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

with two different sgRNAs targeting MTHFD1 ( Fig. 2a-2b) . Similarly, in HEK293T cells with 12 MTHFD1 knocked out by two sgRNAs, influenza replication is also inhibited, suggesting a 13 conserved requirement for this gene by influenza virus in human cells (Fig. 2c) . Further virus 14 titration assay demonstrated that MTHFD1 knockdown significantly inhibits viral replication for 15 mumps virus in PaKi cells ( Fig. 2d and 2e) . The effect is similar to the knockdown of SEC61B, a 16 well-studied host protein required for viral replication and a top hit in our RNAi screen ( Fig. 2d 17 and 2e) 23 . We additionally tested Melaka virus (PRV3M), a pteropine orthoreovirus that causes 18 acute respiratory disease when transmitted by bats to humans 28 . PRV3M titer was also significantly 19 reduced by MTHFD1 knockdown ( Fig. 2e and 2f) . Infection of Zika virus, a member of the 20 flavivirus family, was also inhibited by MTHFD1 knockdown using siRNA (Fig. 2g) . Alternative 21 knockdown with stable expression of two shRNAs validated the requirement of MTHFD1 for the 22 replication of mumps virus and Zika virus (Fig. 2h, 2i and 2j) . Importantly, we also show that this 23 inhibition is completely rescued by overexpression of MTHFD1 from a construct resistant to the 24 shRNA, ruling out potential off-target effects (Fig. 2k) . 25 26