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

卡波西肉瘤相关疱疹病毒（KSHV）是在免疫抑制受试者中引起特定发病率和死亡率的人类病原体。KSHV导致三种类型的癌症。其中之一，卡波西氏肉瘤，是一种内皮细胞肿瘤，它排列在血管和淋巴管的内表面。KSHV感染的严重健康并发症通常见于HIV阳性个体，其免疫系统受到抑制，无法正常发挥功能。例如，在没有采取有效的抗HIV治疗的艾滋病患者中，卡波西肉瘤经常扩散到肺或胃肠道等器官，并且具有很高的死亡率。当细胞感染KSHV时，绝大多数细胞都携带所谓的潜伏或休眠病毒。当病毒重新激活时，KSHV会产生新的病毒颗粒，这些病毒颗粒会传播感染邻近的细胞，并最终感染其他个体。作为伪装大师，KSHV特别擅长逃避宿主免疫系统的识别。例如，许多KSHV基因负责保护受感染的细胞免受宿主淋巴细胞的识别和杀伤。我们在这个项目中的目的是使用定量蛋白质组学技术，以确定如何在内皮细胞和B细胞的蛋白质被KSHV改变，以确定介导这些变化的病毒基因，并了解它们如何影响宿主的免疫系统，并有助于KSHV感染的发病机制。我们的初步结果已经发现了一些新的KSHV靶点-参与重要细胞过程如免疫防御和细胞粘附的宿主蛋白。我们现在希望获得一个定量的公正的概述所有的蛋白质调制后KSHV，在潜伏期和裂解期感染。我们将获得一套全面的病毒诱导的KSHV感染细胞蛋白质的变化，并进一步研究这些新靶点的选定子集。目前没有KSHV特异性药物治疗或疫苗。事实上，目前卡波西肉瘤的治疗主要依赖于抗逆转录病毒和细胞抑制药物用于AIDS-KS治疗。在阻断其他疱疹病毒（即人巨细胞病毒和单纯疱疹病毒）复制的药物中观察到有限的KSHV疗效。未来针对KSHV的治疗策略的目标是通过从潜伏期重新激活病毒并同时阻断裂解期感染来杀死潜伏感染的细胞。因此，在我们的研究中鉴定的可能在维持病毒潜伏期中起作用的细胞蛋白质（即被KSHV降解以促进再活化的那些蛋白质）可以作为这种治疗方法的靶点。此外，全面鉴定新的KSHV靶标和免疫逃避策略将为开发新的抗病毒药物提供途径。

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.