coronavirus, the crystal structure at 2.8 Å resolution of Nsp9,
an RNA SSB protein unique in the RNA virus world, that may have a role in several
processes such as the regulation of SARS-CoV replication/transcription. Likewise,
the structure of the histone macro domain protein has been solved at 1.8 Å
resolution, and shown to carry an ADP-1"-phosphate hydrolase activity, usually
occuring in tRNA splicing pathways. The NendoU endonuclease hexameric structure
has been determined at 2.6 Å resolution and shown to carry an uncommon Mn 2+dependent RNase-A like activity. More recently, we have found that the nsp7/8
complex is endowed with RNA-dependent RNA primase activity, a feature consistent
with the classification of primer-dependent replication of Nidovirales in the
19 | P a g e
VIZIER / SPINE2 Workshop on Structural Virology
Picornavirus superfamily. The last gene product of the orf1b has been demonstrated
to be a Cap-0 dependent 2'-O-methyltransferase, resembling that of DNA viruses
rather than the phylogenetically closer Flavivirus. In Arteriviridae, we have
demonstrated that the nsp9 protein is an RNA dependent RNA polymerase, and that
a primase system resembling that of the coronavirus might be present as well.
I will present the current status of the coronavirus replicase complex based on
current structural and enzymatic results. Although the SARS crisis has acted as a
powerful boost in structural and functional studies, it is essential to re-affirm that the
whole Nidovirales order is of equal scientific interest, and that fundamental
knowledge acquired on this order is also expected to bring stunning discoveries
translastable into efficient drug design should a new Nidovirus emerge in animal or
man.
20 | P a g e
VIZIER / SPINE2 Workshop on Structural Virology
Structure of negative strand RNA virus nucleocapsids
Rob RUIGROK, Marc Jamin and Martin Blackledge
Unit for Virus Host Cell Interactions
UJF-EMBL-CNRS UMR 5233
Institut de Biologie Structurale Jean-Pierre Ebel
CEA; CNRS; UJF UMR 5075
Grenoble
France
Non-segmented negative strand RNA viruses have their viral RNA tightly associated
with the viral nucleoprotein. Such N-RNA complexes form irregular and flexible
helices. The structure of recombinant N-RNA from rabies virus and vesicular
stomatitis virus (VSV) was determined at atomic resolution. Nucleocapsids from
other viral families have only been studied by EM. The viral polymerase is attached
to these structures through the polymerase cofactor, the phosphoprotein (P). During
the last two years we have concentrated our work on the structures of the
phosphoproteins from the rhabdovirus (rabies virus and VSV) and paramyxovirus
(Sendai virus) families. These proteins all have two or three structured domains
(from N- to C-terminus: A domain that binds to RNA-free N (N0), an oligomerisation
domain and an N-RNA binding domain) that are connected by disordered or flexible
linkers.
For Sendai virus we have determined the structure of the N-RNA binding domain of
P and we have characterized the flexibility in this domain by NMR. The peptide of N
that binds to P (NTAIL) was also identified and by an analysis of NMR Residual
Dipolar Couplings we were able to describe the molecular recognition element of
NTAIL as a population of three specific overlapping helical conformers. The dynamic
structure of both partners may explain the rapidly making and breaking of contacts
between P and N in N-RNA during transcription or replication by the polymerase.
For rabies virus we have defined the borders of the ordered domains and their
function and for VSV we have determined the structure of the N-RNA binding
domain. The structure of this domain will be compared with that of rabies virus.
The people from our labs and from other labs that have contributed to this work are:
Francine Gerard, Euripedes Ribeiro Jr., Malene Ringkjøbing Jensen, Klaartje
Houben, Cédric Leyrat, Ivan Ivanov, Ewen Lescop, Laurence Blanchard, Danielle
Blondel, Sonia Longhi, Aurélie Albertini, Manos Mavrakis, Bernhard Brutscher,
Adrien Favier.
21 | P a g e
VIZIER / SPINE2 Workshop on Structural Virology
THE STRUCTURES OF TWO RHABDOVIRUS MATRIX (M) PROTEINS
REVEAL A NOVEL MODE OF SELF ASSOCIATION
Stephen C. GRAHAM1, René Assenberg1, Olivier Delmas2, Anil Verma1, Raymond J.
Owens1, David I. Stuart1, Jonathan M. Grimes1 and Hervé Bourhy2
1Division
of Structural Biology and Oxford Protein Production Facility, Wellcome
Trust Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, UK
2UPRE Lyssavirus Dynamics and Host Adaptation, WHO Collaborating Centre for
Reference and Research on Rabies, Institut Pasteur, 75724 Paris CEDEX 15, France
The Rhabdoviridiae family of viruses, typified by Rabies virus (a lyssavirus and the
etiologic agent of lethal meningoencephalitis) and vesiculostomatitis virus (VSV)
possess a negative-sense single-stranded RNA genome that encodes five proteins.
The rhabdovirus matrix (M) protein is a multifunctional protein implicated in
regulating the switch between transcription and replication of the viral genome,
modulating transcription of host genes, and in virus assembly and budding. To date,
the only structural information available on rhabdovirus M proteins is the structure of
a thermolysin-stable M core of VSV, which lacks the N-terminal 47 residues
(including a PPXY ‘late domain’ thought to be essential for virus budding) and a
surface-exposed hydrophobic loop due to proteolysis. To clarify the various roles of
M throughout the rhabdovirus lifecycle we attempted to solve the crystal structures of
full-length lyssavirus and vesiculovirus M proteins.
We successfully cloned, expressed and purified the M proteins from three
lyssaviruses (Lagos bat virus (LAG), Mokola virus and Thailand dog virus) and two
VSV viruses (VSV Indiana and VSV New Jersey). Crystals were obtained for fulllength M proteins of LAG and VSV New Jersey. Successful crystallization depended
on a number of factors, in particular the addition of an N-terminal SUMO fusion tag to
the protein to increase solubility.
Residues 48-202 of LAG M form a globular domain closely resembling VSV M
despite the aligned residues sharing only 9% sequence identity. Whilst no electron
density is evident for residues 1–30 and 38–47, residues 30–37 (which overlap with
the PPEY ‘late domain’ of LAG M; residues 35–38) form a short stretch of lefthanded polyproline-II helix and are bound to a hydrophobic groove on the surface of
the globular domain. Strikingly, the structure of VSV M also reveals an ordered
segment of the otherwise disordered N-terminus bound to the main globular domain.
While the general location of the binding site for the ordered segment in VSV M is
similar to that observed for LAG M, the nature of the interaction, including the
orientation of the bound segment and the nature of the amino acids at the interface,
differs significantly. The implications of these novel self-associations for virus
morphogenesis and for mediating interactions between M and host proteins will be
discussed.
22 | P a g e
VIZIER / SPINE2 Workshop on Structural Virology
VIRION ARCHITECTURE AND PACKAGING SYSTEM AS PROBES
FOR VIRUS EVOLUTION AND CLASSIFICATION
Dennis H. BAMFORD
Institute of Biotechnology and
Department of Biological and Environmental Sciences
University of Helsinki
Biocenter 2, P.O. Box 56
Finland
Viruses are the most abundant living entities in the biosphere outnumbering their
host organisms by one to two orders of magnitude. It is conceivable that they cause
the highest selective pressure their hosts encounter. As obligate parasites viruses
are dependent on their hosts but their origins seem to deviate from that of cellular
life.
What are the possible principles to build viruses is an open question. However,
structural studies on virus capsids and coat proteins propose that there are only a
limited number of ways to construct a viral protein coat. Consequently, relatedness
of viruses is not connected to the type of cells they infect and the same architectural
principle of the capsid has been observed in viruses infecting bacteria as well as
humans. Using the viral capsid structure it is possible to group viruses to several
lineages that may have existed
before the three cellular domains of life (bacteria, archaea and eukarya) were
separated.
In addition to the capsid proteins putative (packaging) ATPases form the “self
elements” that allow to trace such elements in bacterial, archaeal and eukaryotic
genomes.
This would mean that viruses are ancient and that early cells were infected with
many viruses proposing that the origin of viruses is polyphyletic opposing the
monophyletic origin of cellular life.
23 | P a g e
VIZIER / SPINE2 Workshop on Structural Virology
DNA PACKAGING AND CAPSID-BASED SIGNALING AS REGULATORS
OF VIRUS MATURATION.
