Orthogonal genome-wide screenings in bat cells identify MTHFD1 as a target of broad antiviral therapy

Danielle E Anderson; Jin Cui; Qian Ye; Baoying Huang; Wenhong Zu; Jing Gong; Weiqiang Liu; So Young Kim; Biao Guo Yan; Kristmundur Sigmundsson; Xiao Fang Lim; Fei Ye; Peihua Niu; Xuming Zhou; Wenjie Tan; Lin-Fa Wang; Xu Tan

and have previously been previous identified as key host factors for influenza virus entry in several 23 screens performed in human cells (Fig. 1b) 21 . Among the other hits were two genes, COPZ1 and 24 SEC23B, involved in the protein secretory pathway, which are also known to be required for the 25 replication of many viruses including influenza virus and flavivirus (Fig. 1b) [21] [22] [23] . The enrichment 26 of previously known host factors in the identified hits in the CRISPR screen validated our library 27 and the screening methodology. The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020. 03.29.014209 doi: bioRxiv preprint We synthesized the sgRNA library in an array-based oligonucleotide library and cloned them into 1 the lentiGuide-Puro lentivirus expression vector 24 . A custom bat siRNA library was designed to 2 target 18,328 genes using 40,016 siRNA duplexes. We performed RNAi screening in PaKi cells 3 for identification of host factors for the infection of mumps virus, a paramyxovirus (Fig. 1c) . The 4 screen was performed in 384-well format with a mumps virus strain expressing EGFP from an 5 additional open reading frame 25 . PaKi cells were infected 48 hours after transfection of the siRNA 6 library. High-content imaging analysis was utilized to quantitate the infection rate as well as the 7 total cell number each well, indicating cytotoxicity of the siRNA. Excluding siRNAs with 8 significant cytotoxicity, we selected 45 host dependency factors whose knockdown resulted in 9