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Identification of novel KSHV immune evasion mechanisms using a quantitative temporal viromics analysis

Periodic Reporting for period 1 - KSHV QTV (Identification of novel KSHV immune evasion mechanisms using a quantitative temporal viromics analysis)

Reporting period: 2015-06-15 to 2017-06-14

Kaposi’s Sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi’s sarcoma and two B cell malignancies: primary effusion lymphoma and multicentric Castelman’s disease. Kaposi’s Sarcoma is a tumour of endothelial cells which is inactive in subjects with healthy immune system, but disseminates as an aggressive tumour in immunosuppressed e.g. HIV-infected individuals, particularly affecting the lungs and gastrointestinal tract. In fact, Kaposi’s Sarcoma is one of the commonest tumours of AIDS patients, with an often fatal outcome. The number of KSHV-seropositive individuals varies depending on geographical location, with a maximum of more than 50% in sub-Saharan Africa, where Kaposi’s Sarcoma is the second largest contributor to the cancer burden (Plummer, Lancet 2016). Investigation of the molecular mechanisms of KSHV infection will contribute to our knowledge on KSHV pathogenesis and may open new perspectives for therapeutic treatment of KSHV-associated malignancies.
KSHV manipulates its host cell environment to enable replication and evade the host immune response, but these changes have been predominantly analysed at the transcriptional level. Changes in mRNA may not accurately reflect changes in cellular proteins, nor do they reflect the post-translational changes (such as degradation) induced by viral infection. How KSHV infection alters cellular proteins has only been analysed at the individual protein level or through proteomic studies of individual viral genes e.g. K5. During lytic stage infection, KSHV mediates degradation of several host proteins, including adhesion molecules, immune receptors, transcription and restriction factors, but an unbiased systematic analysis of the KSHV-induced changes in the host cell proteome is lacking. Therefore, the goal of this study was to perform a quantitative proteomics analysis of KSHV-infected cells to unravel virus-induced changes in host cells and determine how these changes impact the host immune system.
The objectives of this project were to: (1) establish a KSHV reactivation model in human endothelial cells, a physiologically relevant cell type for KSHV infection; (2) apply systematic and unbiased approaches to resolve changes in both the host and viral proteomes in endothelial cells upon KSHV reactivation; (3) identify host molecules modulated (i.e. degraded) by KSHV and (4) investigate these novel aspects of virus-host interaction.
For the reported period (24 months) the following results were obtained:
- A proteomics-compatible model for KSHV reactivation from human endothelial cells has been established;
- 69 viral proteins (85% of KSHV proteome) were resolved, including predicted, but previously undetected open reading frames (Fig.1);
- In total, 7300 host cell proteins have been resolved;
- 182 host cell surface and cytosolic proteins are found to be more than 2-fold downregulated, 25 proteins are known targets of KSHV K5, the main immunomodulatory KSHV protein (Fig.2);
- Validation and preliminary characterisation of novel targets has been performed;
- The results of this study were presented at the international KSHV workshop in Berlin.
This study has opened up a number of important leads. It is ongoing and I am currently focused on the following aspects of this project: (1) characterisation of the newly identified targets; (2) Identification of the viral gene(s) responsible for the observed phenotypes; (3) Analysis of the impact of the observed changes on viral reactivation and modulation of the host’s immune response.
At the end of the project I expect to obtain the following results:
- The key phenotypic changes in host cell proteome will be attributed to the responsible viral genes;
- A comprehensive investigation of the novel virus-host interactions will have been performed;
- The impact of the observed changes on viral reactivation and modulation of the host’s immune response will be analysed;
- At least one major publication in high-impact peer-reviewed, open access journal will be submitted;
- An interactive open access database for both the KSHV proteome and host cell proteins manipulated by the virus, will be generated and uploaded to a publicly available repository.
The study is ongoing and the results will significantly advance our understanding of how KSHV manipulates the host cell,. It has the potential to enable development of novel therapeutic strategies to combat KSHV-induced malignancies. Therefore this study has the capacity to improve human health in the areas with high KSHV seroprevalence, such as Mediterranean Europe and Sub-Saharan Africa. Furthermore, the properties of the newly identified KSHV-encoded immune evasins may be further developed in an industrial partnership within Europe. Commercialization of these findings will enrich the European economy and help the EU remain competitive within the global market.