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)是一种人类病原体，在免疫抑制的受试者中会导致特殊的发病率和死亡率。KSHV导致三种类型的癌症。其中一种叫卡波西肉瘤，是一种内皮细胞瘤，排列在血管和淋巴管的内表面。KSHV感染的严重健康并发症经常见于艾滋病毒阳性者，他们的免疫系统受到如此抑制，以至于无法正常运作。例如，在没有采取有效抗艾滋病毒治疗的艾滋病患者中，卡波西肉瘤经常扩散到肺部或胃肠道等器官，死亡率很高。当细胞感染KSHV时，绝大多数细胞携带所谓的潜伏或休眠病毒。在病毒重新激活时，KSHV会产生新的病毒颗粒，这些病毒颗粒会传播到邻近的细胞，并最终传播给其他人。作为伪装高手，KSHV尤其擅长躲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。我们的目标是利用定量蛋白质组学技术来确定KSHV如何改变内皮细胞和B细胞中的蛋白质，以确定介导这些改变的病毒基因，并了解它们如何影响宿主的免疫系统，并在KSHV感染的发病机制中发挥作用。我们的初步结果揭示了一些新的KSHV靶点-宿主蛋白，参与重要的细胞过程，如免疫防御和细胞黏附。我们现在想要对KSHV之后所有在潜伏期和裂解期感染中调节的蛋白质进行一个定量的、公正的概述。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，并进一步表征这些新靶点的精选子集。目前还没有针对KSHV的药物疗法或疫苗。事实上，目前对卡波西氏肉瘤的治疗主要依赖于抗逆转录病毒和细胞抑制药物来治疗艾滋病-KS。在阻止其他疱疹病毒(即人类巨细胞病毒和单纯疱疹病毒)复制的药物中，KSHV的疗效有限。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止裂解期感染来杀死潜伏感染的细胞。因此，在我们的研究中发现的可能在维持病毒潜伏期(即那些被KSHV降解以促进重新激活的蛋白)中可能起作用的细胞蛋白可能成为这种治疗方法的靶标。此外，全面识别新的KSHV靶点和免疫逃避策略将为开发新型抗病毒药物提供一条途径。

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.