Abstract
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.
Highlights
Infectious agents account for approximately 15% of all human cancers per year worldwide [1,2,3,4]
In order to identify the genome-wide K-RTA binding sites on the Kaposi’s sarcoma-associated herpesvirus (KSHV) and human genomes, chromatin immunoprecipitation (ChIP) with K-RTA and generation sequencing (ChIPseq) of the DNA was performed on KSHV-infected tetracycline-inducible TRExBCBL-1/RTA stable cells
Mapping of the K-RTA Chromatin Immunoprecipitation (ChIP) reads to the host cellular genome showed specific binding and a total of 21 K-RTA binding sites were identified on the host genome
Summary
Infectious agents account for approximately 15% of all human cancers per year worldwide [1,2,3,4]. 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. 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 reactivate from latency in response to different stimuli resulting in the expression of >80 genes culminating in the progeny infectious virions [7]
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