Fig 1: KSHV CRISPR Library Screen Identifies K5 as ORF Responsible for Downregulation of the NK Cell Receptor Ligands Nectin-2 and CD155(A) Immunoblot analysis of HuAR2T.rKSHV.219 Cas9 cells harboring sgRNAs specific for indicated viral ORFs untreated or treated with reactivation mix and probed with the indicated antibodies. A non-specific band is labeled with an asterisk.(B) Schematic flowchart of the genetic screen with the KSHV CRISPR library. HuAR2T.rKSHV.219 Cas9 cells were transduced with the sgRNA library followed by lytic KSHV cycle induction and stained with CD155- or Nectin-2-specific antibody. Lytic (RFP+) and CD155- or Nectin-2-high cells were selected by FACS and used for DNA extraction and sequencing.(C and D) Genetic screens with the KSHV CRISPR library identify K5 as the ORF responsible for downregulation of Nectin-2 and CD155. Each dot represents a single KSHV ORF or miRNA plotted by the statistical significance (-log p value) of the sgRNA enrichment.(E) CRISPR-mediated K5 knockout leads to full (Nectin-2) or partial (CD155) rescue of lytic KSHV-induced downregulation of the proteins from the cell surface. Flow cytometry analysis of HuAR2T.rKSHV.219 Cas9 cells harboring control or K5-specific sgRNAs untreated or treated with reactivation mix and stained with the indicated antibody.(F) Flow cytometry analysis of HuAR2T cells transduced with LV K5 or control LV and probed with the indicated antibody.