A structure analysis of the intact virion and replicative complexes of human respiratory syncytial virus

Respiratory syncytial virus (RSV) infects very young babies and children causing severe respiratory disease that often requires hospitalisation and is sometimes fatal. RSV also infects the elderly and people with weak immune systems. Scientists have not yet been able to invent vaccines or medicines for this virus. It is very important then to build our understanding of this dangerous disease to find new ways of attacking the virus. This project aims to achieve this by using a very powerful electron microscope to look, in three dimensions (3D), at the shape of the virus and the different protein molecules it is made from. We have started this work by growing cells on a special support that allows us to put them into the microscope. These cells can be infected with RSV, which reproduces inside them. New virus particles grow out of the cells as long filaments. We can freeze the infected cells very quickly, to preserve their structure, and then examine them at very high magnification in the microscope. We then use computers to process images recorded in the microscope to build 3D maps of the virus that help us understand the shape of the molecules it is made from. By understanding how these different molecules stick together we hope that researchers will be able to invent new medicines that will interfere with the virus and stop it reproducing.
In this study we will focus on two key elements of the virus. Firstly we will look at a protein molecule called the matrix protein, or M. This protein directs the assembly of the virus by binding to the internal viral components and bringing them to the cell surface where the virus forms. M also binds to special viral proteins called F and G that are found on the outer-surface of the virus. These proteins are important for the newly formed virus particles to be able to infect new cells. Thus M is the coordinator of virus assembly and its interactions with other viral proteins are key targets for researchers looking to prevent the spread of infection.The internal components of the virus include a structure known as the nucleocapsid, which contains the virus's genetic code. Bound to this is another structure called the 'polymerase' a protein that copies the genetic code so that more virus particles can be made. This assembly is the second component of RSV that we are interested in solving the structure of. We may be able to achieve this by looking for the polymerase bound onto the nucleocapsid in the virus itself. Another approach is to make the structure artificially in the laboratory. This has proven very difficult but co-investigator Dr Rachel Fearns of Boston University has managed to achieve this recently. Together we will purify large quantities of the polymerase which we can examine in the electron microscope to determine its structure. The higher resolution we can achieve by this approach should provide us with a more detailed understanding of how the polymerase works.
The final component of this project will be to develop new methods for looking at the process of virus assembly at the cell surface. It is presently very difficult to look at whole cells in the electron microscope as they are too thick for the electrons to penetrate. We will use a new technique - Focussed Ion Beam milling (FIB) to prepare thin sections of frozen cells that will allow us to image RSV as it assembles. This will allow us to see whether certain components of the cell itself are co-opted by the virus to help its assembly and may also highlight possible targets for new medicines.

Respiratory syncytial virus (RSV) is a pneumovirus in the family Paramyxoviridae. The viral genome is a negative sense RNA that is encapsidated by a viral protein N, to form a helical ribonucleoprotein (RNP) called the nucleocapsid (NC). The NC is the template for RNA synthesis by the viral RNA dependent RNA polymerase (RdRp - comprised of three viral proteins, L, P and M2-1), together the NC and RdRp make up the holonucleocapsid (hNC). RSV is an enveloped virus and assembly is directed by the M protein that binds the hNC, trafficking the complex to the plasma membrane, where interactions with the envelope glycoproteins F and G promote virion morphogenesis.
The aim of this project is to use cryo-electron microscopy and tomography (CEM and CET) to characterise RSV at intermediate (10-30 angstroms) to high (6-10 angstroms) resolution. We have established methods for the propagation of cells on TEM grids for CEM. RSV infected cells produce long filamentous virions which we imaged by CEM and CET without incurring preparation artefacts. Previous CET of purified paramyxoviruses has shown poorly ordered bag-like particles with tangled NCs inside. Our data show that RSV filaments are highly ordered. CET and Fourier analysis of CEM images shows that RSV exhibits helical symmetry, possibly extending throughout the envelope from the M layer to the glycoproteins. Helical reconstruction will give insights into M oligomerisation and interactions with the viral envelope and F/G proteins. Sub-tomogram averaging of CET data will build on our X-ray and CET studies of the NC by providing data on the terminal structures and possibly the RdRp. Purification of recombinant RdRp and hNC will allow higher resolution CEM and single-particle studies. Finally cryo-sectioning of RSV infected cells using focussed ion beam milling (FIB) will allow CET of virus assembly at the plasma membrane, potentially providing structural insights into the involvement of host-cell factors such as actin.

Beyond the impact of this work on the CVR structural biology groups and University of Glasgow researchers, in the immediate short term the principal beneficiaries of this research will be the research community and in particular those working on negative sense RNA containing viruses and structural electron microscopists. Further short-term beneficiaries are the general public and local public engagement agencies (science centres, galleries and museums).

In the longer term we expect that the outcome of these studies will be of interest to researchers working on development of vaccines and anti-virals to prevent and treat RSV disease, both in industry and in the academic community. Ultimately if the promise of these outcomes is fully realised, the work has the potential to benefit the global population by improving the health of neonates, the elderly and immunocompromised.

The primary outcome of this research will be a collection of structure data on RSV, an important respiratory pathogen. In addition we will develop a capability for cryogenic imaging of virus infected cells. The former will contribute significantly to fundamental understanding of this virus, changing our view of paramyxovirus morphology. These data will inform future experiments by researchers working on the biology of paramyxoviruses, testing putative interactions by site directed mutagenesis for example. Identification of key interactions between M and F/G proteins will provide potential targets for rational drug design to inhibit virus assembly. Intermediate or high resolution data on the polymerase in various states of function will likewise be hugely informative. We have previously solved the X-ray structure of RSV N-RNA, and combining these data with tomography of recombinant helical nucleocapsids generated a model of the viral NC. Docking these data into reconstructions of the holo-nucleocapsid will identify important targets, potentially leading to the development of polymerase inhibitors. The project set out in this proposal is basic rather than translational research, we consider that by applying ourselves to the investigation of two distinct aspects of virus biology (morphogenesis and RNA synthesis) we will provide data on two credible therapeutic targets. Taken to their conclusions such analyses may then result in two or more modes of intervention, a favourable outcome that would reduce the risk of resistance.

To build our understanding of RSV filament morphogenesis, we will develop and implement focussed-ion beam milling (FIB-SEM also known as ion abrasion microscopy) for cryo-sectioning of virus-infected cells. Whole mammalian cells are plunge-frozen resulting in the formation of vitreous ice, thus biological assemblies are preserved in a frozen hydrated state. Cells are then prepared for imaging in the cryo-transmission electron microscope using the FIB SEM. This is achieved by etching the cells into lamellae that are ~3-400 nm thick. This new technique will be of wide interest to the UK structural biology community. We will consolidate the considerable impact of this work through the delivery of a workshop on FIB-SEM cryo-sectioning and presenting our work at the Scottish Microscopy Group symposium.

Calculation of tomographic reconstructions and models of the RSV filamentous virion will produce eye-catching imagery on an important respiratory pathogen. This will be of interest to the media. Such imagery may be included in future public and schools engagement activities, in particular in partnership with Glasgow Science Centre. These data will be incorporated in future art exhibitions building on our previous project Molecular Machines, images from virus research (www.molecularmachines.org.uk). Public exposure to these data will raise awareness of this important virus and improve public understanding of the nature of viruses and virus research. It will also enhance the reputation and raise public awareness of MRC and the CVR.