KSHV genome harbors both constitutive and lytically induced enhancers

Kaposi's sarcoma-associated herpesvirus (KSHV) belongs to the gamma-herpesvirus family and is a well-known human oncogenic virus. In infected cells, the viral genome of 165 kbp is circular DNA wrapped in chromatin. The tight control of gene expression is critical for latency, the transition into the lytic phase and the development if viral-associated malignancies. Distal cis-regulatory elements (CRE), such as enhancers and silencers, can regulate gene expression in a position and orientation-independent manner. Open chromatin is another characteristic feature of enhancers. To systematically search for enhancers, we cloned all the open chromatin regions in the KSHV genome downstream to the luciferase and tested their enhancer activity in infected and uninfected cells. A silencer was detected upstream of the latency promoter (LANAp). Two constitutive enhancers were identified in the K12p-OriLyt_R and ORF29 Intron, where the latter is a tissue-specific enhancer. The following promoters: OriLyt_L, PANp, ALTp and the Terminal Repeats (TRs) acted as lytically induced enhancers. Expression of the Replication and Transcription Activator (RTA), the master regulator of the lytic cycle was sufficient to induce the activity of the lytic enhancers in the uninfected cells. We propose that the TRs that span about 24kbp region, serve as a 'viral super-enhancer' integrating the repressive effect of the latency protein LANA with the activating effect of RTA.To find out the functional significance of the KSHV genomic enhancers, rKSHV.219 infected SLK cells (SLK.219) were transduced with lentiviruses expressing dCas9 tethering either strong activation domain (CRISPRa; dCas9-10XSunTag and ScFv-2ERT2-VPH) or strong repression domain (CRISPRi; dCas9-KRAB-MeCP2) along with specific sgRNAs targeting the viral enhancers as follows: sg_5 for OriLyt_L, sg_6 for PANp, sg_8 for ORF29 Intron, sg_17 for K12p-OriLyt_R-ALTp and sg_21 for TRs. To induce CRISPRa or CRISPRi, the transduced cells were treated with 4-hydroxytamoxifen or doxycycline respectively. Additionally, during CRISPRi, the cells were treated with TPA for KSHV lytic reactivation. The cells were harvested upon the completion of the treatments and followed by total RNA isolation and RNA sequencing.