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

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. We integrated the ORF59-GFP reporter into the AAVS1 safe harbor locus of HCT116 cells. Using the Brunello knockout CRISPR lentivirus library (Doench et al. 2016; Addgene #73179), we looked for genes that, upon knockout, enhanced GFP. We performed three replicates of the screen and compared the sgRNAs found in sorted cells (those with high GFP signal) to sgRNAs found in the unselected (Input) pool of cells .The sequencing data were then analyzed using MAGeCK (Li et al. 2014).