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Open Access
Abstract: Enterovirus 71 (EV71) and Coxsackievirus A16 (CVA16) are the two major causative agents of hand, foot and mouth disease (HFMD), for which there are currently no licenced treatments. Here, the acquisition of resistance towards two novel capsid-binding compounds, NLD and ALD, was studied and compared to the analogous compound GPP3. During serial passage, EV71 rapidly became resistant to each compound and mutations at residues I113 and V123 in VP1 were identified. A mutation at residue 113 was also identified in CVA16 after passage with GPP3. The mutations were associated with reduced thermostability and were rapidly lost in the absence of inhibitors. In silico modelling suggested that the mutations prevented the compounds from binding the VP1 pocket in the capsid. Although both viruses developed resistance to these potent pocket-binding compounds, the acquired mutations were associated with large fitness costs and reverted to WT phenotype and sequence rapidly in the absence of inhibitors. The most effective inhibitor, NLD, had a very large selectivity index, showing interesting pharmacological properties as a novel anti-EV71 agent.
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Dec 2015
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M01-Polara at OPIC (Oxford)
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Ling
Zhu
,
Xiangxi
Wang
,
Jingshan
Ren
,
Claudine
Porta
,
Hannah
Wenham
,
Jens-Ola
Ekström
,
Anusha
Panjwani
,
Nick J.
Knowles
,
Abhay
Kotecha
,
C. Alistair
Siebert
,
A. Michael
Lindberg
,
Elizabeth E.
Fry
,
Zihe
Rao
,
Tobias J.
Tuthill
,
David I.
Stuart
Open Access
Abstract: Picornaviruses are responsible for a range of human and animal diseases, but how their RNA genome is packaged remains poorly understood. A particularly poorly studied group within this family are those that lack the internal coat protein, VP4. Here we report the atomic structure of one such virus, Ljungan virus, the type member of the genus Parechovirus B, which has been linked to diabetes and myocarditis in humans. The 3.78-Å resolution cryo-electron microscopy structure shows remarkable features, including an extended VP1 C terminus, forming a major protuberance on the outer surface of the virus, and a basic motif at the N terminus of VP3, binding to which orders some 12% of the viral genome. This apparently charge-driven RNA attachment suggests that this branch of the picornaviruses uses a different mechanism of genome encapsidation, perhaps explored early in the evolution of picornaviruses.
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Dec 2015
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I03-Macromolecular Crystallography
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Luigi
De Colibus
,
Xiangxi
Wang
,
Aloys
Tijsma
,
Johan
Neyts
,
John
Spyrou
,
Jingshan
Ren
,
Jonathan
Grimes
,
Gerhard
Puerstinger
,
Pieter
Leyssen
,
Elizabeth E.
Fry
,
Zihe
Rao
,
David I.
Stuart
Open Access
Abstract: The replication of enterovirus 71 (EV71) and coxsackievirus A16 (CVA16), which are the major cause of hand, foot and mouth disease (HFMD) in children, can be inhibited by the capsid binder GPP3. Here, we present the crystal structure of CVA16 in complex with GPP3, which clarifies the role of the key residues involved in interactions with the inhibitor. Based on this model, in silico docking was performed to investigate the interactions with the two next-generation capsid binders NLD and ALD, which we show to be potent inhibitors of a panel of enteroviruses with potentially interesting pharmacological properties. A meta-analysis was performed using the available structural information to obtain a deeper insight into those structural features required for capsid binders to interact effectively and also those that confer broad-spectrum anti-enterovirus activity.
