Characterisation of novel virus-host cell interactions essential for herpes simplex virus envelopment

All viruses grow inside the cells that make up the body, manufacturing thousands of copies of themselves that can go on to spread to new individuals. To do this, they hijack cellular machinery to bring together their individual components, assemble them into new viruses, and release these viruses, making them available to infect new cells. A wide range of human viruses, including human immunodeficiency virus, influenza virus and ebola virus are enwrapped in a special membrane coat called an envelope, which is picked up from existing membranes within the cell. Without these envelopes, the viruses are dead, so the ability to block their formation is a realistic and novel target for killing virus infection.

This proposal aims to take one such enveloped virus - the human herpesvirus herpes simplex virus (HSV) to discover the cellular machines that are required for its envelope formation. HSV is an often-disregarded human infection that nonetheless causes significant disease, infecting 24 million annually, and resulting in considerable morbidity and mortality. We aim to use HSV as a model for how viruses make their envelopes, and investigate the individual steps of envelope formation to uncover essential cellular and viral components. However, like all viruses, HSV is minuscule - one 10,000th of a millimetre in diameter - and impossible to see without the help of cutting edge technology. So we will use viruses that we have engineered to shine fluorescently when we look at them with specialist microscopes, and will track their movement through the inside of single cells. Time-lapse movies of travelling virus will be captured under normal conditions and compared to movies captured in cells where individual cell or virus parts are missing. Finally, we will use state-of-the-art microscopes to look deep inside infected cells and produce highly magnified pictures of single viruses as they put themselves together. In this way we aim to identify the building blocks of the machines that are required.

The discoveries that we make will highlight cellular and virus components involved in HSV1 envelope formation. They will also provide information to help understand how other human viruses take advantage of cellular activities. Ultimately, they will lead to the development of new drugs to block the virus envelope - drugs that may be applicable to a wide range of viruses. Our discoveries will benefit the health and wellbeing of the global population and will have a significant impact on the suffering of patients with HSV disease and other virus infections, and the cost of these infections to the health service in the UK and beyond.

Many clinically important human and animal viruses, ranging from human immunodeficiency virus to ebola virus, are enclosed in envelopes derived from cellular membranes. These viruses utilize both the secretory and endocytic transport pathways to direct their envelope proteins to specific sites of virus envelopment. Hence, enveloped viruses subvert vesicle transport in their morphogenesis, and interact with specific cellular factors in the process. These transport factors represent tangible, and potentially broad-spectrum, antiviral targets. This proposal aims to take one such enveloped virus - the human herpesvirus herpes simplex virus (HSV1) - and dissect the essential virus-host interactions involved in its envelopment process. HSV1 is an often-disregarded human infection that nonetheless causes significant disease, infecting 24 million annually, and resulting in considerable morbidity and mortality. Our recent studies showing specific roles for three Rab GTPases in HSV1 trafficking means that we already have a broad understanding of HSV1 envelopment that can act as a model for many other enveloped viruses. We will take three approaches to discover novel factors essential for envelope protein trafficking: (1) a large-scale but rational siRNA screening of cellular transport factors; (2) a directed analysis of Rab6-specific trafficking to the plasma membrane; and (3) a focused proteomic study to find cellular binding partners of gK and UL20, the only two glycoproteins that are essential for envelopment. This work will be underpinned by confocal imaging studies and ultrastructural analyses of virus trafficking. Taken together, the results will provide vital new information on general virus exploitation of cellular trafficking pathways, and will contribute to future rational treatment design, potentially in a broad-spectrum fashion.

Impact on Health and Wellbeing.
The goal of this research is to identify new targets for antiviral treatment of HSV infection which, because of their intervention with cellular pathways, will also have potential application to infections of other clinically important enveloped viruses. Our studies will impact on the health and wellbeing of several groups of patients:

(1) Patients with HSV: HSV is the major cofactor in the transmission of HIV, and so prevention of HSV would be predicted to greatly reduce HIV infection rates. HSV is also the major viral cause of encephalitis, resulting in severe brain damage or death. The ability to block reactivation of HSV could greatly reduce the occurrence of this serious outcome of HSV infection. New treatments of HSV could also lead to a reduction in levels of blindness caused by HSV keratitis. Death or serious disease from neonatal herpes, a result of transmission from mother to baby at birth, could be prevented. Immunosuppressed transplant or cancer pateients will be less susceptible to serious complications of HSV infection. Although not life-threatening, the quality of life of individuals who suffer from frequent reactivation of HSV resulting in cold sores or genital herpes would be greatly improved by new anti-HSV treatment.

(2) Patients with other herpesvirus infections: Because we have shown that other alphaherpesviruses use the same envelopment pathway as HSV, our research will also provide information on how to target other important human herpesvirus infections, such as varicella zoster virus or cytomegalovirus, thereby also contributing to the health of the population.

(3) Patient with other virus infections: In the longer term, targets identified for antiviral intervention in HSV infection could be applied to infections with other important enveloped viruses including HIV, RSV, influenza, dengue and ebola.

Impact on Clinicians
All of the above will impact on the work of clinical virologists, who are involved in treating and caring for individuals with serious complications of HSV, other herpesvirus infections, and in the longer term potentially serious infections with other enveloped viruses.

Impact on Public Engagement
Our proposed research lends itself to engaging with the public and explaining the concepts of virus assembly. We will develop our data into a resource that can be presented on the Internet, or taken into schools to introduce children to current ideas in virology. School-age students will be introduced to virology directly through laboratory experience opportunities in my research group.

Impact on Health Services
Development of new treatments for HSV and potentially other virus infections will have an economic impact on health services both in the UK and worldwide. Fewer patients will present with serious HSV-related illness, or other infections, so therefore fewer medical staff and resources will be required to care for them. A reduction in virus transmission through the population would result in fewer drugs being needed to treat individuals, relieving the economic burden of these drugs on the health service. Globally, a reduction in HIV transmission would reduce the cost to health services of caring and treating HIV patients, and would relieve the economic burden of anti-retroviral drugs.

Impact on Pharma Companies
This project would contribute to the economic competitiveness of the UK through the development of drugs that target envelopment of HSV or other important human and animal virus infections. Our discoveries would be commercialized by existing companies in the UK, with the potential for enhanced wealth generation and employment opportunities.

Impact on Future Virology Research Leaders
The project will employ a new researcher who will be trained in specialist transferable skills such as confocal microscopy, proteomics and siRNA technology. This individual has the potential to become a virology leader of the future.