Citation
- Authors: Wang R. et al.
- Year: 2021
- Journal: Cell 184 106-119
- Applications: in vitro / DNA / jetOPTIMUS
- Cell types:
- Name: HEK-293FT
- Name: Huh-7.5.1
Description: Human hepatocarcinoma cells
Known as: Huh7,5,1, Huh 7,5,1
Method
To rescue the VSVdG-CoV2-S(AA), 293FT cells were co-cultured with Huh7.5.1 cells at a ratio of 1:2 in a 6-well plate to be 80%–90% confluent the next day. Cells were transfected using JetOptimus (Polyplus) with pCAGGS-T7 (200ng), pCAGEN-CoV2-N (300ng), pCAGEN-CoV2-P (500ng), pCAGEN-CoV2-L (200ng), pCMV-VSV-G (800ng), and VSV-eGFP-CoV2-S(AA) (650ng). Cells were trypsinized and passed to a 10cm plate at 4 days post-transfection. At 10 days post-transfection, syncytia formation was seen and at 11 days post-transfection most of the cells had strong green fluorescence and supernatant was collected and frozen at −80°C. Huh7.5.1 cells were infected with supernatant and passaged 6 times every 3-4 days.
Abstract
The Coronaviridae are a family of viruses that cause disease in humans ranging from mild respiratory infection to potentially lethal acute respiratory distress syndrome. Finding host factors common to multiple coronaviruses could facilitate the development of therapies to combat current and future coronavirus pandemics. Here, we conducted genome-wide CRISPR screens in cells infected by SARS-CoV-2 as well as two seasonally circulating common cold coronaviruses, OC43 and 229E. This approach correctly identified the distinct viral entry factors ACE2 (for SARS-CoV-2), aminopeptidase N (for 229E), and glycosaminoglycans (for OC43). Additionally, we identified phosphatidylinositol phosphate biosynthesis and cholesterol homeostasis as critical host pathways supporting infection by all three coronaviruses. By contrast, the lysosomal protein TMEM106B appeared unique to SARS-CoV-2 infection. Pharmacological inhibition of phosphatidylinositol kinases and cholesterol homeostasis reduced replication of all three coronaviruses. These findings offer important insights for the understanding of the coronavirus life cycle and the development of host-directed therapies.