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

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.