Single-cell image-based genetic screens systematically identify regulators of Ebola virus subcellular infection dynamics.
Rebecca J CarlsonJ J PattenGeorge StefanakisBrian Y SoongAdityanarayanan RadhakrishnanAvtar SinghNaveen ThakurGaya K AmarasingheNir HacohenChristopher F BaslerDaisy W LeungCaroline UhlerRobert A DaveyPaul C BlaineyPublished in: bioRxiv : the preprint server for biology (2024)
Ebola virus (EBOV) is a high-consequence filovirus that gives rise to frequent epidemics with high case fatality rates and few therapeutic options. Here, we applied image-based screening of a genome-wide CRISPR library to systematically identify host cell regulators of Ebola virus infection in 39,085,093 million single cells. Measuring viral RNA and protein levels together with their localization in cells identified over 998 related host factors and provided detailed information about the role of each gene across the virus replication cycle. We trained a deep learning model on single-cell images to associate each host factor with predicted replication steps, and confirmed the predicted relationship for select host factors. Among the findings, we showed that the mitochondrial complex III subunit UQCRB is a post-entry regulator of Ebola virus RNA replication, and demonstrated that UQCRB inhibition with a small molecule reduced overall Ebola virus infection with an IC50 of 5 μM. Using a random forest model, we also identified perturbations that reduced infection by disrupting the equilibrium between viral RNA and protein. One such protein, STRAP, is a spliceosome-associated factor that was found to be closely associated with VP35, a viral protein required for RNA processing. Loss of STRAP expression resulted in a reduction in full-length viral genome production and subsequent production of non-infectious virus particles. Overall, the data produced in this genome-wide high-content single-cell screen and secondary screens in additional cell lines and related filoviruses (MARV and SUDV) revealed new insights about the role of host factors in virus replication and potential new targets for therapeutic intervention.
Keyphrases
- genome wide
- single cell
- deep learning
- dna methylation
- high throughput
- rna seq
- small molecule
- protein protein
- sars cov
- copy number
- induced apoptosis
- transcription factor
- binding protein
- cell cycle arrest
- amino acid
- oxidative stress
- randomized controlled trial
- convolutional neural network
- healthcare
- poor prognosis
- optical coherence tomography
- molecular dynamics simulations
- nucleic acid
- artificial intelligence
- cell therapy
- gene expression
- risk assessment
- big data
- disease virus
- resistance training
- endoplasmic reticulum stress
- signaling pathway
- crispr cas
- electronic health record
- human health
- cell death
- high intensity
- protein kinase
- genome editing