Alasdair C. STEVEN
Laboratory of Structural Biology
National Institute of Arthritis, Musculoskeletal and Skin Diseases
National Institutes of Health
Bethesda MD 20892
USA
Despite their entirely disparate host ranges, the tailed bacteriophages and the
herpesviruses share many features in their capsid assembly pathways, probably
reflecting common albeit distant evolutionary origins (1). In both cases, the capsid is
first assembled as a precursor procapsid into which the linear double-stranded DNA
genome is packaged from a replicating concatemer by a viral ATPase/DNA
translocase/endonuclease (aka terminase). This sequence of events must be
regulated at several levels: how is packaging initiated? How is it determined that
packaging must cease? What signals that a phage nucleocapsid is ready to bind its
tail or that a herpesvirus nucleocapsid should exit the nucleus and proceed on its
envelopment pathway? This talk will discuss observations on herpes simplex virus
type 1 and coliphage HK97 that indicate that, in both cases, a key regulatory event is
a relatively subtle conformational change in the capsid shells induced by the
pressure of packaged DNA on the inner capsid surface late in the packaging
process. This transition is distinct from the relatively dramatic conformational
changes that are the hallmark of procapsid maturation but is sufficient to facilitate
protein-protein interactions that allow maturation to be completed.
1. Steven, A.C., Heymann, J.B., Cheng, N., Trus, B.L., and Conway, J.F. (2005).
Curr. Op. Struct. Biol. 15, 227-236.
24 | P a g e
VIZIER / SPINE2 Workshop on Structural Virology
VIRAR : A STRUCTURAL GENOMICS PROJECT ON
ARCHAEAL VIRUS PROTEINS
Consortium group leaders:
H. VAN TILBEURGH1, C Cambillau2 and D. Prangishvilli3
1 IBBMC,
2 AFMB,
Université de Paris Sud, Orsay, France
Université de Aix Marseille 2, CNRS, Marseille, France
3
Institut Pasteur, Paris, France
Samples collected from natural habitats that contain predominantly archaea have
revealed viruses with totally novel morphotypes. Enormous progress has recently
been made in the characterization and genomic sequencing of these ds DNA
viruses. Most of the archaeal viruses are unrelated to other known viruses or
phages, suggesting that they may have different evolutionary origins. In order to
better understand the biology and evolution of these fascinating viruses, we decided
to launch a structural proteomics project on a few archaeal virus families. We
focused our efforts on viruses infecting Crenarchaeota. We cloned and
overexpressed putative proteins from linear particle viruses belonging to the rod-like
Rudiviridae and filamentous Lipothrixviridae families. We have obtained up till now 8
new protein structures, most of them with new folds. These structures and some
corresponding functional data will be presented.
25 | P a g e
VIZIER / SPINE2 Workshop on Structural Virology
SOME RECENT DEVELOPMENTS IN INSTRUMENTATION, PROTOCOLS AND SOFTWARE
AIMED AT CHALLENGING STRUCTURE DETERMINATION PROJECTS
Gérard BRICOGNE
Global Phasing Ltd.
Sheraton House, Castle Park
Cambridge CB3 0AX
United Kingdom
There are numerous typical difficulties associated with challenging structure
determination projects: small crystals, often with multiple lattices and unfavourable
morphologies, showing anisotropic diffraction, too radiation sensitive for a single
sample to yield a complete dataset, while non-isomorphism between different
crystals precludes a simple-minded merging of partial datasets from several crystals.
Several ongoing developments have the potential of alleviating these difficulties, a
few of which will be described in this talk.
A proper statistical analysis of radiation damage effects, capable of representing the
gradual drift of diffraction data with increasing radiation dose, is still lacking. When
such effects are dominated by site-specific damage, their use for phasing through
the available methods is predicated on the assumption that all sources of nonisomorphism are uncorrelated between different observations of a given reflection. A
treatment of such damage by a dose-dependent parametrisation of anomalous
scatterers [1] overcomes the limitations of this assumption as far as the site-specific
component of non-isomorphism is concerned. However, the problem remains for the
general damage to the macromolecule, which requires that the foundations of
scaling (with or without merging), phasing (in the structure of the likelihood functions)
and refinement (of which structure) be re-examined from first principles. This is
possible by means of multivariate likelihood functions for arbitrary covariances of
uncertainties on complex-valued structure factors [2]. The relevance of this approach
to the successive steps of data reduction and structure determination will be
discussed.
An intuitive and already well-known practical conclusion is that data for SAD or MAD
phasing should be collected in such a way that all measurements relevant to the
phasing of a given unique reflection be recorded as close in dose as possible. Such
experimental designs were used in the early days of the MAD method but have
unfortunately been largely overlooked since. Combined with new implementations of
kappa goniometry and suitably adapted data processing, they constitute an effective
counter-measure against radiation damage.
Another option is to increase the amount of phase information extracted per unit
dose inflicted on a crystal. It will be shown that the rather obscure phenomenon of
AAS (anisotropy of anomalous scattering), once thought to possibly undermine the
very viability of the MAD method [3], provides such an opportunity [4].
References:
[1] Schiltz, M., Dumas, P., Ennifar, E., Flensburg, C., Paciorek, W., Vonrhein, C. & Bricogne, G., Acta
Cryst. D60, 1024-1031, 2004.
[2] Bricogne, G., Proceedings of the Workshop on Advanced Special Functions and Applications,
Melfi (PZ), Italy, 9-12 May 1999, edited by D. Cocolicchio, G. Dattoli & H. M. Srivastava, pp 315-321.
Rome: Aracne Editrice 2000.
[3] Fanchon, E. & Hendrickson, W.A., Acta Cryst. A46, 809-820, 1990.
[4] Schiltz, M. & Bricogne, G., Acta Cryst. D64, 711-729.
26 | P a g e
VIZIER / SPINE2 Workshop on Structural Virology
POSTER
ABSTRACTS
27 | P a g e
VIZIER / SPINE2 Workshop on Structural Virology
P.01
STRUCTURES OF CORONA- AND TOROVIRUS HEMAGGLUTININ-ESTERASES OFFER
INSIGHT IN RECEPTOR SPECIFICITY AND EVOLUTION
Martijn A. Langereis2, Qinghong Zeng1, Arno L.W. van Vliet2,
Raoul J. de Groot2 and Eric G. HUIZINGA1
1Crystal
and Structural Chemistry, Bijvoet Center for Biomolecular Research, Faculty
of Science and 2Virology Division, Department of Infectious Diseases & Immunology,
Faculty of Veterinary Medicine
Utrecht University
1Padualaan
8, 3584 CH Utrecht
The Netherlands
The hemagglutinin-esterases (HEs) are a family of viral envelope glycoproteins that
mediate reversible attachment to O-acetylated sialic acids by acting both as lectins and
as receptor-destroying enzymes (RDEs). This dual activity prevents virions from being
trapped by receptors located on nonpermissive cells, the heavy sialylated mucins of the
mucus barrier, or other virus particles. Related HEs occur in influenza C, toro- and
coronaviruses apparently as a result of relatively recent lateral gene transfer events. We
determined crystal structures of toro- and coronavirus HEs that display a preference for
9-O- or 7,9-O-acetylated sialic acid. Key to obtaining sufficient amounts of protein
suitable for crystallization was the use of codon-optimized plasmids and expression in
N-acetylglucosaminyltrans-ferase-I deficient HEK293S cells. The crystal structures show
that toro- and coronavirus HEs arose from an influenza C-like hemagglutinin-esterase
fusion protein (HEF). In the process, HE was transformed from a trimer into a dimer,
while remnants of the fusion domain were adapted to establish new monomer-monomer
contacts. The structural design of the RDE-acetylesterase domain remained essentially
unaltered. Its catalytic pocket shows limited variation that correlates with differences in
substrate preference. In contrast the HE receptor-binding domain underwent extensive
remodeling and is markedly different even in HEs with shared specificity: the 9-O
specific corona- and torovirus HEs bind the receptor in an orientation that is reversed
with respect to 9-O specific HEF. The enormous variation in the receptor binding site is
surprising as the architecture of the HEF site was preserved in influenza A
hemagglutinin (HA) over a much larger evolutionary distance. Apparently, HA and HEF
are under more stringent selective constraints than HE, limiting their exploration of
alternative binding-site topologies. We attribute the plasticity of the CoV HE receptorbinding site to evolutionary flexibility conferred by functional redundancy between HE
and its companion spike protein S. Our findings offer unique insights into the structural
and functional consequences of independent protein evolution following interviral gene
exchange and open new potential avenues to broad-spectrum antiviral drug design.
