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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 of viral-associated malignancies. Distal cis-regulatory elements, 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 of the luciferase gene and tested their enhancer activity in infected and uninfected cells. A silencer was detected upstream of the latency-associated nuclear antigen promoter. Two constitutive enhancers were identified in the K12p-OriLyt-R and ORF29 Intron regions, where ORF29 Intron is a tissue-specific enhancer. The following promoters: OriLyt-L, PANp, ALTp, and the terminal repeats (TRs) acted as lytically induced enhancers. The expression of the replication and transcription activator (RTA), the master regulator of the lytic cycle, was sufficient to induce the activity of lytic enhancers in uninfected cells. We propose that the TRs that span about 24 kbp region serve as a “viral super-enhancer” that integrates the repressive effect of the latency-associated nuclear antigen (LANA) with the activating effect of RTA. Utilizing CRISPR activation and interference techniques, we determined the connections between these enhancers and their regulated genes. The silencer and enhancers described here provide an additional layer to the complex gene regulation of herpesviruses. IMPORTANCE In this study, we performed a systematic functional assay to identify cis-regulatory elements within the genome of the oncogenic herpesvirus, Kaposi’s sarcoma-associated herpesvirus (KSHV). Similar to other herpesviruses, KSHV presents both latent and lytic phases. Therefore, our assays were performed in uninfected cells, during latent infection, and under lytic conditions. We identified two constitutive enhancers, one of which seems to be a tissue-specific enhancer. In addition, four lytically induced enhancers, which are all responsive to the replication and transcription activator (RTA), were identified. Furthermore, a silencer was identified between the major latency promoter and the lytic gene locus. Utilizing CRISPR activation and interference techniques, we determined the connections between these enhancers and their regulated genes. The terminal repeats, spanning a region of about 24 kbp, seem like a “viral super-enhancer” that integrates the repressive effect of the latency-associated nuclear antigen (LANA) with the activating effect of RTA to regulate latency to lytic transition. [ABSTRACT FROM AUTHOR]

The Kaposi's sarcoma-associated herpesvirus latency-associated Herpesviru nuclear antigen (LANA) protein interacts with glycogen synthase kinase 3 (GSK-3) and relocalizes GSK-3 in a manner that leads to stabilization of β-catenin and upregulation of β-catenin-responsive cell genes. The LANA-GSK-3 interaction was further examined to determine whether there were additional downstream consequences. In the present study, the nuclear GSK-3 bound to LANA in transfected cells and in BCBL1 primary effusion lymphoma cells was found to be enriched for the inactive serine 9-phosphorylated form of GSK-3. The mechanism of inactivation of nuclear GSK-3 involved LANA recruitment of the extracellular signal-regulated kinases 1 and 2 (ERK1/2) and the ribosomal S6 kinase 1 (RSK1). ERK1/2 and RSK1 coprecipitated with LANA, and LANA was a substrate for ERK1 in vitro. A model is proposed for the overall inactivation of nuclear GSK-3 that incorporates the previously described GSK-3 phosphorylation of LANA itself. Functional inactivation of nuclear GSK-3 was demonstrated by the ability of LANA to limit phosphorylation of the known GSK-3 substrates C/EBPβ and C/EBPα. The effect of LANA-mediated ablation of C/EBP phosphorylation on differentiation was modeled in the well-characterized 3T3L1 adipogenesis system. LANA-expressing 3T3L1 cells were impaired in their ability to undergo differentiation and adipogenesis. C/EBPβ induction followed the same time course as that seen in vector-transduced cells, but there was delayed and reduced induction of C/EBPβ transcriptional targets in LANA-expressing cells. We conclude that LANA inactivates nuclear GSK-3 and modifies the function of proteins that are GSK-3 substrates. In the case of C/EBPs, this translates into LANA-mediated inhibition of differentiation. [ABSTRACT FROM AUTHOR]