What is your background in structural biology and how did you become involved in researching ribonucleic acid (RNA) viruses and, in particular, the human metapneumovirus (hMPV)?

I started to develop an interest for structural biology and RNA viruses during my Master’s studies at the University of Grenoble, France, in 2006. At the time, I began to appreciate that some RNA viruses could infect and even kill human beings using the information contained in a genome of approximately 10,000 nucleotides (while the human genome is over 3 billion nucleotides in length). I wanted to understand the succession of molecular events that could enable such minimal organisms to defeat larger and much more complex life forms.

With my background in chemistry, I naturally became attracted to the fields of biology

that most resembled chemistry, such as structural biology, biochemistry and biophysics. I completed my PhD in 2010 on the structure of the rabies virus replication complex at the Unit of Virus Host Cell Interactions in Grenoble. After this, I began work on hMPV at the University of Oxford, UK, with Dr Jonathan Grimes on the European Commission-funded Small-molecule Inhibitor Leads Versus Emerging and neglected RNA viruses (SILVER) project.

Could you briefly explain why hMPV is such a serious threat?

hMPV was first identified in 2001 in Dutch children with bronchiolitis. It is an RNA virus in the Paramyxoviridae family and is most closely related to respiratory syncytial virus. Since its initial identification in 2001, hMPV has been isolated from individuals with acute respiratory tract infections (RTIs) in virtually every continent. With a seasonal distribution similar to influenza viruses, hMPV can cause severe infections and is responsible for 5-10 per cent of hospitalisations of children suffering from acute RTIs. This makes hMPV a serious threat to human health, in particular when you consider the high potential that RNA viruses have to mutate, evolve and quickly adapt to new hosts; and the structural and genomic relatedness of viruses like hMPV, respiratory syncytial virus and Ebola virus.

What are the key goals and activities of the SILVER project?

SILVER is a global consortium arising from the coordination of Europe and Asia’s leading molecular virologists, structural biologists,

medicinal chemists and bioinformaticians, which aims to generate a state-of-the-art drug design programme to tackle emerging diseases caused by RNA viruses. Its activities range from the structural characterisation of viruses and viral proteins to the screening and identification of lead compounds in vitro and in vivo, and their optimisation through medicinal chemistry and structure-based drug design approaches.

You use a wide variety of cutting-edge structural biology methods in your research. What novel techniques have you found to be particularly effective?

Understanding macromolecular structure, dynamics and interactions at the atomic level increasingly requires the use of hybrid methods. This is because each particular technique comes with its own set of limitations, requiring macromolecules to be either small, large or very rigid, and many proteins are actually only loosely structured or even intrinsically disordered. There have been a number of exciting developments in the field of small angle X-ray scattering (SAXS) with the ability to model macromolecular dynamics using ensembles of atomic structures without limitations in size or conformational flexibility. This involves combining information from atomic

Dr Cedric Leyrat explains how controlling the function of RNA viruses through their complete structural characterisation could lead to the safer and cheaper development of effective drugs and vaccines

Structural biology: a route to revelationD

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New targets forviral vaccinationAt the University of Oxford’s Division of Structural Biology, a team of researchers is using state-of-the-art methodologies to reveal the structure and dynamics of the human metapneumovirus for the fi rst time

CHARACTERISED BY A strong capacity for mutation and host jumping, RNA viruses are familiar to many as the cause of devastating outbreaks of disease, most recently experienced with the Ebola epidemic in West Africa. Their rapid evolution and adaptation has the added effect of making RNA viruses a major challenge to the design and development of effective drugs and vaccines. Structurally similar to Ebola, measles, mumps and rabies, the human metapneumovirus (hMPV) is responsible for a huge proportion of bronchiolitis and pneumonia cases across the globe. By the age of fi ve, almost all children have been exposed to the negative-strand RNA virus with reinfections occurring throughout adult life.

Despite lacking the visceral impact of a major disease outbreak, bronchiolitis and pneumonia nevertheless present a serious threat to human health with infants, elderly and immune-compromised individuals particularly vulnerable to lower respiratory tract infections. Until recently, little has been known about the mechanisms underlying the transcription, replication and morphogenesis of hMPV and more than a few of its closely related viral cousins, putting them in the same untreatable league. Now, the European Commission-funded Small-molecule Inhibitor Leads Versus Emerging and neglected RNA viruses (SILVER) project is attempting, for the fi rst time, to unravel the secrets of a whole host of RNA viruses and herald a new stage of drug design and vaccine development.

