Modification of the host lipidome by Rhinovirus infection

Picornaviruses are a family of small viruses that are important pathogens in animals and man. Members of the family include polioviruses, foot and mouth disease virus, hepatitis A virus and the common cold viruses (rhinoviruses). Rhinovirus (RV) infections are the most common infection afflicting mankind and there are no effective therapies or vaccines. Although the symptoms of the common cold are usually mild in healthy individuals, common colds are still an enormous burden to society. In patients with asthma or Chronic Obstructive Pulmonary Disease (COPD) RV infections can provoke acute worsening of their disease. This is a major cause of serious illness and death and represents an enormous health care cost. Viruses need to hijack the host cell's biological systems in order to replicate. RV like all picornaviruses are very simple but despite years of study there is still relatively little known about how they interact with the host cell's biological systems in order to replicate themselves, or how they get around the host's defence mechanisms. Following entry of the virus into a host cell, the viral genome is translated into a single long protein that subsequently gets cut into the 4 structural coat proteins and 7 non-structural proteins that participate in viral replication. These 7 non-structural proteins perform the functions required to replicate the viral genome and to assemble new virus particles inside the host cell. In order to perform these functions, the viral proteins interact with proteins and biological systems in the host cell to turn the cell into a factory for producing virus. Understanding how these 7 viral proteins turn the host cell into a virus producing factory is crucial if we are to discover drugs that block the virus. Historically we have tried to make antiviral drugs that target the virus, but viruses are very good at mutating to escape the effects of drugs targeted at the virus itself. However some of the host proteins that the virus hijacks may be drug targets themselves. The advantage being that the virus may not easily be able to mutate to avoid the effects of drugs targeted at a host protein. The aim of the project is to find host targets to which we can make antiviral drugs.

To do this we must first define how the virus replicates on membranes inside the cell. These membranes are made of lipids (or fats) and proteins and we believe the virus changes the lipid and protein composition of these membranes for its replication. Experimentally we will infect cultured human lung cells with the virus and then extract fats from the infected cells and measure any changes in fat content using state of the art biochemistry methods. Using computational techniques we anticipate that these results will tell us about host systems essential for viral replication. We can confirm these results by knocking out these host systems or using chemical inhibitors to block them and measuring the effect on viral replication. As we are working in a hospital setting we will have the ability in future to see if these biological systems are disturbed in patients with asthma or COPD, so translating our findings from the lab to the bedside. If the results look promising, we will start drug discovery programmes based on our findings, either ourselves or by partnering with Industry. The project is a partnership between Imperial College and the Babraham Institute and will create a multi-disciplinary team across two centres of excellence tackling a challenging biological problem of major medical importance.

This project aims to examine the effects of Rhinoviruses (RV) upon lipid metabolism in human primary bronchial epithelial cells (BECs) isolated from four distinct healthy individuals. BECs will be infected in triplicate at a high MOI of HRV16 under optimised conditions that give maximal and synchronised infection. A 6 point time course will be performed and the lipids extracted and identified and quantified by HPLC-MS/MS thereby determining the cellular lipidomes, including the signalling lipids. Bioinformatic analysis will identify virally-induced changes in individual lipid molecular species and further through pathway analysis we will identify which enzyme activities have been affected. Since many of these changes will affect cellular membranes, effects upon the secretory pathway will be examined using cell biological methodologies. Using this data we will identify candidate host targets we believe to be important for viral replication and the host inflammatory response. These will be validated by siRNA knockdown, overexpression or the use of small molecule inhibitors where available. As part of our ongoing studies we can analyse samples and data from human healthy and asthma volunteers infected with RV to confirm pathway involvement.

Who will be impacted?
a) Our studies will generate new data describing the interaction between a relevant human cell and a pathogenic virus. This will benefit academic virologists and cell biologists. Because RV infection represents a serious health burden to asthmatics and COPD patients, this research will be of interest to patients and respiratory clinicians.
b) 50% of the health care costs associated with asthma are due to exacerbations caused by RV infection. Respiratory viral infections, particularly RV, interact powerfully with allergy in early life, substantially increasing (3 to 30-fold) the risk of development of asthma and allergies later in life. Currently there are no vaccines or chemotherapy to treat this virus and discovery of novel targets would represent a significant impact to Industry who will benefit from the discovery of new drug targets and patients would benefit from new efficacious drugs.
c) Because RV is related to other important pathogens discoveries made may be relevant for other pathogens with impact to both human and animal health.
d) Combining our ongoing transcriptomics and proteomics with lipidomics is a powerful systematic approach to studying complex biological problems. If successful we believe combining such orthogonal methodological approaches could become widely adopted.
e) The postdoctoral researchers employed on the grant will learn new skills and expertise, which will be utilised in this research programme and benefit their future, thus enabling the transfer of knowledge between individuals and institutes. They will be offered the opportunity to take part in personal and research specific training courses to promote their professional development, in keeping with the principles set out in the Concordat to Support the Career Development of Researchers. Additionally, they will share their data at lab meetings, present research at international conferences and be encouraged to take part in public engagement activities.

Defining the route to impact.
i) The targets we discover will be overexpressed or knocked out (siRNA) in human BECs and assessed for their ability to halt viral replication and their impact on the host inflammatory and anti-viral response. Any previously known chemical modifiers of these targets will also be tested at this stage.
ii) Beyond this 3 year grant, targets may be assessed in a variety of mouse models of RV infection developed in the Johnston lab. If there is already an appropriate small molecule against the target, this will be done pharmacologically. We may also use genetic means to knock out or overexpress the in vitro validated target.
iii) Findings from the proposed in vitro studies can be directly translated to humans. As part of ongoing studies the Imperial group has stored samples from healthy and asthmatic volunteers experimentally infected with RV. Changes in lipid metabolism can be analysed in epithelial cells and changes in lipid mediators in BAL.
iv) If the in vitro or in vivo validated target is novel and considered "druggable" we will initiate a small molecule drug discovery campaign. RS has over 25 years' experience in small molecule drug discovery. Small molecule inhibitors or activators of the target will be used to test the hypothesis both in vitro and in vivo. At this stage it may be possible to partner with a Pharma company or create a spinout company based on these discoveries.
v) If the validated target is known and there is already an approved medicine against that target, we could progress to human clinical studies. Johnston has substantial experience of RV challenge clinical trials.
vi) RS has significant experience in creating impact from basic research. As well as working in the Pharma industry and having taken targets from discovery phase through to clinical trials, he has been involved with the creation of 4 start-up biotechnology companies in the UK and was CEO of MRCT