Date Published: April 18, 2019
Publisher: Public Library of Science
Author(s): Rajeev Kaul, Pravinkumar Purushothaman, Timsy Uppal, Subhash C. Verma, Ke Lan.
The oncogenic Kaposi’s sarcoma-associated herpesvirus (KSHV) has two distinct life cycles with lifelong latent/non-productive and a sporadic lytic-reactivating/productive phases in the infected immune compromised human hosts. The virus reactivates from latency in response to various chemical or environmental stimuli, which triggers the lytic cascade and leads to the expression of immediate early gene, i.e. Replication and Transcription Activator (K-RTA). K-RTA, the latent-to-lytic switch protein, activates the expression of early (E) and late (L) lytic genes by transactivating multiple viral promoters. Expression of K-RTA is shown to be sufficient and essential to switch the latent virus to enter into the lytic phase of infection. Similarly, the virus-encoded bZIP family of protein, K8 also plays an important role in viral lytic DNA replication. Although, both K-RTA and K8 are found to be the ori-Lyt binding proteins and are required for lytic DNA replication, the detailed DNA-binding profile of these proteins in the KSHV and host genomes remains uncharacterized. In this study, using chromatin immunoprecipitation combined with high-throughput sequencing (ChIP-seq) assay, we performed a comprehensive analysis of K-RTA and K8 binding sites in the KSHV and human genomes in order to identify specific DNA binding sequences/motifs. We identified two novel K-RTA binding motifs, (i.e. AGAGAGAGGA/motif RB and AGAAAAATTC/motif RV) and one K8 binding motif (i.e. AAAATGAAAA/motif KB), respectively. The binding of K-RTA/K8 proteins with these motifs and resulting transcriptional modulation of downstream genes was further confirmed by DNA electrophoretic gel mobility shift assay (EMSA), reporter promoter assay, Chromatin Immunoprecipitation (ChIP) assay and mRNA quantitation assay. Our data conclusively shows that K-RTA/K8 proteins specifically bind to these motifs on the host/viral genomes to modulate transcription of host/viral genes during KSHV lytic reactivation.
Infectious agents account for approximately 15% of all human cancers per year worldwide [1–4]. Kaposi’s sarcoma-associated herpesvirus or human herpesvirus-8 (KSHV/HHV-8), an oncogenic herpesvirus, is one of the seven DNA tumor viruses associated with several human malignancies. KSHV is the causative agent of Kaposi’s Sarcoma/KS, a common malignancy in HIV/AIDS patients, primary effusion lymphoma/PEL, and multicentric Castleman’s disease/MCD [5, 6]. KSHV’s life cycle involves prolonged, persistent latent phase and a short-lived lytic reactivation. During latency, the quiescent state of KSHV’s life cycle, only a limited set of viral genes/latent genes are expressed with no virion production. The virus can however reactivate from latency in response to different stimuli resulting in the expression of >80 genes culminating in the progeny infectious virions .
KSHV reactivation is an extremely complex process that involves a combination of both viral and cellular factors. KSHV-encoded K-RTA is a master regulator for the lytic reactivation from viral latency. Expression of K-RTA and K-bZIP/K8 is essential for KSHV reactivation. Additionally, K8 directly binds to K-RTA through K-bZIP’s basic domain and a specific K-RTA region to modulate gene expression. To explore the genome-wide binding sites of K-RTA and K8 on KSHV and host cell genomes during KSHV reactivation, chromatin immunoprecipitation of K-RTA and K8 were carried out, followed by parallel sequencing (ChIP-seq) in doxycycline-induced KSHV-positive TRExBCBL-1/RTA stable cells. We identified 21 K-RTA binding sites and 38 K8 binding sites on host genome and 7 K-RTA binding sites and 32 K8 binding sites on the KSHV genome, respectively (Tables 3–6). Our data demonstrated that out of all the K-RTA binding sites identified on host cell genome, majority were either in intergenic region (38%) or within the gene body (24%), whereas remaining were in distal promoter region (38%). Similarly, for all the K-RTA binding sites on KSHV genome, 71% were found in proximal promoter region (-1 to +1 Kb from TSS) whereas 29% were mapped in distal promoter region (-1 to -10 Kb from TSS) (Fig 2A and 2B).