28 | P a g e
VIZIER / SPINE2 Workshop on Structural Virology
P.02
HOST RECOGNITION OF BACTERIOHPHAGE K1F:
THE PRODUCT COMPLEX OF EndoNF
Eike - Christian SCHULZ1, Katharina Stummeyer2, Achim Dickmanns1,
Rita Gerardy-Schahn2 and Ralf Ficner1
1: Abteilung für Molekulare Strukturbiologie, Institut für Mikrobiologie und Genetik,
Georg-August-Universität Göttingen, Justus-von-Liebig-Weg 11, 37077 Göttingen,
Germany
2: Medizinische Hochschule Hannover, MHH, Abteilung Zellulaere Chemie, OE 4330
Carl-Neuberg-Strasse 1, 30625 Hannover, Germany
Alpha-2,8-linked polysialic acid (polySia) is an important mediator of cellular motility
and functional plasticity in the vertebrate brain and has implications in tumor
metastasis. PolySia is also a common cell wall modification of pathogenic
prokaryotes like Escherichia coli K1 and Neisseria meningitides serogroup B that
cause meningitis and severe sepsis in humans. The cell wall modification serves as
a protective barrier against harmful influences such as viral infections. The only
known source for enzymes that specifically degrade polySia are E. coli K1 specific
bacteriophages. They carry endosialidases as host specificity determining tailspike
proteins required to digest the bacterial polySia capsule during infection.
We now determined three crystal structures of active site mutants of the
endosialidase cloned from bacteriophage K1F (endoNF) in complex with oligomeric
sialic acid. The structures have been refined to resolutions up to 1.4 Å. A well
defined electron density map of oligomeric sialic acid could be observed for three
binding sites, one of which is located in the active site cleft. The complex structures
confirm the helical conformation of α-2,8-linked polySia and allow new mechanistic
conclusions.
29 | P a g e
VIZIER / SPINE2 Workshop on Structural Virology
P.03
NATIVE 3D ANALYSIS OF MEMBRANE FUSION IN HERPES SIMPLEX VIRUS 1 ENTRY:
FROM A COMPLEX VIRUS-HOST SYSTEM TO SINGLE PLAYERS
Ulrike E. MAURER°, Florent C. Bender*, J. Charles Whitbeck*, Brian Hannah*,
Roselyn J. Eisenberg*, Gary H. Cohen*, Juha T. Huiskonen §°, Beate Sodeik# , Kay
Grünewald°
*Department of Microbiology, School of Dental Medicine, and Department of
Pathobiology, School of Veterinary Medicine, University of Pennsylvania,
Philadelphia, PA 19104.
°Department of Molecular Structural Biology, Max-Planck-Institute of Biochemistry,
Martinsried, Germany
§Institute of Biotechnology, University of Helsinki, Finland
#Institute of Virology, Hannover Medical School, Germany
Glycoprotein B (gB) from herpes simplex virus 1 (HSV-1) is an essential envelope
protein that is highly conserved among the members of the Herpesviridae. Together
with gD and gH/gL, it mediates the entry of the virus into host cells. Recently, we
captured in situ snapshots of the virus fusion at the plasma membrane from adherent
cells and synaptosomes using cryo electron tomography [1]. The incoming capsid
separated from the tegument and was closely surrounded by the cortical cytoskeleton.
After entry, the viral membrane curvature changed concomitantly with a reorganization
of the envelope glycoprotein spikes. Individual glycoprotein intermediates were
revealed in different conformational stages during entry, thus showing the complex viral
fusion mechanism in action. To further analyze the structural changes occurring in gB
domains in the course of interaction with the membrane, we incubated the soluble
truncated form of gB (gB730t, residues 31-730) with liposomes. This form of gB lacks
the transmembrane domain, but was still able to bind to liposomes, and completely
covered the liposome surface. We determined the structure of liposome-bound gB730t
by averaging subvolumes extracted from the tomographic reconstructions. By fitting the
x-ray structure of gB [2] into our structure, we were able to determine the lipid binding
site. We are currently expanding this approach by including other truncated forms of gB
as well as gB mutants to further characterize functional domains of the protein, which
play a crucial role in membrane fusion during HSV-1 entry.
[1]
[2]
Maurer UE, Sodeik B and Grünewald K (2008) Native 3D Intermediates of Membrane Fusion in
Herpes Simplex Virus 1 Entry. PNAS (accepted)
Heldwein EE, Lou H, Bender FC, Cohen GH, Eisenberg RJ and Harrison SC (2006) Crystal
structure of glycoprotein B from herpes simplex virus 1. Science 313:217-220.
30 | P a g e
VIZIER / SPINE2 Workshop on Structural Virology
P.04
STRUCTURES OF AVIAN REOVIRUS FIBRE SIGMA-C AND OF ITS
DOUBLE-STRANDED RNA-BINDING PROTEIN SIGMA-A
Pablo Guardado-Calvo1, X. Lois Hermo-Parrado1, Gavin C. Fox3, Antonio L. LlamasSaiz2, José Martinez-Costas1, J. Benavente1,
Mark J. VAN RAAIJ1,4
1Department
of Biochemistry and Molecular Biology and 2X-ray Unit, University of
Santiago de Compostela, E-15782 Santiago de Compostela, Spain
3Spanish
CRG Beamline BM16, European Synchrotron Radiation Facility, 6 rue
Jules Horowitz, F-38043 Grenoble, France
4Department
of Structural Biology, Institute of Molecular Biology of Barcelona-CSIC,
c/Josep Samitier 1-5, E-08028 Barcelona, Spain
Avian reovirus fibre, a homo-trimer of the sigmaC protein, is responsible for primary
host cell attachment. Partial proteolysis yielded a C-terminal protease-stable
receptor-binding domain that could be crystallised. We have solved its structure
using two-wavelength anomalous diffraction. The C-terminal globular domain has a
beta-barrel fold with the same overall topology as mammalian reovirus fibre
(sigma1). The monomers show a more splayed-out arrangement than in the sigma1
structure. Also resolved are two triple beta-spiral repeats of the stalk. Recently, the
structure could be extended further towards the N-terminus, showing the shaft
domain also contains a triple alpha-helical coiled coil.
The avian reovirus protein sigmaA plays a dual role, it is a structural protein, part of
the transcriptionally active core, but it is also implicated in the virus' resistance to
interferon by binding dsRNA and thus preventing the activation of the dsRNAdependent protein kinase. We have crystallised the protein in absence of RNA and
solved its structure by molecular replacement, using the mammalian reovirus sigma2
structure. Twelve crystallographically independent molecules were located in the P1
unit cell. The supramolecular structure suggests a single-helical model for dsRNA
binding; site-directed mutagenesis, analytical ultracentrifugation and electron
microscopy results are consistent with this model.
31 | P a g e
VIZIER / SPINE2 Workshop on Structural Virology
P.05
STRUCTURAL BASED INHIBITION OF FLAVIVIRUS REPLICATION ENZYMES:
HELICASE AND METHYLTRANSFERASE
Mario MILANI1, Eloise Mastrangelo1, Michela Bollati1, Etienne Decroly2, and Martino
Bolognesi1
1Department
of Biomolecular Sciences and Biotechnology, CNR-INFM, University of
Milano, Via Celoria 26, 20133-Milano, Italy
Architecture et Fonction des Macromolécules Biologiques, UMR 6098,
AFMB-CNRS-ESIL, Case 925, 163 Avenue de Luminy, 13288-Marseille, France
Unité des Virus Emergents, Faculté de Médecine, 27 Bd Jean Moulin, 13005Marseille, France
2Laboratoire
The genus Flavivirus comprises over seventy RNA viruses, many of which are
important human pathogens. The Flavivirus genome consists of 11 kb capped at the
5’ terminus encoding a 370 kDa polyprotein precursor. The polyprotein is processed
into three structural proteins and seven non structural proteins involved in the viral
replication (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) (Fields et al., 2001).
During viral replication the nascent transcripts must be unwound from their
complementary template RNA: such role is displayed by the C-terminal domain of
the NS3 protein (helicase, Hel domain). In addition, proper translation requires the
capping of viral RNA and among other enzymes is essential the function of the NS5
protein N-terminal domain (methyltransferase activity, Mtase). Consequently, the
inhibition of flaviviral Hel and Mtase might provide a tool against flaviviral infection. In
this work we used virtual docking approach (autodock4) to examine the affinity of
various commercial compounds against the two viral enzymes. After identification of
the best candidate inhibitors, the compounds have been tested by in vitro
activity/inhibition assays showing IC50 from 2 to 0.01 uM.
The development of mechanistic hypothesis for the activity of both the enzymes,
which we based on our previous crystal structure analysis, was instrumental to the
achievement of these results. In fact, based on such hypothesis, novel sites for
inhibitor binding could be proposed and subsequently validated through the above
described approach
32 | P a g e
VIZIER / SPINE2 Workshop on Structural Virology
P.06
Structural study of human astrovirus protease and Rabbit
hemorrhagic disease virus protease
Daniele BONIVENTO1, Silvia Speroni1, Jacques Rohayem2, Simone Nenci1,
Bruno Coutard3, Bruno Canard3, and Andrea Mattevi1
1 Dept.
of Genetics and Microbiology, University of Pavia, via Ferrata 1, Pavia,
27100 Italy
2 The
Calicilab, Institute of Virology, Dresden University of Technology,
Fiedlerstrasse 42, 01307 Dresden, Germany
3AFMB
UMR 6098 CNRS/UI/UII ESIL Case 932 163 Avenue de Luminy 13288
Marseille cedex 9, France
Different classes of RNA viruses adopt a translational strategy based on the
proteolytic processing of a polyprotein, with subsequent release of the viral
replicative enzymes. The proteases involved in the processing of the polyprotein
thus constitute a key element in the post-translational regulation of the viral
machinery and represent relevant drug targets. In order to gain more insight into the
mode of action of these enzymes we are actually investigating the structure of two
proteases from different viral classes: human astrovirus, belonging to Picornaviruses
and Rabbit hemorrhagic disease virus which belongs to Caliciviruses.
