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A genome-wide arrayed CRISPR screen identifies PLSCR1 as an intrinsic barrier to SARS-CoV-2 entry that recent virus variants have evolved to resist [1]
['Jérémie Le Pen', 'Laboratory Of Virology', 'Infectious Disease', 'The Rockefeller University', 'New York', 'United States Of America', 'Gabrielle Paniccia', 'Volker Kinast', 'Department Of Medical Microbiology', 'Virology']
Date: 2024-10
Interferons (IFNs) play a crucial role in the regulation and evolution of host–virus interactions. Here, we conducted a genome-wide arrayed CRISPR knockout screen in the presence and absence of IFN to identify human genes that influence Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection. We then performed an integrated analysis of genes interacting with SARS-CoV-2, drawing from a selection of 67 large-scale studies, including our own. We identified 28 genes of high relevance in both human genetic studies of Coronavirus Disease 2019 (COVID-19) patients and functional genetic screens in cell culture, with many related to the IFN pathway. Among these was the IFN-stimulated gene PLSCR1. PLSCR1 did not require IFN induction to restrict SARS-CoV-2 and did not contribute to IFN signaling. Instead, PLSCR1 specifically restricted spike-mediated SARS-CoV-2 entry. The PLSCR1-mediated restriction was alleviated by TMPRSS2 overexpression, suggesting that PLSCR1 primarily restricts the endocytic entry route. In addition, recent SARS-CoV-2 variants have adapted to circumvent the PLSCR1 barrier via currently undetermined mechanisms. Finally, we investigate the functional effects of PLSCR1 variants present in humans and discuss an association between PLSCR1 and severe COVID-19 reported recently.
Funding: The Laboratory of Virology and Infectious Disease was supported by the National Institutes of Health (P01AI138398-S1, 2U19AI111825, R01AI091707-10S1, and R01AI161444 to CMR); a George Mason University Fast Grant to CMR; the G. Harold and Leila Y. Mathers Charitable Foundation to CMR; the Meyer Foundation to CMR; the Pilot Project Robertson Therapeutic Development Fund at The Rockefeller University to CMR; and the Bawd Foundation to CMR. The Laboratory of Human Genetics of Infectious Diseases was supported by the Howard Hughes Medical Institute to JLC, the St. Giles Foundation to JLC, the National Institutes of Health (R01AI163029 and UL1TR001866 to JLC), the French National Research Agency (ANR) under the “Investments for the Future” program (ANR-10-IAHU-01 to JLC), the Integrative Biology of Emerging Infectious Diseases Laboratory of Excellence (ANR-10-LABX-62-IBEID to JLC), the French Foundation for Medical Research (FRM) (EQU201903007798 to JLC), ANR GENVIR (ANR-20-CE93-003) to JLC, ANR AAILC (ANR-21-LIBA-0002) to QZ, ANR GENFLU (ANR-22-CE92-0004) to QZ, the ANR-RHU COVIFERON Program (ANR-21-RHUS-08) to JLC, the French Foundation for Medical Research (FRM) (EQU201903007798) to JLC, the European Union’s Horizon 2020 research and innovation program under grant agreement No. 824110 (EASI-genomics) to JLC, the HORIZON-HLTH-2021-DISEASE-04 program under grant agreement 101057100 (UNDINE) to JLC, the Square Foundation to JLC, William E. Ford, General Atlantic’s Chairman and Chief Executive Officer, Gabriel Caillaux, General Atlantic’s Co-President, Managing Director and Head of Business at EMEA, and the General Atlantic Foundation to JLC, the French Ministry of Higher Education, Research, and Innovation (MESRI-COVID-19) to JLC, and REACTing-INSERM to JLC. JLP was supported by the Francois Wallace Monahan Postdoctoral Fellowship at The Rockefeller University and the European Molecular Biology Organization Long-Term Fellowship (ALTF 380-2018 to JLP). GP was supported by the James H. Gilliam Fellowship for Advanced Study from the Howard Hughes Medical Institute and the Graduate Research Fellowship Program from the National Science Foundation (FAIN 1946429 to GP). VK was supported by a travel grant of the Boehringer Ingelheim Fonds (BIF) and a scholarship of the German Liver Foundation. MB was supported by a Swiss National Science Foundation fellowship (P500PB_203007 to MB). DL was supported by the European Society for Immunodeficiencies bridge grant and a fellowship from the FRM. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Data Availability: mRNA-seq data is available on the Sequence Read Archive (SRA), hosted by the National Center for Biotechnology Information (NCBI), under BioProject PRJNA1138251 and BioSamples SAMN42689767-SAMN42689770 corresponding to S2 and S3 Tables, BioSamples SAMN42689771- SAMN42689774 corresponding to S4 and S5 Tables, BioSamples SAMN42689775- SAMN42689776 corresponding to S14 and S15 Tables, BioSamples SAMN42689777- SAMN42689778 corresponding to S16 and S17 Tables. Code and Supporting Information are available on Dryad (DOI: 10.5061/dryad.6q573n65k ).
