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

We developed the first bat genome-wide RNAi and CRISPR libraries and performed two screens 8 with two different RNA viruses. Although the screening methodologies are quite different, we 9 uncovered similar pathways essential for viral replication, namely the endocytosis pathway and 10 protein secretory pathways. The different genes in these two pathways identified from the screens 11 reflect the complementarity of the methodologies, with RNAi screening useful for observing acute 12 effects on viral infection and CRISPR screening for long-term effects on cell survival. Other than 13 technical differences between screening methods, viral kinetics, sensitivity of the viruses to loss- 20 Both screens identified MTHFD1 as an essential viral host factor. MTHFD1 is a key enzyme 21 in the C1 metabolism responsible for production of purine, dTMP and methyl group, the first two 22 of which are building blocks for DNA and RNA synthesis. Interestingly, all previous RNAi and 23 CRISPR screens using human cells have not identified MTHFD1 as a host factor. Moreover, we 24 found bats cells and tissues have lower MTHFD1 expression levels than their human counterparts 25 (Supplementary Fig. 5a, 5b and 5c ). This might be related to the difference in the promoter 26 regions between bats and humans, notably a lack of CCAAT box 34 in the promoter of two bat 27 species (Supplementary Fig. 5d) The copyright holder for this preprint (which was not peer-reviewed) is the .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 converts the whole cellular machinery into a factory to massively produce progeny viruses, which 1 explains the vital dependence on MTHFD1. This is analogous to cancer cell's addiction to non-2 oncogenic gene functions such as many metabolic enzymes 35 . Similar to the therapeutic targeting 3 of these non-oncogenic addictions for developing cancer drugs, these viral addictions to metabolic 4 enzymes such as MTHFD1 could open doors to many new opportunities for developing broad-5 spectrum antiviral drugs 36 . These drugs could be readily applied in emerging epidemics, as 6 demonstrated by our evidence here that MTHFD1 inhibitor carolacton can suppress the infection 7 of SARS-CoV-2. We envision the functional genomics tools we provided here will be instrumental 8 to further discoveries by better understanding the bat-virus interaction. 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 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 Influenza A virus (IAV) for 12h. The infection rates were quantified with immunofluorescence 1 staining of HA. Data shown as the mean ±standard deviation (n=3). b. Representative images 2 from a. (scale bar=100 µm). c. MTHFD1 knock out 293T cells were generated from two 3 independent sgRNAs and infected with IAV for another 12h. Then the cells were collected and 4 analyzed by Western blotting. Data shown are representative of three independent experiments. d-5