THE HYBRID APPROACH

As part of the SILVER team, Dr Cedric Leyrat is working closely with Dr Jonathan Grimes and PhD student Max Renner at the University of Oxford’s Division of Structural Biology (STRUBI), UK, to

produce a detailed atomic-level understanding of hMPV and allow for unprecedented insights into the mechanisms by which viruses are able to infect cells. In order to elucidate the structural and dynamic characteristics of hMPV proteins, Leyrat’s contribution to SILVER relies on a combination of state-of-the-art structural biology and biophysical techniques, and the use of powerful X-rays produced at the nearby national synchrotron facility, Diamond Light Source.

Among the individual components of these hybrid methods are cryo-electron microscopy, small angle X-ray scattering (SAXS), X-ray crystallography, multi-angle laser light scattering, dynamic light scattering, fl uorescence-based thermal shift assays and surface plasmon resonance. Coupled with advanced computation methods such as classical and coarse-grained molecular dynamics, protein docking and ab

models derived from crystallography or nuclear magnetic resonance and applying conformational sampling methods in order to analyse their solution structure using SAXS data. These hybrid approaches provide unprecedented insights into the structure and dynamics of these systems, in many cases explaining their functional properties.

Why is it important to know the complete structural characterisation of viruses? What can be gained from understanding the mechanisms of hMPV at the molecular and atomic level?

This knowledge is fundamental because all biological processes originate from the molecular properties of proteins, nucleic acids etc. Ultimately, these processes can all be traced back to the structure, dynamics and interactions between macromolecules, and between macromolecules and small molecules such as drugs and hormones. Reaching a complete atomic-level description of a biological system provides an in-depth understanding that enables researchers to eventually control its function, predict the results of perturbations or re-design it to perform differently. For hMPV, this means being able to design drugs that block specifi c viral functions or improve host immunity and create safe and effective vaccines.

Crystal structure of the dimeric, diamond-shaped matrix protein of human metapneumovirus.

Crystal structure of the human metapneumo-virus transcriptional anti-terminator M2-1, revealing an asymmetric tetramer with a modular architecture.

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MOLECULAR AND ATOMIC DETERMINATION OF HUMAN METAPNEUMOVIRUS

OBJECTIVES

To develop a molecular, atomic-level understanding of the mechanisms underlying the transcription, replication and morphogenesis of metapneumovirus and evolutionary related viruses. To facilitate this, experimental and computational structural biology methods are used to capture atomistic snapshots of viral proteins and to study their dynamics in order to understand their functions.

KEY COLLABORATORS

Dr Bernadette van den Hoogen; Professor Ron Fouchier, ERASMUS MC, Netherlands

Dr Hervé Bourhy, Institute Pasteur, France

Dr Etienne Decroly, Laboratory for the Architecture and Function of Biological Macromolecules, CNRS, France

PARTNERS

University of Oxford

Diamond Light Source Ltd

INSTRUCT (www.structuralbiology.eu)

FUNDING

European Union Seventh Framework Programme (FP7)

CONTACT

Dr Cedric Leyrat

The Division of Structural BiologyUniversity of OxfordHenry Wellcome Building forGenomic MedicineRoosevelt Drive Oxford, OX3 7BN UK

T +44 1865 287 547E cedric@strubi.ox.ac.uk

www.strubi.ox.ac.uk

CEDRIC LEYRAT has a Master’s in Life Sciences and a PhD in Structural Biology from the Université Joseph Fourier, France, and is now a postdoctoral researcher in the Division of Structural Biology at the University of Oxford, UK, working within the large-scale collaborative European Union project SILVER. He has published 18 peer-reviewed journal articles and one book chapter.

initio protein modelling, it is possible to collate the information generated through these experimental techniques.

