The PNUTS-protein phosphatase 1 complex acts as an intrinsic barrier to KSHV replication [ChIP-Seq II]

In this study, we found that the host protein PNUTS suppressed Kaposi's sarcoma-associated herpesvirus (KSHV) gene expression during lytic reactivation and from heterologous viral reporters. To gain insights into the mechanism, we performed ChIP-seq of the pol II subunit RPB3 in cells with an integrated reporter containing the KSHV ORF59 gene. Our results were consistent with previous reports showing that depletion of PNUTS leads to global loss of pol II slow down and termination after polyadenylation sites. In contrast, PNUTS depletion appeared to decrease promoter-proximal pausing on our integrated reporter suggesting a distinct mode of gene regulation on viral genes. Pol II ChIP-seq using antibodies against RPB3 was performed on samples treated with either a nontargeting siRNA (siNT) or one of two siRNAs targeting PNUTS (siP1 or siP2). The cells were iSLK cells latently infected with the BAC16 KSHV bacmid. Pellets and inputs were analyzed for each sample; two biological replicates were perforemd (R1 and 2).