A genome-wide CRISPR screen identifies interactors of the autophagy pathway as conserved coronavirus targets
Fig 4
LC3-GFP puncta formation is impaired in TMEM41B, FBKP8, and MINAR1-KO cells.
(A) Upon starvation, the mTORC1 complex is blocked and activation of the PI3K complex, as well as the ULK1 complex, leads to the initiation of phagophore formation, as an initial step in the autophagy pathway. MERS-CoV and HCoV-229E top scoring CRISPR KO screen hits FKBP8, MINAR1, TMEM41B, and VMP1 are involved in this early pathway. Furthermore, the ATG8 system containing among others LC3, which is recruited by VPM1 or FBKP8, is necessary for targeting cellular cargo to the autophagosome. PPP3R1 is up-regulated and initiates TFEB translocalization to the nucleus, where it catalyzes transcription of ATGs. MERS-CoV or conserved HDFs are indicated in respective colors. Inhibitor intervention in this pathway is shown in red. (B) Heatmap of canonical autophagy genes showing how host factors, including Beclin-1, ATG5, ATG7, and ATG14 are regulated in this CRISPR KO screen. MERS-CoV–specific host factors are shown in purple, HCoV-229E–specific host factors are indicated in green, and common host factors are marked in orange. Raw data for (B) can be found in Supporting information S1 Data, tab 1. (C) Immunofluorescence staining of LC3-GFP expressing Huh7, TMEM41B-KO, FKBP8-KO, and MINAR1-KO cells upon rapamycin treatment and HCoV-229E infection. LC3-GFP is depicted in green, dsRNA is shown in red and DAPI in blue, and scale bar is 20 μm. Representative images of 1 out of 4 independent replications are shown. Images were acquired using an EVOS FL Auto 2 imaging system with a 20× air objective and processed using Fiji. (D) Quantification of (C) shows LC3 puncta-positive cells upon rapamycin treatment and HCoV-229E infection in native Huh7, as well as TMEM41B-KO, FKBP8-KO, and MINAR1-KO cells. LC3-postive puncta are indicated by arrows in HCoV-229E–infected WT cells. Five images per condition in 3 independent experiments were acquired using an Evos FL Auto 2 imaging system with a 4× air objective, analyzed and quantified in Fiji. Each cell that depicted LC3-positive puncta was counted as a puncta positive cell, independent of the actual number and size of puncta. Statistical significance determined using the Holm–Sidak method, with alpha = 0.05 in GraphPad Prism 8.3.1. Each row was analyzed individually, without assuming a consistent SD. Raw data can be found in Supporting information S1 Data, tab 11. Used reagents are listed in detail in Table 1. FKBP8, FK506 binding protein 8; HCoV, human coronavirus; HDF, host dependency factor; KO, knockout; MERS-CoV, Middle East Respiratory Syndrome Coronavirus; MINAR1, Membrane Integral NOTCH2 Associated Receptor 1; TMEM41B, transmembrane protein 41B; VMP1, vacuole membrane protein 1; WT, wild-type.