FIG 2.

ISG-expression screening reveals novel candidate genes with anti-BUNV activity. (A) Schematic representation of H. sapiens and M. mulatta ISG libraries. The libraries are in a 96-well plate format with one ISG per well. (B) Simplified schematic of the pSCRPSY-DEST lentiviral vector (the dotted line indicates mRNA splicing). (C) Schematic representation of the ISG screen. MT4 cells were transduced with the ISG libraries. After 48 h, cells were challenged with BUNV-EGFP, and the infectivity of BUNV-EGFP in ISG-expressing MT4 cells was quantified by flow cytometry at 48 hpi. (D) Results of the screen. Each dot represents one ISG. Infectivity measured for each ISG-expressing well was normalized to the average for the screen, which is indicated as 100%. (E) Confirmatory assays for the selected top 20 H. sapiens and M. mulatta candidate inhibitory ISGs. New lentiviral stocks independent of the primary screen were made and used to transduce MT4 cells. BUNV-EGFP infection was performed as in the primary screen. Titers are presented as numbers of GFP infectious units (IU) per milliliter and were calculated for triplicate wells.