The astrovirus protease is a serine protease with a trypsin-like fold; we solved the
crystal structure of this enzyme to 2.0 Å resolution, revealing some unexpected
features: i) a peculiar conformation of the catalytic Asp-His-Ser triad in which the
aspartate side-chain is oriented away from the histidine, being replaced by a water
molecule; ii) an unusual conformation and composition of the S1 pocket; and iii) the
lack of the typical picornavirus surface β-ribbons together with a “featureless” shape
of the substrate-binding site.
The Rabbit hemorrhagic disease virus is a 3C-like cysteine protease; despite
several efforts we did not manage to crystallize it, therefore we are now exploring
different approaches to encourage crystallization: use of different constructs, surface
mutagenesis and cocrystallization with substrate peptides.
33 | P a g e
VIZIER / SPINE2 Workshop on Structural Virology
P.07
CORONAVIRUS NONSTRUCTURAL PROTEIN 16 IS A CAP-0 BINDING ENZYME
POSSESSING (NUCLEOSIDE-2’O)-METHYLTRANSFERASE ACTIVITY
Etienne Decroly, Isabelle Imbert, Bruno Coutard, Mickaël BOUVET, Barbara Selisko,
Karin Alvarez, Alexander E. Gorbalenya, Eric J. Snijder, and Bruno Canard
Department of Structural Virology & Drug design
AFMB/ESIL
163 Avenue de Luminy Case 925 13288 Marseille Cedex 09
France
The coronavirus family of positive-strand RNA viruses includes important pathogens
of livestock, companion animals, and humans, including the SARS-coronavirus that
was responsible for a worldwide outbreak in 2003. The unusually complex
coronavirus replicase/transcriptase is comprised of 15 or 16 virus-specific subunits
that are autoproteolytically derived from two large polyproteins.
In line with bioinformatics predictions, we now show that Feline coronavirus (FCoV)
nonstructural protein (nsp) 16 possesses an AdoMet-dependent RNA (nucleoside2’O)-methyltransferase (2’O-MTase) activity that is capable of cap-1 formation.
Purified recombinant FCoV nsp16 selectively binds to short capped RNAs.
Remarkably, an N7-methyl guanosine cap (7MeGpppAC3-6) is a prerequisite for
binding. HPLC analysis demonstrated that nsp16 mediates methyl transfer from
AdoMet to the 2’O position of the first transcribed nucleotide, thus converting
7MeGpppAC
7MeGpppA
3-6 into
2’OMeC3-6. The characterization of eleven nsp16 mutants
supported the previous identification of residues K45, D129, K169, and E202 as the
putative K-D-K-E catalytic tetrad of the enzyme. Furthermore, residues Y29 and
F173 of FCoV nsp16, which may be the functional counterparts of aromatic residues
involved in substrate recognition by the vaccinia virus MTase VP39, were found to
be essential for both substrate binding and 2’O-MTase activity. Finally, the weak
inhibition profile of different AdoMet analogues indicates that nsp16 has evolved an
atypical AdoMet binding site. Our results suggest that coronavirus mRNA may carry
a cap-1 onto which 2'O methylation follows an order of events in which 2’O-methyl
transfer must be preceded by guanine N7 methylation, with the latter step being
performed by a yet unknown N7-specific MTase.
34 | P a g e
VIZIER / SPINE2 Workshop on Structural Virology
P.08
Computational Methods in Structural Virology for Investigating Drug
Resistance, Viral Entry and Viral-Host Interactions
Francisco S. DOMINGUES, Christoph Welsch, Oliver Sander,
Hongbo Zhu, Ingolf Sommer, Thomas Lengauer
Computational Biology and Applied Algorithmics
Max-Planck-Institut Informatik
Campus E1 4, Saarbrücken
Germany
We developed several structure-based computational approaches for investigating
viral drug resistance, viral entry and viral-host protein interaction. We describe three
medically relevant applications that involve HCV and HIV proteins.
First, we have investigated the mechanisms of drug resistance to telaprevir in
HCV[1]. Recent phase 1b clinical trial in patients infected with HCV genotype 1
revealed mutations in the NS3-4A protease that confer varying degrees of drug
resistance. In particular, the mutated positions in two variants associated with drug
resistance are located in the protein interior and not in the ligand binding pocket.
Based on the available experimental structures of NS3-4A, we analysed the binding
mode of different ligands and the network of non-covalent interactions within the
protease structure to identify possible mechanisms of drug resistance.
We then describe a method developed for predicting HIV-1 coreceptor usage upon
cell entry[2]. HIV-1 cell entry commonly uses receptor CD4, as well as one of the
chemokine receptors CCR5 or CXCR4 as coreceptor. Knowledge of coreceptor
usage is critical for monitoring disease progression as well as for supporting therapy
with the novel drug class of coreceptor antagonists. We show that using structural
information on the V3 loop in combination with sequence features of V3 variants
improves prediction of coreceptor usage. In particular, we propose a distance-based
descriptor of the spatial arrangement of physico-chemical properties that increases
discriminative performance. A detailed analysis and interpretation of structural
features important for classification shows the relevance of several specific
hydrogen-bond donor sites and aliphatic side chains to coreceptor specificity.
Finally, we describe an approach for comparing protein-protein interactions that
aligns vector representations of non-covalent interactions from different protein
interfaces[3]. The method was applied to analyse the interaction between HIV gp120
and a CD4 mimic, demonstrating how this tool can assist in the development of
mimetic antagonists.
[1] Welsch C, Domingues FS, Susser S, Antes I, Hartmann C, Mayr G, Schlicker A, Sarrazin C,
Albrecht M, Zeuzem S, Lengauer T. Molecular basis of telaprevir resistance due to V36 and T54
mutations in the NS3-4A protease of the hepatitis C virus. Genome Biol. 2008 9:R16.
[2] Sander O, Sing T, Sommer I, Low AJ, Cheung PK, Harrigan PR, Lengauer T, Domingues FS.
Structural descriptors of gp120 V3 loop for the prediction of HIV-1 coreceptor usage. PLoS Comput
Biol. 2007 3:e58.
[3] Zhu H, Sommer I, Lengauer T, Domingues FS. Alignment of non-covalent interactions at proteinprotein interfaces. PLoS ONE. 2008 3:e1926.
35 | P a g e
VIZIER / SPINE2 Workshop on Structural Virology
P.09
BENZIMIDAZOLES: POTENT FLAVIVIRIDAE INHIBITORS TARGETED
AT THE RDRP
IBBA C., Casula L., Collu D., Loddo R., Busonera B., Giliberti G.,
Iuliano F., Shukla S., Asthana S., PEZZULLO M.
Department of Biomedical Science and Technology
Section of General Microbiology and Virology & Microbial Biotechnologies
University of Cagliari
Italy
We here report the highly potent and selective antiviral activity of four
benzimidaziole derivatives (I, II, III, IV). In cell-based assays the above compounds
showed EC50s in the range 0.8-1.5 μM against Bovine Viral Diarrhea Virus (BVDV),
whereas they were inactive against Yellow Fever, Dengue and other positive-sense
RNA viruses (such as poliovirus 1 and Coxsackie B2), negative-sense RNA viruses
(Respiratory Syncyctial and Vesicular Stomatitis) and a double-stranded RNA virus
representative (Reo 1).
To get some insights into the target of the above compounds, drug-resistant viruses
were selected by serial passages of wild type BVDV in the presence of stepwise
doubling drug concentrations, starting from cell cultures infected with an m.o.i. of
0.01 and treated with drug concentrations equal to the EC50s.
Sequence analysis of resistant cDNAs revealed the presence of single aminoacid
substitutions in the coding region of the RNA-dependent RNA polymerase (RdRp). I
selected for the I261M mutation; II selected for the N264D mutation; III selected for
both I261M and N264D mutations; IV selected for the A392E mutation.
Target validation was obtained with a newly developed fluorescence-based RdRp
assay, in which all benzimidazoles inhibited the BVDV enzyme in a dose-dependent
manner. They also inhibited the HCV1b RdRp, although with much lower potency.