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Oct 2015
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I24-Microfocus Macromolecular Crystallography
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Abhay
Kotecha
,
Julian
Seago
,
Katherine
Scott
,
Alison
Burman
,
Silvia
Loureiro
,
Jingshan
Ren
,
Claudine
Porta
,
Helen M
Ginn
,
Terry
Jackson
,
Eva
Perez-Martin
,
C Alistair
Siebert
,
Guntram
Paul
,
Juha T
Huiskonen
,
Ian M
Jones
,
Robert
Esnouf
,
Elizabeth
Fry
,
Francois F
Maree
,
Bryan
Charleston
,
Dave
Stuart
Diamond Proposal Number(s):
[10627]
Abstract: Virus capsids are primed for disassembly, yet capsid integrity is key to generating a protective immune response. Foot-and-mouth disease virus (FMDV) capsids comprise identical pentameric protein subunits held together by tenuous noncovalent interactions and are often unstable. Chemically inactivated or recombinant empty capsids, which could form the basis of future vaccines, are even less stable than live virus. Here we devised a computational method to assess the relative stability of protein-protein interfaces and used it to design improved candidate vaccines for two poorly stable, but globally important, serotypes of FMDV: O and SAT2. We used a restrained molecular dynamics strategy to rank mutations predicted to strengthen the pentamer interfaces and applied the results to produce stabilized capsids. Structural analyses and stability assays confirmed the predictions, and vaccinated animals generated improved neutralizing-antibody responses to stabilized particles compared to parental viruses and wild-type capsids.
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Sep 2015
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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Jingshan
Ren
,
Xiangxi
Wang
,
Ling
Zhu
,
Zhongyu
Hu
,
Qiang
Gao
,
Pan
Yang
,
Xuemei
Li
,
Junzhi
Wang
,
Xinliang
Shen
,
Elizabeth
Fry
,
Zihe
Rao
,
Dave
Stuart
,
K.
Kirkegaard
Diamond Proposal Number(s):
[10627]
Open Access
Abstract: Enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) are the primary causes of the epidemics of hand-foot-and-mouth disease (HFMD) that affect more than a million children in China each year and lead to hundreds of deaths. Although there has been progress with vaccines for EV71, the development of a CVA16 vaccine has proved more challenging, and the EV71 vaccine does not give useful cross-protection, despite the capsid proteins of the two viruses sharing about 80% sequence identity. The struc- tural details of the expanded forms of the capsids, which possess nonnative antigenicity, are now well understood, but high resolution information for the native antigenic form of CVA16 has been missing. Here, we remedy this with high resolution X-ray structures of both mature and natural empty CVA16 particles and also of empty recombinant viruslike particles of CVA16 produced in insect cells, a potential vaccine antigen. All three structures are unexpanded native particles and antigenically identical. The recombinant particles have recruited a lipid moiety to stabilize the native antigenic state that is different from the one used in a natural virus infection. As expected, the mature CVA16 virus is similar to EV71; however, structural and immunogenic com- parisons highlight differences that may have implications for vaccine production.
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Sep 2015
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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Xiangxi
Wang
,
Jingshan
Ren
,
Qiang
Gao
,
Zhongyu
Hu
,
Yao
Sun
,
Xuemei
Li
,
David J.
Rowlands
,
Weidong
Yin
,
Junzhi
Wang
,
David I.
Stuart
,
Zihe
Rao
,
Elizabeth E.
Fry
Abstract: Hepatitis A virus (HAV) remains enigmatic, despite 1.4 million cases worldwide annually. It differs radically from other picornaviruses, existing in an enveloped form and being unusually stable, both genetically and physically, but has proved difficult to study. Here we report high-resolution X-ray structures for the mature virus and the empty particle. The structures of the two particles are indistinguishable, apart from some disorder on the inside of the empty particle. The full virus contains the small viral protein VP4, whereas the empty particle harbours only the uncleaved precursor, VP0. The smooth particle surface is devoid of depressions that might correspond to receptor-binding sites. Peptide scanning data extend the previously reported VP3 antigenic site, while structure-based predictions suggest further epitopes. HAV contains no pocket factor and can withstand remarkably high temperature and low pH, and empty particles are even more robust than full particles. The virus probably uncoats via a novel mechanism, being assembled differently to other picornaviruses. It utilizes a VP2 'domain swap' characteristic of insect picorna-like viruses and structure-based phylogenetic analysis places HAV between typical picornaviruses and the insect viruses. The enigmatic properties of HAV may reflect its position as a link between ‘modern’ picornaviruses and the more ‘primitive’ precursor insect viruses; for instance, HAV retains the ability to move from cell-to-cell by transcytosis.
