Quantitative proteomic and CRISPR-based genetic approaches to latent and active KSHV infection

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Kaposi's sarcoma associated herpesvirus (KSHV) is a human pathogen that causes particular morbidity and mortality in immunosuppressed subjects. KSHV causes three types of cancer. One of these, Kaposi's sarcoma, is a tumour of endothelial cells, which line the interior surface of blood and lymphatic vessels. Severe health complications of KSHV infection are often seen in HIV-positive individuals, whose immune system is so suppressed it does not function correctly. For instance, in AIDS patients who are not taking effective anti-HIV treatment, Kaposi's Sarcoma often spreads to organs such as the lungs or gastrointestinal tract and has a high mortality. When cells become infected with KSHV, the vast majority harbour so-called latent or dormant virus. Upon viral reactivation, KSHV makes new virus particles, which spread to infect neighbouring cells and ultimately, to other individuals. As a master of disguise, KSHV is particularly adept at evading recognition by the host immune system. For example, many KSHV genes are responsible for protecting infected cells from recognition and killing by host lymphocytes. Our aim in this project is to use quantitative proteomic techniques to determine how proteins in endothelial and B cells are altered by KSHV, to identify the viral genes which mediate these changes and to understand how they affect the immune system of the host and contribute to the pathogenesis of KSHV infection. Our preliminary results have uncovered a number of novel KSHV targets - host proteins that are involved in important cellular processes such as immune defence and cell adhesion. We now want to gain a quantitative unbiased overview of all the proteins modulated following KSHV, in both latent and lytic-phase infection. We will obtain a comprehensive set of virus-induced changes in the proteins of KSHV-infected cells and further characterise a selected subset of these novel targets. No KSHV-specific drug therapies or vaccines are at present available. Indeed the current therapy for Kaposi's Sarcoma relies mostly on anti-retroviral and cytostatic drugs for AIDS-KS treatment. Limited KSHV efficacy is seen in drugs that block replication of other herpesviruses (i.e Human Cytomegalovirus and Herpes Simplex virus). The goal of future therapeutic strategies against KSHV is to kill latently infected cells by reactivating the virus from latency and simultaneously blocking lytic stage infection. Cellular proteins identified in our study which likely play a role in the maintenance of virus latency (i.e. those that are degraded by KSHV to facilitate reactivation) may therefore serve as targets for such therapeutic approaches. Furthermore, a comprehensive identification of novel KSHV targets and immune evasion strategies will provide a route towards the development of novel antiviral drugs.