Automated Docking and Molecular Dynamics methods were used to help us to
clarify issues related to binding site and binding modes of the most potent
benzimidazoles, thus allowing to identify features undetectable by other means.
Molecular modelling studies revealed that all the above NNI mutations are located
in the fingers domain of the BVDV RdRp, thus suggesting that, in this virus, the NNI
binding site is present in a domain different from that (thumb) reported for the same
class of NNIs in HCV.
36 | P a g e
VIZIER / SPINE2 Workshop on Structural Virology
P.10
USING REPLICATIVE INFIDELITY TO TEST VIRAL PROTEASES
M. KURZ, J. Zhu, N. Stefan, C. Mandl and T. Skern
Department of Medical Biochemistry
Max F. Perutz Laboratories
Dr.-Bohr-Gasse 9/3, A-1030 Vienna
Austria
Tick-borne encephalitis virus (TBEV), a member of the flavivirus family, is endemic in
large parts of Europe as well as in parts of Russia, China and Japan. It causes an
acute infection of the nervous system. Cases of TBE do still occur in Central Europe,
despite the availability of an effective vaccine.
TBEV has a single stranded positive sense RNA genome. The genomic information
is translated into a polyprotein which is then cleaved into the mature proteins by both
viral and cellular proteases. We want to establish a TBEV-replicon system, in which
the formation of viral particles depends on only one specific cleavage event of a
heterologously expressed foreign proteinase. Due to the error prone nature of the
viral RNA-polymerase, random mutations are introduced into this proteinase during
replication. Proteinase mutants that still have cleavage activity can be distinguished
from inactive ones by the formation of virus like particles. Therefore, this system can
be used to study structure-function relationships and inhibitor resistance.
We constructed a modular proteinase containing both the relevant parts of the
cofactor NS2B and the viral proteinase NS3, connected by a flexible linker and
provided with a His-tag for purification. The substrate C-prM was translated in vitro
utilizing rabbit reticulocyte lysate and 35S labeled Methionine. It provides a natural
cleavage site for the NS2B3 and was used to check the activity of the recombinant
protease. We successfully expressed and purified NS2B3.
An inactive form with a mutation in the active site, NS2B3 S192A, is used for
crystallization assays which are still ongoing. The active enzyme cleaves its natural
substrate C-prM. To make our replicon system work, we need a substrate that is
cleaved by the heterologously expressed proteinase but not by NS2B3. Therefore,
the natural TBEV cleavage site was substituted by various cleavage sites of the
HIV1 proteinase. Whether these substrates meets this demand is currently under
investigation.
37 | P a g e
VIZIER / SPINE2 Workshop on Structural Virology
P.11
ARRANGEMENTS FOR THE REGULATED PROCESSING OF
ALPHAVIRAL NONSTRUCTURAL POLYPROTEIN
Aleksei LULLA1 and Andrey Golubtsov2
1Institute
2Institute
of Technology, University of Tartu, Nooruse 1, 50411 Tartu, Estonia.
of Biotechnology,P.O. Box 56 (Viikinkaari 9), University of Helsinki, 00014
Helsinki, Finland.
Semliki Forest virus (SFV) is a type member of Alphavirus genus, which produces its
replicase proteins in a form of ns-polyprotein precursor P1234 and their maturation is
performed in a temporally controlled manner by papain-like protease activity of nsP2
protein. A large body of evidence suggests that template preference and enzymatic
capabilities of alphaviral replication complex have very important connection with its
composition irreversibly altered by proteolysis. Final cleavage within polyprotein at the
late stages of infection leading to maturation of nsP2 and nsP3 proteins is believed to
denote the “point of no return” for viral replication, since plus-strands are no more
accepted as templates and this puts emphasis on virions production in place of
replicase self-amplification. Numerous previous studies devised rules for when and how
ns-protease acts. Nevertheless, the understanding of the molecular bases for these
processing rules and the triggers of specific cleavage of particular site at the right
moment of replication remained rather illusive.
The data obtained in our study aimed at investigating the molecular details of the
regulation of the processing of 2/3 site in SFV ns-polyprotein provided evidence that socalled macro-domain (170 aa) of nsP3 protein is used for precise positioning of a
substrate recognition sequence at the catalytic center of the protease and this process
is coordinated by exact amino-terminal end of the N-terminal domain of nsP2 protein,
thus representing the unique regulation mechanism used by alphaviruses. Although
maturation of alphaviral replicase was always considered to be an attractive model for
studies of fine mechanisms of proteolytic processing, it can now be concluded that even
in such a seemingly simple system the efficiencies and the character of the cleavages
performed by the same protease are obviously different and all three sequentially
organized processing events are regulated by use of different mechanisms, only some
of which are common for both most studied alphaviruses, SFV and Sindbis virus.
Experimental data also disclosed the involvement of viral RNA in the regulation of the
processing of 2/3 site suggesting the multimeric nature of the alphaviral replication
complex residing on a viral RNA in a specifically ordered manner. Based on a newly
available data a model for replicase complex formation was proposed, which may apply
for replicases of several positive-strand RNA viruses.
38 | P a g e
VIZIER / SPINE2 Workshop on Structural Virology
P.12
STRUCTURAL AND FUNCTIONAL STUDIES OF FLAVIVIRAL PROTEINS NS3 AND NS5.
Eloise MASTRANGELO#, Mario Milani#, Michela Bollati#,
Graziella Sorrentino# and Martino Bolognesi#
#Dept.
of Biomolecular Sciences and Biotechnology
University of Milan
Via Celoria 26, 20133-Milan, Italy
The development of new antiviral drugs is a priority in view of the worldwide viral
threats to human health. In the context of the EC Integrated Project “Vizier” we are
characterizing the Flavivirus replicative machinery through the study of the Viral
Replication Complex.
In particular we have performed structural and functional studies of the NS3 and NS5
proteins that are the main actors of viral replication as components of a multiprotein
complex. During the replication, the viral genome is transcribed into a negativestrand RNA, used as template to synthesise the daughter viral genomic RNA.
Besides the evident role of the RNA-dependent RNA polymerase (RdRp, NS5 Cterminal domain, Malet et al., 2007), to maintain proper viral replication the nascent
transcript must be unwound from its complementary template RNA. Such role is
performed by the C-terminal domain of NS3 (helicase domain). We analysed
different NS3 constructs showing that full length NS3 displays increased helicase
activity. Such results suggest that the protease domain (i.e. NS3 N-terminal domain)
plays an assisting role in the RNA unwinding process. We investigated the
interaction between the helicase and protease domains in NS3 by small angle X-ray
scattering, producing a low resolution model showing scarce interaction between the
two domains. These results suggest that domain rearrangements affecting the
protease/helicase contact interface may occur in the RNA-bound protein
(Mastrangelo et al., 2007).
Once synthesized, RNA must be properly capped (cap I structure). This process
requires almost three enzymes: a RNA-triphosphatase (again NS3 C-terminal
domain), a guanylyltransferase and a N7 and 2'O MTase (the NS5 N-terminal
domain). We recently demonstrated a possible involvement of NS5 also during the
second step of RNA capping process. In fact, N-terminal domain of NS5 can bind
covalently GMP. Such reaction may be the first step in the achievement of the 5' 5'
bond between RNA diphosphate and GMP during the guanylyltransferase reaction
(Bollati et al., in preparation).
Following the characterization of single components of the viral replication
machinery, we performed several experiments (pull-down, cross-linking, coexpressions) to obtain part of the poliprotein complex, involving the helicase and the
RdRp domains, however without noticeable success so far.
- Malet H, Egloff MP, Selisko B, Butcher RE, Wright PJ, Roberts M, Gruez A, Sulzenbacher G,
Vonrhein C, Bricogne G, Mackenzie JM, Khromykh AA, Davidson AD, Canard B (2007). J Biol Chem.
14: 10678-89.
- Mastrangelo E, Milani M, Bollati M, Selisko B, Peyrane F, Pandini V, Sorrentino G, Canard B,
Konarev PV, Svergun DI, de Lamballerie X, Coutard B, Khromykh AA, Bolognesi M. (2007) J. Mol.
Biol. 372, 444-55.
39 | P a g e
VIZIER / SPINE2 Workshop on Structural Virology
P.13
ACTIVE SITE JUMPING IN TAILSPIKE PROTEINS OF THE PODOVIRIDAE FAMILY OF
TEMPERATE BACTERIOPHAGES AND PHAGE HEAD-BINDING MIMICRY IN CRYSTALS
J. J. MÜLLER*, S. Barbirz§, A. Seul§, R. Seckler§, U. Heinemann*
*Max-Delbrück-Centrum
für Moleculare Medizin, 13125 Berlin, Robert-Rössle-Straße
10, Germany
§Physikalische Biochemie, Universität Potsdam, 14476 Golm, Karl-LiebknechtStraße 24-25, Germany
Sf6, HK620, and P22 belong to the Podoviridae family of bacteriophages that infect
gram-negative bacteria by insertion of their double-stranded DNA. With their up to
six tailspike proteins (TSP) attached to the phage head, they recognize and cleave
their host cell receptor, i.e. the O-antigen. The trimeric TSP have highly homologous
N-terminal head binding domains but no homology in their receptor binding parts,
designated TSPN.
