GSE201045
The PNUTS-protein phosphatase 1 complex acts as an intrinsic barrier to KSHV replication [eCLIP]
Control of RNA Polymerase II (pol II) elongation is a critical component of gene expression in mammalian cells. The PNUTS-protein phosphatase 1 (PP1) complex controls elongation rates, slowing pol II after polyadenylation sites to promote termination. The Kaposi's sarcoma-associated herpesvirus (KSHV) co-opts pol II to express its genes, but little is known about its regulation of pol II elongation. We identified PNUTS as a suppressor of a KSHV reporter gene in a genome-wide CRISPR screen. PNUTS depletion also enhances global KSHV gene expression and overall viral replication. Reflecting its host gene activities, PNUTS binds viral RNAs downstream of polyadenylation sites, restricts transcription readthrough of viral genes, and requires PP1 interaction. Surprisingly, PNUTS represses the KSHV reporter by decreasing productive elongation at the 5´-end of the gene.  From these data, we conclude that PNUTS' activity forms an intrinsic barrier to KSHV replication likely by suppressing pol II elongation at promoter-proximal regions. Overall design: We reactivated iSLK cells latently infected with wild-type BAC16 KSHV. The virus was reactivated with sodium butyrate and doxycycline and cells were harvested 24 hours later. After lysis and immunoprecipitation with anti-PNUTS ab (A300-439A-T; Bethyl), samples were run on an SDS-PAGE, transferred to PVDF membrate, and cut from the gel from the 115-190kD region. Total lysate was run on the gel and a size-matched input (SMI) was processed in parallel. RNA was isolated from both SMI and IP samples, libraries were made for sequencing. The protocol followed that of van Nostrand et al (2016). Two independent biological replicates were performed.