Characterising the Mode of Action of VP22 - a Novel Herpes Simplex Virus Virulence Factor Important for In Vivo Replication.

Herpes simplex virus (HSV) is well known for its role in oral and genital herpes. However, it is not widely publicised that infection with HSV can also have some very serious outcomes. For example, it has the ability to cause extreme brain damage, blindness or even death. It is also a major driving force behind HIV infection. Once HSV has infected the human body it cannot be eliminated, and lies dormant in the nervous system for life, ready to strike again with new symptoms when the person is especially vulnerable. People, whose immune systems are suppressed, such as transplant, cancer chemotherapy, and AIDS patients, are particularly susceptible to complications of HSV. With 24 million new cases globally each year, this virus is a huge economic burden on our health systems. However, despite extreme effort from the pharmaceutical industry, no successful HSV vaccine has yet been developed. Moreover, although drug treatments for HSV have existed for decades, their use has made no impact on this HSV epidemic. It is therefore vital that new HSV treatments are found.

To develop new treatments, it is important to understand how HSV causes disease. When a virus enters a cell in the body, a battle begins between the attacking virus and the defending cell to see which one can win. If the virus wins, the result is disease. During this process, both the cell and the virus attack each other with proteins they have made. In our work to investigate how HSV causes disease, we have discovered that one virus protein called VP22, which is not needed for the virus to grow well in cells of animal origin, is required for the virus to grow in natural cells of human origin. We propose that VP22 is required because it attacks a particular defence mechanism that only the human cells can put in place during HSV infection, and that in the absence of VP22, this defence mechanism wins. Using state-of-the art, large-scale experimentation, we will identify all cellular genes that are switched on in HSV infected cells when VP22 is missing, to investigate the cellular defence mechanisms that VP22 might block. We will then find out if VP22 binds specific cellular proteins and work out if this is the method by which VP22 allows HSV to grow in these human cells. Finally, as VP22 is packaged into the structure of the virus, we will ask if the virus delivers a powerful attacker of cellular defence mechanisms at the very first stage of virus entry into a cell.

If we prove that VP22 is a potent inhibitor of a cellular defence in human cells, this will open up new avenues for HSV vaccine and drug development. In the case of vaccines, it is conceivable that virus lacking VP22 will be sufficiently weak to cause limited disease while invoking a good immune response to protect against future infections in the human. Furthermore, if we discover how VP22 functions, it will be possible to develop drugs that bind and inhibit the activity of VP22, thereby stopping the ability of HSV to cause disease. Our research will benefit the health and wellbeing of the global population - those who suffer serious complications from HSV infection, those who contract HIV as a result of genital HSV infection, and those that suffer frequent and debilitating outbreaks of cold sores or genital herpes. Ultimately, this work will have a significant impact on both the suffering of patients with HSV and the cost of HSV infection to the health service in the UK and beyond.

Herpes simplex virus (HSV) is an often-disregarded human infection that nonetheless causes considerable disease, infecting 24 million annually, and resulting in significant morbidity and mortality. No vaccine has yet been developed for HSV, and although HSV antivirals have existed for decades, their use has made no impact on HSV infection rates. This proposal concerns a major structural protein of HSV named VP22, and its role in pathogenesis. Work in animal models has shown that HSV replication in the host is severely attenuated in the absence of VP22, suggesting that this protein is important for disease. Paradoxically, VP22 is not required for HSV replication in tissue culture, and despite much work, its role in virus infection has remained elusive. However, we have recently found that VP22-negative HSV is restricted for growth in primary human fibroblasts, and this cell type is therefore an excellent model to uncover the activity of VP22. Unlike most immortalised cells, primary fibroblasts are fully competent for antiviral responses, leading us to hypothesize that VP22 is required to counteract an antiviral response. We propose to use transcriptomic analysis to determine how human fibroblasts respond to HSV infection in the absence of VP22. We will also use proteomic analysis of VP22-containing complexes from infected fibroblasts to identify candidate factors through which VP22 may act to overcome cellular restriction. Potential partners of VP22 will be confirmed by in vitro interaction studies, and their role in virus restriction determined by RNAi depletion studies. Finally, we will assess the ability of varicella zoster virus and bovine herpesvirus 1 homologues of VP22 to rescue the restriction of VP22-negative HSV, and test the species specificity of VP22. By determining the mode of action of VP22 and enhancing our understanding of the HSV disease process, this work will contribute to future rational approaches to vaccine and antiviral design for HSV.

Impact on Health and Wellbeing
The goal of this research is twofold - to develop new treatments for HSV infection and to engineer more efficient oncolytic HSV for treatment of cancers. If VP22 proves to be a good target for inhibition of HSV replication in humans, then our studies will impact on the health and wellbeing of several groups of HSV infected patients:
1. Prevention of HSV spread would be predicted to greatly reduce HIV infection rates, particularly in Africa. As a consequence, deaths from HIV would be significantly reduced.
2. The ability to block reactivation of HSV could greatly reduce the occurrence of fatal encephalitis - a serious outcome of HSV infection.
3. New treatments of HSV could lead to a reduction in levels of blindness caused by HSV keratitis.
4. Death or serious disease from neonatal herpes, a result of transmission from mother to baby at birth, could be prevented.
5. Immunosuppressed transplant or cancer pateients will be less susceptible to serious complications of HSV infection.
6. 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.
Our research may also provide information on how to target other important human herpesvirus infections, such as varicella zoster virus, thereby further contributing to the health of the population.

If knocking out VP22 proves to create an oncolytic virus that is highly selective for replication in tumour cells, then our work will impact on future treatments for cancer patients.

Impact for 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 and other herpesvirus infections, and clinicians treating transplant patients and cancer patients. It will also impact on the work of oncologists treating patients with cancer.

Impact on Public Engagement
Our proposed research lends itself to engaging with the public and explaining how humans fight virus infection. 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, 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 for individuals with recurrent cold sores or genital herpes, 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.
New treatments for cancer will also have an economic impact on health services both in the UK and worldwide.

Impact on Pharma Comapanies
This project would contribute to the economic competitiveness of the UK through the development of drugs that target the activity of VP22, attenuated vaccines lacking the VP22 gene, or oncolytic viruses with enhanced selectivity for cancer cells. Our discoveries would be commercialized by existing companies in the UK, with the potential for enhanced wealth generation and employment opportunities.

Impact on Future Research Leaders
The postdoctoral researcher employed on this project will be trained in new transferable skills such as student supervision, scientific writing and presentations. This individual will have the have the potential to become a research leader of the future.