We have determined the crystal structures of the TSPN of Shigella flexneri phage
Sf6 to 1.25 Å (1), of Escherichia coli phage HK620 to 1.38 Å (2), and the first
complete P22 tailspike structure to 1.65 Å resolution. All three tailspikes show a
conserved architecture of the receptor binding part, i.e. a central, right-handed 
helix, flanked N-terminally by a mostly -helical region, and C-terminally by diverse β
folds. The Sf6 TSP C-terminal domain consists of a β sandwich reminiscent of viral
capsid proteins.
Complex structures of the TSPs with their O-antigen fragments and corresponding
biochemical analysis show a Shigella cell wall O-antigen fragment to bind to an
endorhamnosidase active site located between two β-helix subunits each anchoring
one catalytic carboxylate. The functionally and structurally related bacteriophage P22
TSP (3) and HK620 TSP have their active sites on single subunits. Sf6 TSP may
serve as an example for the evolution of different host specificities on a similar
general architecture.
By introduction of the point mutation Y108W into the P22 TSP, the structure of a
complete tailspike consisting of head binding domain, neck, and receptor binding
part has been determined for the first time. With the help of trimeric structures of
some additional chimeric constructs, made of the tailspike head binding domain and
a long α helix, we could explain the mechanics of the neck kinking during biological
processes which has been predicted from cryo-EM data. By moderate mechanical
stress, the tryptophans Y108 will be drawn out of their hydrophobic pockets
positioned between two subunits. Thereby, the chains are extended by about 5Å,
which renders possible a kink within the head binding domain and receptor domain
connecting neck. Crystallographic contacts provide forces which mimic the kinking
and bend the neck by about 6°, thereby destroying the crystallographic threefold
symmetry found so far in all TSPN trimers.
(1)
(2)
(3)
Müller, J.J., Barbirz, S., Heinle, K. Freiberg, A., Seckler, R., Heinemann, U. (2008)
Structure, 16, 766-775.
Barbirz, S., Müller, J.J, Uetrecht, C., Clark, A.J., Heinemann, U., Seckler, R. (2008)
Mol. Microbiol., in press.
Steinbacher, S., Baxa, U., Miller, S., Weintraub, A., Seckler, R., Huber, R. (1996).
Proc. Natl. Acad. Sci. USA 93, 10584-10588.
40 | P a g e
VIZIER / SPINE2 Workshop on Structural Virology
P.14
RESIDUE L143 OF THE FOOT-AND-MOUTH DISEASE VIRUS LEADER PROTEINASE IS A
DETERMINANT OF CLEAVAGE SPECIFICITY
David NEUBAUER, Christina Mayer, Aloysius T. Nchinda, Regina Cencic,
Katja Trompf and Tim Skern
Department of Medical Biochemistry
Max F. Perutz Laboratories
Dr. Bohrgasse 9/3, A-1030 Vienna
Austria
The foot-and-mouth disease virus (FMDV) leader protease (Lpro) is a specific papainlike cysteine proteinase for which only three substrates have been as yet identified.
These are the viral polyprotein which is cleaved by the proteinase in an
intramolecular self-processing reaction and the two homologues of eukaryotic
initiation factor 4G (eIF4G). The self-processing reaction at the Lpro/VP4 junction
(LysLeuLys*GlyAlaGly) is severely impaired by replacing leucine at P2 with
phenylalanine. Molecular modeling and energy minimization identified the L pro
residue L143 as being responsible for this discrimination. The mutation L143A in L pro
was sufficient to restore the efficiency of the self-processing reaction to wild type
levels on a substrate with P2 leucine whereas the mutation L178A was not. The
mutant L143A also showed improved self-processing on the sequence
AspPheGly*ArgGlnThr present on eIF4GII. Only leucine and methionine can be
found at position 143 of all sequenced FMDV serotypes. The fact that the variant
L143M also showed delayed self-processing on the cleavage sites containing
phenylalanine at P2 implies that these two bulky side chains are a determinant of the
restricted specificity of Lpro.
41 | P a g e
VIZIER / SPINE2 Workshop on Structural Virology
P.15
THE ROLE(S) OF GENOMIC RNA IN MS2 CAPSID ASSEMBLY
Óttar ROLFSSON and Peter Stockley
Astbury Centre for Structural Molecular Biology
University of Leeds
Leeds, LS2 9JT
United Kingdom
Viral capsids are composed of protein subunits that in the complex environment of
the cell are able to selectively package their genome and self-assemble into a
structure of defined shape and size. How viruses achieve this is a major problem
within structural virology. The possible role of the genome in this process is poorly
understood.
We are focusing on the role(s) of the viral genome in ssRNA virus capsid assembly
employing the phage MS2 as our model system. MS2 is a bacteriophage comprised
of a 3569 nt ssRNA genome protected by a T = 3 icosahedral capsid and a single
maturation protein necessary for cell entry. The phage capsid itself is composed of
180 coat protein subunits.
Our goal is to define the contributing effect of genomic RNA in MS2 capsid
assembly. Our approach involves monitoring the efficiency of capsid assembly using
gel shift assays, EM and sedimentation velocity analysis by comparing capsid
assembly reactions in which different segments of the MS2 genome have been used
to initiate capsid assembly.
These investigations suggest that the coat protein subunits act to fold the MS2 RNA
into the volume of the viral capsid and that RNA sequence/structure is therefore an
important factor for efficient capsid assembly.
42 | P a g e
VIZIER / SPINE2 Workshop on Structural Virology
P.16
INVESTIGATING THE INFLUENCE OF DIMERISATION ON THE ACTIVITY OF THE LEADER
PROTEASE OF FOOT AND MOUTH DISEASE VIRUS
Jutta STEINBERGER and Tim Skern
Department of Medical Biochemistry
Max F. Perutz Laboratories
1030 Vienna, Dr.-Bohr-Gasse 9/3
Austria
The leader protease (Lbpro) of foot and mouth disease virus frees itself from the
growing polypeptide chain during translation by cleavage between its own Cterminus and the N-terminus of the subsequent protein VP4. Previous
crystallography and NMR studies revealed that the C-terminal extension (CTE) of
Lbpro is bound by the substrate-binding site of a neighbouring molecule resulting in
the formation of a stable dimer in solution.
The amino acid residues W105 and T117, located at the interface between the
globular domains of the two Lbpro molecules, are potentially involved in dimer
formation. Therefore, we used PCR mutagenesis to substitute these residues with
alanine. Both single mutations as well as the double mutation W105A T117A were
fully active in self-processing. Nevertheless, the mutation W105A was not sufficient
to disturb dimer formation, as assayed by size exclusion chromatography. Therefore,
we tried to provoke repulsion between dimerized Lb pro molecules by substituting
W105 with the positively charged amino acid arginine. However, Lb pro bearing the
W105R mutation showed full activity in self-processing. Analysis of dimer formation
of this and other mutated proteins will be presented.
Supported by the Austrian Science Foundation.
43 | P a g e
VIZIER / SPINE2 Workshop on Structural Virology
P.17
THE ROLE OF SPECIFIC HISTIDINES AS PH SENSORS IN FLAVIVIRUS
MEMBRANE FUSION
Karin STIASNY, Richard FRITZ, and Franz X. HEINZ
Institute of Virology
Medical University of Vienna
1095 Vienna, Kinderspitalgasse 15
Austria
Flaviviruses enter cells by receptor-mediated endocytosis and fusion of the viral with
the endosomal membrane. The fusion event is triggered by the acidic pH of this
compartment and is mediated by the major envelope protein E, a class II viral fusion
protein. At acidic pH the native metastable E dimers dissociate leading to the
exposure of a previously buried functional element (fusion peptide, FP) that initiates
fusion by interacting with the target membrane. Further conformational changes
result in the generation of a trimeric postfusion structure.
Several histidines are absolutely conserved in the E protein of all flaviviruses and
because of their pKa have been hypothesized to function as molecular switches for
fusion. To provide experimental evidence for the role of these residues in flavivirus
fusion, we studied the effect of their replacement on membrane fusion in the context
of recombinant subviral particles of tick-borne encephalitis virus. Surprisingly,
replacement of three of the absolutely conserved histidines had virtually no effect on
fusion-related processes. However, mutating a histidine (H323) located at the
interface between domains I and III led to a strong impairment of E dimer
dissociation, FP exposure, and binding to target membranes; i.e. the early stages of
fusion. These results suggest that histidine 323 forms part of the low-pH trigger
required for initiating flavivirus membrane fusion.