The effectiveness of their approach is clearly visible in the group’s penetrating structural analyses of four hMPV proteins – its phosphoprotein, viral attachment glycoprotein, matrix protein and antiterminator protein. In the case of hMPV phosphoprotein, Leyrat has achieved an accurate structural model through the combination of four techniques. The Rosetta fold-and-dock protocol for protein structure prediction (recently developed through the Berkeley Open Infrastructure for Network Computing) predicts atomistic models, while SAXS is used to select only those models that best represent the data. Partially unstructured proteins can make modelling tricky but this has been dealt with by enhanced fi ltering through ensemble analysis. Finally, the implementation of classical molecular dynamics allows for an even higher selectivity of correct models. Together, these techniques have resulted in a detailed, cross-validated picture of hMPV phosphoprotein structure and dynamics in solution. With such high accuracy, these hybrid techniques are expected to become increasingly relevant in structural biology, with this method alone providing a promising approach to general protein structure determination.

SOLVING STRUCTURES

Through such high resolution models, the team’s research has begun to unveil the inner workings of hMPV that are integral to the development of future treatments. Investigations of hMPV’s viral attachment glycoprotein, and comparisons with the Ebola virus’ surface glycoprotein, are indicative of similar immune evasion strategies used by pneumoviruses and fi loviruses. The glycoprotein does not elicit much of an immune response, unlike hMPV’s fusion protein, but its deletion has been shown to increase the activity of immunologic CD4+ T cells. “The glycoprotein is able to reduce the amount of neutralising antibodies binding to the fusion protein, leading to a transient immunity that may explain incidences of reinfection,” explains Leyrat. Further characterisation of the glycoprotein has enabled the group to demonstrate its properties to be intrinsic disorder, sequence hypervariability and heavy O-glycosylation, which contrasts to the more structured attachment glycoproteins in other viruses from the same family.

Performing a central role in the assembly of viral particles and their budding from the cells is hMPV’s matrix protein. By solving its X-ray crystallographic structure at a resolution of 2.8 angstrom, Leyrat has discovered a high-affi nity binding site for the calcium ion (Ca2+): “This was surprising because Ca2+ has not been observed in any matrix protein from other negative strand RNA viruses,” enthuses Leyrat. A closer look at the conservation of the Ca2+ binding site indicates that calcium may in fact play a pivotal role in the replication and morphogenesis of the pneumovirus subfamily, apparently crucial to the stability of the matrix protein. This means it could be involved in regulating key processes such as viral entry, uncoating, assembly and

budding. Ca2+ binding pockets, therefore, may be potential targets for the development of small-molecule inhibitors.

Excitingly, the team has also solved the structure of the hMPV antiterminator protein M2-1, which must be present for hMPV to infect humans. Antiterminator proteins are critical for viral replication, ensuring that the enzymes ignore termination signals when building the RNA to facilitate the completion of synthesis. Medicines that target these genes could prove an important avenue for arresting the spread of viral infections. Together with Grimes and Renner, Leyrat has uncovered an important characteristic of the protein: “We are able to explain how the protein regulates viral transcription and replication by fl ipping between open and closed forms”. Their model shows the atomic details of both forms revealing novel molecular surfaces when open, that could be targeted by antiviral drugs.

DESIGNER DRUGS

Leyrat and his colleagues’ work in the context of SILVER has signifi cantly added to current understanding of hMPV and its close viral relations, opening up the possibility to pursue small molecule drug design and genetically engineered proteins with altered functions. However, the development of such antiviral drugs is burdened with a host of requirements that act as a barrier between successful in vitro development and production by the pharmaceutical industry. Among these challenges is the ability to design a molecule that can be chemically synthesised, bind to the target with high affi nity, is soluble, relatively specifi c to the target, non-toxic to healthy cells and able to retain suffi cient bioavailability while being metabolisable by the human body. “In my opinion, because diseases usually result from the activities of proteins, the best way to overcome these challenges is to use macromolecules instead,” states Leyrat.

This path will, however, remain untrodden until a more fundamental understanding of structural, cellular and systems biology is achieved. Challenging though small molecule drug design may be, there have been successes. In generating complete structural characterisations of viruses and their host interactions – thereby gaining control of all of its functions – it is possible that therapeutics can be designed that are safer and cheaper. Indeed, it is anticipated that by the time SILVER draws to a close, at least one inhibitor lead will have proven to exhibit all the properties required to justify development by the pharmaceutical industry.

From left to right: Max Renner, Cedric Leyrat and Jonathan Grimes at the crystallisation facility in STRUBI, Oxford.

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INTELLIGENCE