Single cell analysis to identify genetic factors associated with spontaneous Kaposi's sarcoma-associated herpesvirus (KSHV) reactivation

这个项目的重点是卡波西肉瘤相关疱疹病毒(KSHV)，一种人类肿瘤病毒，卡波西肉瘤(KS)和两种淋巴增生性疾病的发展所需的人类肿瘤病毒。非洲艾滋病的流行使KS成为一种流行性疾病，KS现在是撒哈拉以南非洲最常见的成人肿瘤。此外，KS是实体器官移植后肿瘤发展的常见并发症。目前，还没有特效的KSHV抗病毒药物或疫苗。像所有疱疹病毒一样，KSHV有两个截然不同的生命周期，潜伏期和裂解复制。然而，与其他致癌疱疹病毒不同，潜伏的基因表达驱动癌症的发展，KSHV裂解复制对于KSHV介导的肿瘤发生是必不可少的。因此，研究调节KSHV复活和裂解复制的分子机制对于全面了解KSHV的致病机理具有重要意义。值得注意的是，抑制KSHV的裂解复制是有效的KSHV靶向治疗的关键目标。目的由于KSHV的激活和裂解复制在KSHV介导的肿瘤发生中起着至关重要的作用，令人惊讶的是，KS肿瘤中只有一小部分细胞经历了自发的重新激活，启动了裂解基因的表达。是什么调节了如此小比例的细胞在肿瘤中重新激活，目前尚不清楚。然而，白宫实验室已经证明，在感染KSHV的细胞中观察到基因组不稳定，这表明宿主细胞基因组风险因素可能参与其中(Jackson等人，2014，PLoS Pathogens，10(5)：e1004098)。实验计划为了解决这种可能性，我们将对细胞进行单细胞分析，比较潜伏和自发重新激活KSHV感染的细胞。我们将使用BD FACSseq(最近作为MRC单细胞基因组临床研究能力和技术倡议基金的一部分购买)来选择不同的潜伏和自发重新激活的细胞群体。特定的细胞表面标记将被用来识别潜伏和裂解的种群。将为100个潜伏和100个重新激活的细胞生成单细胞RNA-Seq数据，典型的读取计数为每个细胞100万次读取。在通过BWA与人类参考基因组序列比对后，将使用袖扣和差异表达的转录本来确定原始转录本读取计数，这些转录本与使用Edger从潜伏细胞到重新激活细胞的切换有关。总基因组结构拷贝数变异体(CNV)将通过对从单个细胞基因组扩增的DNA样本创建的全基因组文库进行测序来确定50个潜伏细胞和50个再激活细胞的CNV，每个文库产生约2000万个读数。通过将映射到每个裂解细胞基因组固定间隔的读数的数量与使用内部开发的软件从潜伏细胞的序列数据中确定的值进行比较，可以检测到CNV。拷贝数变化的区域将通过识别在重新激活的与潜伏的细胞池中持续变化的区域来确定。一旦确定了特定的基因变化，将进行RNAi和过度表达研究，通过分析裂解基因表达和感染性病毒粒子的产生来确定这些变化是否增强了感染KSHV的细胞系的自发再激活。因此，该项目将检验这样一种假设，即病毒感染本身不足以促进KS的发展，需要额外的遗传辅助因素。

This project focuses on Kaposi's sarcoma-associated herpesvirus (KSHV), a human tumour virus required for the development of Kaposi's sarcoma (KS) and two lymphoproliferative disorders. The African AIDs epidemic has turned KS into an epidemic disease and KS is now the most common adult tumour in sub-Saharan Africa. Moreover, KS is a frequent complication in cancer development after solid organ transplantation. At present, there is no specific KSHV antiviral or vaccine. Like all herpesviruses KSHV has two distinct life cycles, latency and lytic replication. However, unlike other oncogenic herpesviruses, where latent gene expression drives cancer development, KSHV lytic replication is essential for KSHV-mediated tumourigenesis. Therefore, it is important to study the molecular mechanisms which regulate KSHV reactivation and lytic replication to fully understand KSHV pathogenesis. Notably, inhibiting KSHV lytic replication is a key goal for efficacious KSHV-targeted therapeutics. Aims As KSHV reactivation and lytic replication play a vital role in KSHV-mediated tumourigenesis, it is rather surprising that only a small percentage of cells in the KS tumour undergo spontaneous reactivation initiating lytic gene expression. What regulates such a small percentage of cells to reactivate in the tumour is unknown. However, the Whitehouse lab has demonstrated that genomic instability is observed in KSHV-infected cells which suggests that host cell genomic risk factors may be involved (Jackson et al., 2014, PLoS Pathogens, 10(5): e1004098). Experimental Plan To address this possibility we will perform single cell analysis on cells comparing latent versus spontaneously reactivating KSHV-infected cells. We will select distinct populations of latent and spontaneously reactivating cells using a BD FACSseq (recently purchased as part of the MRC Clinical Research Capabilities and Technologies Initiative Grant for Single Cell Genomics). Specific cell surface markers will be used to identify latent and lytic populations. Single cell RNA-Seq data will be generated for 100 latent and 100 reactivating cells to a typical read count of 1 million reads per cells. After alignment to the human reference genomic sequence by BWA, raw transcript read counts will be determined using Cufflinks and differentially expressed transcripts linked to the switch from latent to reactivating cells using EdgeR. Gross genomic structural copy number variants (CNV) will be determined in 50 latent and 50 reactivating cells, by the sequencing of whole genome libraries created from genome amplified DNA samples from single cells to generate approximately 20 million reads per library. CNVs will be detected by comparing the number of reads mapping to fixed intervals along the genome in each lytic cell to values determined from sequence data from latent cells using software developed in house. Regions of copy number variation will be determined by identifying regions that are consistently altered across the pool of reactivated versus latent cells. Once specific genetic changes have been identified RNAi and overexpression studies will be performed to "reverse" the effect to determine if these changes enhance spontaneous reactivation in KSHV-infected cell lines, monitored by analysing lytic gene expression and infectious virion production. This project will therefore test the hypothesis that viral infection per se is not sufficient for KS development and that additional genetic cofactors are required.