44 | P a g e
VIZIER / SPINE2 Workshop on Structural Virology
45 | P a g e
VIZIER / SPINE2 Workshop on Structural Virology
LIST OF ALL PARTICIPANTS
Carme ARNAN
Structural Biology
IBMB-CSIC
C/Baldiri Reixac 10-12
I-20133 Barcelona, Spain
carcri@ibmb.csic.es
Dennis BAMFORD
Instiute of Biotechnology
University of Helsinki
PL 56 (Viikinkaari 5)
00014 Helsinki, Finland
dennis.bamford@helsinki.fi
Mads BEICH-FRANDSEN
Dept. for Biomolecular Structural Chemistry
University of Vienna
Campus-Vienna-Biocenter 5
1030 Vienna, Austria
mads.beich-frandsen@univie.ac.at
Dieter BLAAS
Dept. for Medical Biochemistry
Medical University of Vienna
Dr.-Bohr-Gasse 9/3
1030 Vienna, Austria
dieter.blaas@meduniwien.ac.at
Martino BOLOGNESI
Dept. for Biomolecular.Sciences & Biotechnology
University of Milano
Via Celoria, 26
I-20133 Milano, Italy
martino.bolognesi@unimi.it
Daniele BONIVENTO
Dept. for Genetics and Microbiology
University of Pavia
Via Ferrata 1
27100 Pavia, Italy
daniele.bonivento@uniroma1.it
Mickael BOUVET
AFMB UMR6098
University of Aix-Marseille I & II
ESIL/AFMB Case 925
13288 Marseille Cedex 09, France
mickael.bouvet@afmb.univ-mrs.fr
Gerard BRICOGNE
Global Phasing Ltd.
Sheraton House
Castle Park
CB3 0AX Cambridge, United Kingdom
Gb10@globalphasing.com
Bruno CANARD
AFMB
University of Méditerranée Marseille
Campus de Luminy
13288 Marseille, France
bruno.canard@afmb.univ-mrs.fr
Siva CHARAN DEVANABOY
Dept. for Structural Biology
University of Graz
Humboldstr. 50/3
8010 Graz, Austria
siva.charan@uni-graz.at
Lionel COSTENARO
Structural & Computational Biology
Institute for Reseachr in Biomedicine
Parc Científic de Barcelona
08028 Barcelona, Spain
lionel.costenaro@irbbarcelona.org
Stephen CUSACK
EMBL
Grenoble Outstation
6 rue Jules Horowitz
38042 Grenoble, France
cusack@embl.fr
Kristina DJINOVIC-CARUGO
Dept. for Biomolecular Structural Chemistry
University of Vienna
Campus Vienna Biocenter 5
1030 Vienna, Austria
kristina.djinovic@univie.ac.at
Francisco DOMINGUES
Computational Biology & Applied Algorithmics
Max-Planck-Institut for Informatics
Campus E1 4
66123 Saarbrücken, Germany
doming@mpi-sb.mpg.de
Aleksandra FLANAGAN
Division of Structural Biology
University of Oxford
Welcome Trust Centre, Roosevelt Drive
OX3 7BN Oxford, United Kingdom
Aleksandra@strubi.ox.ac.uk
Claus FLENSBURG
Global Phasing Ltd.
Sheraton House
Castle Park
CB3 0AX Cambridge, United Kingdom
claus@globalphasing.com
46 | P a g e
VIZIER / SPINE2 Workshop on Structural Virology
Richard FRITZ
Institute of Virology
Medical University of Vienna
Kinderspitalgasse 15
1095 Vienna, Austria
richard.fritz@meduniwien.ac.at
Stephen GRAHAM
Division of Structural Biology
University of Oxford
Welcome Trust Centre, Roosevelt Drive
OX3 7BN Oxford, United Kingdom
stepheng@strubi.ox.ac.uk
Jonathan GRIMES
WTCHG
University of Oxford
Welcome Trust Centre, Roosevelt Drive
OX3 7BN Oxford, United Kingdom
jonathan@strubi.ox.ac.uk
Rolf HILGENFELD
Institute of Biochemistry
University of Lübeck
Ratzeburger Allee 160
23538 Lübeck, Germany
hilgenfeld@biochem.uni-luebeck.de
Eric G. HUIZINGA
Crystal & Structural Chemistry
University of Utrecht
Padualaan 8
3584-CH Utrecht, The Netherlands
e.g.huizinga@uu.nl
Cristina IBBA
Dept. for Biomedical Science & Technology
University of Cagliari
Sesta Strada Ovest
09010 Macchiareddu (Ca), Italy
cristinaibba@microbiologia.ca.it
Anna JANSSON
Dept. for Cell & Molecular/Structural Biology
University of Uppsala
Husargatan 3
75124 Uppsala, Sweden
anna.jansson@icm.uu.se
Jan KADLEC
EMBL
Grenoble Outstation
BP 181, 6 rue Jules Horowitz
38042 Grenoble Cedex 9, France
kadlec@embl.fr
Martina KURZ
Dept. for Medical Biochemistry
Medical University of Vienna
Dr.-Bohr-Gasse 9/3
1030 Vienna, Austria
martina.kurz@meduniwien.ac.at
Hongmin LI
New York State Department of Health
Wadsworth Center
120 New Scotland Ave.
12208 Albany, USA
lih@wadsworth.org
Aleksei LULLA
Institute of Technology
University of Tartu
Nooruse 1
504111 Tartu, Estonia
lulla@ut.ee
Katsumi MAENAKA
Medical Institute of Bioregulation
University of Kyushu
3-1-1 Maidashi, Higashi-ku
812-8582 Fukuoka, Japan
kmaenaka@bioreg.kyushu-u.ac.jp
Ioannis MANOLARIDIS
EMBL
Hamburg Outstation
Notkestrasse 85
22603 Hamburg, Germany
manolari@embl-hamburg.de
Eloise MASTRANGELO
Science Biomolecular & Biotechnology
University of Milano
Via Celoria, 26
20133 Milano, Italy
Eloise.mastrangelo@mi.infm.it
Ulrike MAURER
Molecular Structural Biology
Max-Planck-Institute of Biochemistry
Am Klopferspitz 18
82152 Martinsried, Germany
maurer@biochem.mpg.de
Mario MILANI
Science Biomolecular & Biotechnology
University of Milano
Via Celoria, 26
20133 Milano, Italy
mario.milani@mi.infm.it
Jürgen MÜLLER
Crystallography
Max-Delbrück-Center
Robert-Rössle-Strasse 10
13125 Berlin, Germany
jjm@mdc-berlin.de
David NEUBAUER
Dept. for Medical Biochemistry
Medical University of Vienna
Dr.-Bohr-Gasse 9/3
1030 Vienna, Austria
david.neubauer@univie.ac.at
47 | P a g e
VIZIER / SPINE2 Workshop on Structural Virology
Jan PAESHUYSE
Rega-Institute Virology & Exp. Chemotherapy
K.U. Leuven
Minderbroedersstraat 10
3000 Leuven, Belgium
jan.paeshuyse@rega.kuleuven.be
Karen PANGERL
Institute of Virology
Medical University of Vienna
Kinderspitalgasse 15
1095 Vienna, Austria
karen.pangerl@meduniwien.ac.at
Magnus PERSSON
Dept. for Cell & Molecular/Structural Biology
University of Uppsala
Husargatan 3
75124 Uppsala, Sweden
magnusj@xray.bmc.uu.se
Margherita PEZZULLO
Dept. for Biomedical Science & Technology
University of Cagliari
Sesta strada Ovest, Z.I. di Macchiareddu
09010 Uta (CA), Italy
margheritapezzullo@microbiologia.ca.it
Félix REY
Dept. for Structural Virology
Instiute Pasteur
25 rue de Dr. Roux
75015 Paris, France
rey@pasteur.fr
Ottar ROLFSSON
Astbury Centre for Structural Molecular Biology
University of Leeds
Manton building, Clarendon Way
LS2 9JT Leeds, United Kingdom
bmbor@leeds.ac.uk
Rob RUIGROK
Unit of Virus Host Cell Interactions
University Joseph Fourier
6 rue Horowitz, B.P. 181
38042 Grenoble Cedex 9, France
ruigrok@embl.fr
Oliver SANDER
AG3
Max-Planck-Institute for Informatics
Campus E 1 4
66123 Saarbrücken, Germany
osander@mpi-sb.mpg.de
Eike SCHULZ
Dept. for Molecular Structural Biology
University of Göttingen
Justus-von-Liebig-Weg 11
37077 Göttingen, Germany
eschulz1@gwdg.de
Oliver SMART
Global Phasing Ltd.