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Oct 2014
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I24-Microfocus Macromolecular Crystallography
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Open Access
Abstract: The integral membrane protein LIMP-2 has been a paradigm for mannose 6-phosphate receptor (MPR) independent lysosomal targeting, binding to β-glucocerebrosidase (β-GCase) and directing it to the lysosome, before dissociating in the late-endosomal/lysosomal compartments. Here we report structural results illuminating how LIMP-2 binds and releases β-GCase according to changes in pH, via a histidine trigger, and suggesting that LIMP-2 localizes the ceramide portion of the substrate adjacent to the β-GCase catalytic site. Remarkably, we find that LIMP-2 bears P-Man9GlcNAc2 covalently attached to residue N325, and that it binds MPR, via mannose 6-phosphate, with a similar affinity to that observed between LIMP-2 and β-GCase. The binding sites for β-GCase and the MPR are functionally separate, so that a stable ternary complex can be formed. By fluorescence lifetime imaging microscopy, we also demonstrate that LIMP-2 interacts with MPR in living cells. These results revise the accepted view of LIMP-2–β-GCase lysosomal targeting.
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Jul 2014
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[5968]
Open Access
Abstract: A departure from a linear or an exponential intensity decay in the diffracting power of protein crystals as a function of absorbed dose is reported. The observation of a lag phase raises the possibility of collecting significantly more data from crystals held at room temperature before an intolerable intensity decay is reached. A simple model accounting for the form of the intensity decay is reintroduced and is applied for the first time to high frame-rate room-temperature data collection.
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May 2014
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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Luigi
De Colibus
,
Xiangxi
Wang
,
John A. B.
Spyrou
,
James
Kelly
,
Jingshan
Ren
,
Jonathan
Grimes
,
Gerhard
Puerstinger
,
Nicola
Stonehouse
,
Thomas
Walter
,
Zhongyu
Hu
,
Junzhi
Wang
,
Xuemei
Li
,
Wei
Peng
,
David J.
Rowlands
,
Elizabeth E.
Fry
,
Zihe
Rao
,
David I.
Stuart
Diamond Proposal Number(s):
[8423]
Abstract: Enterovirus 71 (HEV71) epidemics in children and infants result mainly in mild symptoms; however, especially in the Asia-Pacific region, infection can be fatal. At present, no therapies are available. We have used structural analysis of the complete virus to guide the design of HEV71 inhibitors. Analysis of complexes with four 3-(4-pyridyl)-2-imidazolidinone derivatives with varying anti-HEV71 activities pinpointed key structure-activity correlates. We then identified additional potentially beneficial substitutions, developed methods to reliably triage compounds by quantum mechanics enhanced ligand docking and synthesized two candidates. Structural analysis and in vitro assays confirmed the predicted binding modes and their ability to block viral infection. One ligand (with IC50 of 25 pM) is an order of magnitude more potent than the best previously reported inhibitor and is also more soluble. Our approach may be useful in the design of effective drugs for enterovirus infections.
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Feb 2014
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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Jingshan
Ren
,
Xiangxi
Wang
,
Zhongyu
Hu
,
Qiang
Gao
,
Yao
Sun
,
Xuemei
Li
,
Claudine
Porta
,
Thomas
Walter
,
Robert
Gilbert
,
Yuguang
Zhao
,
Danny
Axford
,
Mark
Williams
,
Katherine
Mcauley
,
David J.
Rowlands
,
Weidong
Yin
,
Junzhi
Wang
,
David I.
Stuart
,
Zihe
Rao
,
Elizabeth E.
Fry
Open Access
Abstract: It remains largely mysterious how the genomes of non-enveloped eukaryotic viruses are transferred across a membrane into the host cell. Picornaviruses are simple models for such viruses, and initiate this uncoating process through particle expansion, which reveals channels through which internal capsid proteins and the viral genome presumably exit the particle, although this has not been clearly seen until now. Here we present the atomic structure of an uncoating intermediate for the major human picornavirus pathogen CAV16, which reveals VP1 partly extruded from the capsid, poised to embed in the host membrane. Together with previous low-resolution results, we are able to propose a detailed hypothesis for the ordered egress of the internal proteins, using two distinct sets of channels through the capsid, and suggest a structural link to the condensed RNA within the particle, which may be involved in triggering RNA release.
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Jun 2013
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