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

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.

Kaposi's Sarcoma associated herpesvirus (KSHV) is the causative agent of three human malignancies: Kaposi's sarcoma, a tumour of endothelial cells together with B cell-associated primary effusion lymphoma (PEL) and multicentric Castelman's disease. Kaposi's Sarcoma often leads to life-threatening complications in immunocompromised, particularly HIV-infected individuals. Like other herpesviruses, infection with KSHV is persistent and lifelong, with the virus remaining latent with occasional reactivations. KSHV must therefore remodel the host cell proteome to enable its lifestyle: in latent infection, changes are particularly related to angiogenesis and cell proliferation, while upon reactivation the virus targets host immune molecules to evade recognition from the host immune system. In a preliminary proteomic analysis we identified a number of novel KSHV targets, suggesting that our understanding of KSHV-induced pathogenesis and viral immune evasion strategies are far from complete. Here we propose to apply quantitative proteomic approaches to gain a comprehensive and unbiased overview of how KSHV remodels the host cell proteome in latency and the temporal changes that occur upon viral reactivation. We will initially focus on endothelial cells and then examine B cells, two physiologically relevant cell types for KSHV infection. We will use a novel KSHV genome-wide CRISPR library screening approach to identify the viral genes responsible for the observed phenotypic cellular changes and to understand their mechanism of action. We will assess the impact of these changes on the maintenance of virus latency, the ability of the virus to manipulate the host immune system, and KSHV-induced pathogenesis. Our goal is to generate and disseminate important findings and knowledge about infection with KSHV. This will stimulate further research in this area, and in the longer term will inform the design of novel therapeutic strategies for the treatment of KSHV-associated disease.

We foresee that the proposed project will have an impact on the following parties from academic, commercial and professional sectors:

Academic sector: UK-based and international virologists and immunologists, including, but not limited to, researchers from herpesvirus community; medical doctors working on cancer-related topics. The impact will be achieved mainly through bringing advancement to the fields by: (1) applying the proposed approaches to other viruses (e.g. proteomics and CRISPR-based genetic tools); (2) using datasets from the study as a source material for initiation of new studies (characterization of the novel virus-host protein-protein counteraction); (3) investigation of the newly identified players in KSHV pathogenesis in the broader cancer-related settings, e.g. non-KSHV-induced angiogenesis.

Commercial private sector: UK-based and international biotechnological companies: (1) those that focus on discovery of the novel drug targets and biomarkers. The newly identified immune evasion strategies as well as proteins involved in KSHV reactivation may represent novel drug targets. A comparative analysis of the proteomes of a healthy and KSHV-infected cell is likely to lead to discovery of novel molecules that can represent potential biomarkers; (2) companies that commercialise CRISPR/Cas9-based approaches through: (i) advancing functional genomics tools; (ii) developing therapeutic strategies to target latent viruses in human cells. These novel tools along with potential drug targets and biomarkers can be further investigated and commercially exploited by biotech companies. Introduction of the novel therapeutic strategies will have impact on public health in UK and, if UK-based companies are involved, it will also have a direct impact on the economic competitiveness of UK, for example, through creation of new jobs and profits from international sales. We estimate realistic timelines for such commercial exploitation to occur within five to ten years upon access of the companies to the datasets .

Professional/educational sector: Professional staff (postdoctoral fellows and PhD students) from groups of Paul Lehner and Thomas Schulz, either directly (through performing experiments) or indirectly (participating in discussion of results) involved in the project, will benefit through learning new methods and state-of-the-art techniques and broadening their knowledge in molecular virology. This, in turn, will have an impact on their future career both in academic and industrial sectors, e.g. by enabling them to move to a higher managerial or teaching position within company or University. Postdoctoral fellows will also improve their communication skills through presenting work at local and international meetings.