Sheraton House
Castle Park
CB3 0AX Cambridge, United Kingdom
osmart@globalphasing.com
Vernon SMITH
Bruker AXS B.V.
Technical Sales Manager Structural Biology
Oostsingel 209
2612 HL Delft, The Netherlands
vernon.smitz@brucker-axs.nl
Jutta STEINBERGER
Dept. for Medical Biochemistry
Medical University of Vienna
Dr.-Bohr-Gasse 9/3
1030 Vienna, Austria
jutta.steinberger@univie.ac.at
Alasdair STEVEN
Laboratory of Structural Biology Research
National Institutes of Health
50 South Drive, room 1517
20892 Bethesda, Maryland, USA
dq1n@nih.gov
Karin STIASNY
Institute of Virology
Medical University of Vienna
Kinderspitalgasse 15
1095 Vienna, Austria
Karin.stiasny@meduniwien.ac.at
Paul TUCKER
EMBL
Hamburg Outstation
Notkestrasse 85
22603 Hamburg, Germany
tucker@embl-hamburg.de
Mark VAN RAAIJ
Dept. for Structural Biology
Instituto de Biologia Molecular de Barcelona
c/Josep Samitier 1-5
08028 Barcelona, Spain
vanraaij@usc.es
Herman VAN-TILBEURGH
IBBMC
University of Paris Sud
Bat 430
91405 Orsay, France
herman.van-tilbeurgh@u-psud.fr
Winfried WEISSENHORN
UVHCI c/o CIBB
University Jospeh Fourier
6 rue Jules Horowitz
38042 Grenoble, France
weissenhorn@embl.fr
48 | P a g e
VIZIER / SPINE2 Workshop on Structural Virology
Justyna WOJDYLA
EMBL
Hamburg Outstation
Notkestrasse 85
22603 Hamburg, Germany
tyyna@embl-hamburg.de
Thomas WOMACK
Global Phasing Ltd.
Sheraton House
Castle Park
CB3 0AX Cambridge, United Kingdom
twomack@globalphasing.com
Jürgen ZLATKOVIC
Clinical Institute of Virology
Medical University of Vienna
Kinderspitalgasse 15
1095 Vienna, Austria
juergen.zlatkovic@meduniwien.ac.at
49 | P a g e
VIZIER / SPINE2 Workshop on Structural Virology
Venue
Institute of Molecular Biotechnology, IMBA
Dr.-Bohr-Gasse 3,
A-1030 Vienna
IMBA is in the building number 2 on the map.
The venue will take place in the main lecture hall of IMBA.
50 | P a g e
VIZIER / SPINE2 Workshop on Structural Virology
Travel Directions
IMBA is located in the 3rd district (3. Bezirk; postal code 1030) of Vienna.
Public Transport Stations close to the MFPL/IMBA:
 Railway: S7 St. Marx (100m away)
 Tram: 71 or 18 “St. Marx” (right in front) or
18 “Viehmarktgasse”
 Bus: 74A “St. Marx” (right in front)
 Underground: U3 “Schlachthausgasse” (additional 7 min walk or two
stations with tram 18 to “Viehmarktgasse”)
From the Vienna International Airport Wien Schwechat:
 Railway S7 directly to St. Marx/Vienna Biocenter
 City Airport Train (CAT) to “Wien Mitte”, additionally bus 74A to
“St.Marx”
 Vienna Airport Shuttle Bus to the train station “Südbahnhof”,
additionally tram 18 to “St. Marx”
 Taxi
 Car: motorway A4, Exit Knoten Prater
From the train stations:
 Südbahnhof: tram 18 to “St.Marx”
 Westbahnhof: tram 18 to “St.Marx”
 Franz-Josefs-Bahnhof: U4 to “Landstraße” (= “Wien Mitte”),
additionally railway S7 or bus 74A to “St.Marx”
Arriving by Car:
A4: Exit Knoten Prater
A23: Exit Erdberg
Lectures will take place in the lecture hall of Institute of Molecular
Biotechnology (IMBA) at Campus Vienna Biocenter, Dr. Bohrgasse 3,
1030 Vienna.
The Campus Vienna Biocenter is Austria's largest life-sciencies park,
which encompasses Research Institute of Molecular Pathology (IMP),
Institute of Molecular Biotechnology (IMBA), Gregor Mendel Institute of
Molecular Plant Biology (GMI) and the Max F. Perutz Laboratories (MFPL)
of the University of Vienna and of Medical University.
51 | P a g e
VIZIER / SPINE2 Workshop on Structural Virology
Where to eat in Vienna’s third District
Here just a few of the numerous restaurant suggestions:
Irodion
Landstraßer Hauptstraße 71, 1030 Wien
Greek, daily 11:30 – 24:00
Credit cards will be accepted
Lubin
Hainburger Straße 48, 1030 Wien
Mail: reservierung@lubin.at; www.lubin.at
Fish restaurant; daily 11.00-15.00 and
17.30-24.00
Credit cards will be accepted
Restaurant im Hotel Artis
Rennweg 51, 1030 Wien
Mail: hotel@artis.at
www.artis.at
Mon-Sat 11.00-22.00,
warm meals 11.00-14.00 and 18.00-22.00
Credit cards will be accepted
Salm Bräu
Rennweg 8, 1030 Wien
Mail: office@salmbraeu.com
Viennese and international specialities
Mon-Sun 11.00-24.00
Visa accepted
Akula
1030 Wien, Ungargasse 21 - 23
Indish; daily 11.00-14.30 and00-23.00
CC: Amex, Diners, Mastercard, VISA,
JCB
Amon’s Gastwirtschaft
1030 Wien, Schlachthausgasse 13
E-Mail: office@amon.at
Mon-Sat 10.00-24.00, Sun 10.00-16.00
CC: Amex, Diners, Mastercard, VISA,
Hobex
Asia Restaurant Lou
1030 Wien, Landstraßer Hauptstraße 139
Asian; daily 11.30-15.00 and 17.00-22.30
CC: VISA, Mastercard
Akakiko
1030 Wien, Landstraßer Hauptstraße 59
E-Mail: info@akakiko.at
Japanese; daily 10.30-24.00
Bar-Celona
1030 Wien, Rasumofskygasse 34
E-Mail: bar-celona@gmx.at
Spanish; Mon-Sat 9.00-22.00
Credit cards will not be accepted.
Bieramt
1030 Wien, Am Heumarkt 3
E-Mail: bieramt@bieramt.at
Pub; daily 11.00-1.00. Kitchen until 24.00
CC: Amex, Diners, Mastercard, VISA
Burgenland Vinothek
1030 Wien, Baumannstraße 3
E-Mail: office@burgenland-vinothek.at
Vinotheken / Weinbar; Tue-Fri 13.0019.00, Sat 10.00-17.00
CC: VISA, Mastercard, Bankomat
Asia Running Sushi Paradies
1030 Wien, Landstraßer Hauptstraße 99
Telefon: 01/7151494
Japanese; Mon-Fri 10.00-20.00, Sat
10.00-18.00
Credit cards will not be accepted.
Klabautermann
1030 Wien, Markhofgasse 4
Mon-Fri 9.00-02.00,
Sat & Sun 17.00-24.00
Credit cards will not be accepted.
Modena
1030 Wien, Landstraßer Hauptstraße 133
E-Mail: pizzeria_modena@chello.at
Italian Restaurant; Mon-Fri 11.00-24.00,
Sat 12.00-4.00, Sun 12.00-22.30
CC: VISA, Mastercard, Bankomat
www.pizzeriamodena.at
52 | P a g e
VIZIER / SPINE2 Workshop on Structural Virology
Conference dinner
will take place in “Wiener Rathauskeller”
Wiener Rathauskeller
Rathausplatz 1
A-1010 Wien
Tel.: +43 (1) 405 1210
Fax: +43 (1) 405 1219-27
e-mail: office@wiener-Rathauskeller.at
It can be reached from the venue location by:
- tram 71 from St Marx to Schwarzenberg Platz
and then change to
- tram D from Schwarzenberg Platz to Rathaus
(We will hand out your tram tickets at registration.)
53 | P a g e
VIZIER / SPINE2 Workshop on Structural Virology
Wireless information
These are the usernames and passwords to enter the WLAN-System
at our workshop venue.
Accounts are active
from 14th July 2008 09:00
to 16th July 2008 17:00.
Username
Password
imba/guest4 73354855
54 | P a g e
guest5
35466725
guest6
03333768
guest8
11209378
guest9
87997864
guest10
70686201
guest11
89991773
guest12
15539269
guest13
23149593
guest14
37907579
guest15
96552483
guest16
86328437
guest17
21908131
guest18
86012123
guest19
36833535
guest20
69872184
VIZIER / SPINE2 Workshop on Structural Virology