Fig 1: TMEM41B and VMP1 are host dependency factors for flaviviruses and human coronaviruses, but not alphaviruses.(A) Multiple rounds of virus infection in 293FT WT, TMEM41B KO clone #1, and VMP1 KO clone #1 cells. (Top) Western blotting analysis to detect TMEM41B and VMP1 protein levels in the WT and KO cells. (Bottom) Crystal violet staining to visualize surviving cells after cytolytic virus infection. Non-infected controls are indicated as “mock”. (B, C) Validation of TMEM41B and VMP1 as host dependency factors across all DENV serotypes. For (B), cells were infected by either DENV1 (MOI = 1 PFU/cell), DENV2 (MOI = 0.1 PFU/cell), DENV3 (MOI = 3 PFU/cell), or DENV4 (MOI = 5 PFU/cell) for 72 hours. Accumulation of DENV NS3 proteins was detected by western blot. For (C), cells were infected at MOI of 1 for 72 hours. Released progeny virions in the supernatant were assessed by plaque assay. (D) HCoV-OC43 protein accumulation (Left) and progeny virion levels (Right) in 293FT WT and KO cells. Cells were infected with HCoV-OC43 at MOI of 0.5 for 48 hours. Released progeny virions in the supernatants were harvested for titration by TCID50 assay, and infected cell lysates were analyzed by western blotting. (E) Production of CHIKV progeny virions from WT and KO cells. Cells were infected with CHIKV at MOI of 0.1 for 24 hours, and released virions were harvested for titration by plaque assay. (F) cDNA complementation rescue of DENV infection. (Left) Production of DENV2 infectious particles from KO cells complemented with corresponding cDNAs, i.e., mCherry (control), TMEM41B, or VMP1. (Right) Western blotting analysis of DENV2 NS3 accumulation in WT, KO, and cDNA-complemented cells. Cells were infected at MOI of 0.1 for 72 hours. Released virions in the supernatants were harvested for titration by plaque assay, and cell lysates were analyzed by western blotting. (G) Validation of TMEM41B and VMP1 as DENV host factors in A549 cells. (Left) Production of DENV1 infectious particles from A549 WT, TMEM41B KO clone #1, and VMP1 KO clone #1 cells complemented with corresponding cDNAs. (Right) Western blotting analysis of DENV1 NS3 accumulation in WT, KO, and cDNA-complemented cells. Cells were infected at MOI of 0.5 for 32 hours. GAPDH was used as a loading control for all blots. All data shown represent results from at least two independent experiments. Error bars represent mean +/- SEM, n = 3. * indicates p-value < 0.05 as determined by two-tailed unpaired t-test or one-way ANOVA. LOD indicates the limit of detection.