The structure and function of the human respiratory syncytial virus M2-1 protein and its critical interaction with viral cofactors

Human respiratory syncytial virus (HRSV) is responsible for an extremely common respiratory disease, with most humans on earth being infected at least once before they reach the age of 2. In most adults, the disease is not life threatening. However, in the very young, the elderly and the immunocompromised, HRSV infections often require hospitalization and can be fatal. Recent studies suggest that each year HRSV is responsible for over 34 million lower respiratory tract infections and between 66,000 and 199,000 deaths. In addition, HRSV infection early in life may contribute towards increased incidence of asthma later in adulthood, which affects over 5 million people in the UK alone. There is no HRSV vaccine, and the only current option for preventing HRSV disease is to give vulnerable people repeated injections of a drug called palivizumab. However, this treatment is only moderately effective and is prohibitively expensive, therefore new treatments to combat HRSV disease are required. The experiments described in this proposal will identify several critical weaknesses in HRSV that will allow new drugs to be developed to prevent HRSV disease.
HRSV is made of several different building blocks called proteins that must fit together very precisely in order to do their specific job. One of these building blocks is known as the M2-1 protein, and previous work has shown that it must interact with other pieces of the virus in order for HRSV to multiply. These interactions include: (1) interactions with the viral genetic material, (2) interactions with other copies of the M2-1 protein, and (3) interactions with another virus protein called P. The interactions occur because the shape of these individual components allows them to fit together very precisely, just like pieces of a very complicated jigsaw puzzle.
The aim of this grant is to determine the shapes of the individual components in very high detail. By doing so, we will be able to design drugs that will interfere with the interaction and therefore prevent the virus from growing and causing disease.
To do this work accurately, we will use a technique called X-ray crystallography, which will allow us to determine the precise shape of the N protein in three dimensions. This technique will reveal all the tiny clefts and crevices that allow these interactions, and critically, it will how us how this interaction could be blocked.

Human respiratory syncytial virus (HRSV) is ubiquitous pathogen that is the major cause of severe respiratory tract infections in infants, the elderly and immunocompromised. Recent studies suggest that each year HRSV is responsible for over 34 million infections and between 66,000 and 199,000 deaths. In addition, HRSV infection early in life may contribute towards increased incidence of asthma later in adulthood. There is no HRSV vaccine. The only available specific treatment regime involves prophylactic administration of a humanized anti-HRSV monoclonal antibody, which although being effective, is expensive. Thus there remains a strong need for alternative and complementary anti-HRSV therapies.
This proposal describes experiments that will investigate the structure and function of the HRSV M2-1 protein, which is an essential component of the viral polymerase. M2-1 is a phosphoprotein, and is known to associate with viral RNA and also the HRSV P protein. The ability of M2-1 to be phosphorylated, and to interact with RNA and P are critical for its function.
Our research will use structural biology techniques paired with functional analysis using biochemical and cell-based assays to reveal critical information regarding the role and activities of M2-1 during the virus life cycle. The research we describe will build on our exciting and extensive preliminary data including the first crystal structure of the M2-1 protein at a resolution of 2.4 Å. We propose to determine the structure of M2-1 bound to both RNA and P protein ligands, and also determine the structural significance of phosphorylation at critical residues. This research program will identify RNA and P binding interfaces on the surface of M2-1, and identify critical residues involved in these processes. This work will identify the structural basis of M2-1 function, and also provide promising targets for structure-based antiviral drug design that can be addressed in future translational research.

Human respiratory syncytial virus (HRSV) constitutes a serious and real threat to human health both in the United Kingdom and globally. HRSV is recognized as the most important viral agent of lower respiratory tract infection worldwide, and a recent study reported that yearly, HRSV causes 34 million lower respiratory tract infections, with 10% requiring hospitalization, resulting in between 66,000 and 199,000 deaths. In addition, recent evidence suggests HRSV infection early in life may contribute towards increased incidence of asthma in adulthood. There is no HRSV vaccine. Current treatment options include prophylactic administration of a humanized HRSV-specific monoclonal antibody, which although being effective, is highly expensive. The broad-range antiviral ribavirin is an option for post-exposure treatment, although its efficacy is marginal. Thus there remains a strong need for alternative and complementary anti-HRSV therapies. Thus characterisation of an essential process in the virus life cycle (processivity of transcription mediated by M2-1) has enormous potential impact on the well being of the UK population.

This grant proposal is rooted in basic science, and primary beneficiaries in the short term will be the UK and worldwide scientific community of virologists, structural and cell biologists, who will be able to build on our data. We anticipate this impact will first occur within year one of the proposal, when our initial M2-1 structure is presented at national and international meetings and published in appropriate high ranking journals. We anticipate high productivity from this grant, and so impact in this manner will continue throughout the entire funding period.

In addition to academic beneficiaries, we anticipate our findings will be utilized by the commercial private sector in order to develop anti-viral chemotherapy tools against HRSV. Ultimately this will enhance the economic competiveness of the UK, improve the skill sets of UK based scientists, and improve or safeguard the health of the UK population. To facilitate the transition from basic research to translational research, the University of Leeds has several mechanisms already in place, including seed funding, dedicated personnel with industrial and knowledge transfer expertise, and previously established commercial/academic partnerships. The ways in which we will exploit these resources is detailed in the 'pathways to impact' document that accompanies this proposal.

The final group of beneficiaries is the public. At the University of Leeds (UoL) and Astbury Centre for Structural Molecular Biology we have strong links with local museums, particularly the Thackray Museum (The Museum of Modern Medicine, Leeds). We regularly work with the museum on biologically and medically relevant science undertaken at the UoL. The most recent being an exhibit on structural molecular biology and its role in discovery and the life of Bill Astbury, a former Professor at UoL who was one of the pioneers of applying X-ray imaging to biological material. In this way the public will be made aware of the efforts of academic and government institutions to safeguard the nation's heath.

For details see the Pathways to Impact