Copyright: © 2024 Le Pen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Follow-up experiments revealed that PLSCR1 is a cell intrinsic factor that restricts spike-mediated SARS-CoV-2 entry, independently of the IFN pathway, via currently undetermined mechanisms. Our genetic screen data and meta-analysis provide a valuable resource to broaden our understanding of coronavirus infection and innate immunity. Furthermore, we extend the recent characterization of PLSCR1 as an antiviral against SARS-CoV-2 impacting COVID-19 outcomes ( S1 Fig ) [ 19 , 25 , 26 ].
We then compiled a comprehensive list of genes interacting with SARS-CoV-2, incorporating findings from our own screen as well as existing literature. This meta-analysis revealed several host genes of interest, both previously described and novel. Notably, the ISG product phospholipid scramblase 1 (PLSCR1) emerged as a prominent antiviral factor. PLSCR1 is involved in several biological processes [ 22 ], including regulating the movement of phospholipids between the 2 leaflets of a cell membrane (lipid scrambling) [ 23 ] and IFN signaling in the context of virus infection [ 24 ].
Here, we conducted a human whole-genome arrayed CRISPR KO screen to identify genes that influence SARS-CoV-2 infection in cells with or without pretreatment with a low dose of IFN. The arrayed approach, though logistically challenging, has advantages over the pooled format in capturing both proviral and antiviral genes, genes affecting virus egress, and those coding for secreted products that exert their impact on neighboring cells. It reliably captures genotype–phenotype correlations while also unveiling the effects of single gene perturbation on cell growth and death [ 20 ]. Additionally, the shorter culture time and lack of competition among cells with different gene KO in the arrayed screen allow the inclusion of genes that would be depleted and deemed essential in a pooled format [ 21 ]. The arrayed format thus enables the identification of crucial cellular functions that may be co-opted by the virus or are vital for the cell’s defense against infection.
Several recent studies have identified ISGs restricting SARS-CoV-2. Most of these studies involved gain-of-function genetic screens, overexpressing individual ISGs. The factors bone marrow stromal cell antigen 2 (BST2), cholesterol 25-hydroxylase (CH25H), lymphocyte antigen 6 family member E (LY6E), 2′-5′-oligoadenylate synthetase 1 (OAS1), and receptor transporter protein 4 (RTP4) were notably identified as SARS-CoV-2 antivirals in these studies [ 10 – 15 ]. One advantage of the gain-of-function approach is that it circumvents potential genetic redundancies between ISGs [ 16 , 17 ]. However, this approach is biased towards ISGs that act autonomously when overexpressed and does not mimic the cellular context of the IFN response, where hundreds of genes and gene products are differentially regulated to establish an antiviral state. To counter this limitation, 2 recent publications examined the effects of ISG loss of function in IFN-treated cells. They conducted pooled CRISPR knockout (KO) screens in cells pretreated with IFN before SARS-CoV-2 infection [ 18 , 19 ]. By sorting for cells with high SARS-CoV-2 viral load, they identified SARS-CoV-2 restriction factors such as death domain associated protein (DAXX).
Approximately 1% to 5% of critical Coronavirus Disease 2019 (COVID-19) patients have mutations that compromise the production of or response to type I IFNs, while an additional 15% possess autoantibodies that neutralize type I IFNs [ 2 – 8 ]. This highlights the essential role of type I IFN in the defense against the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) virus that caused the COVID-19 pandemic [ 9 , 10 ]. Consequently, investigating IFN-stimulated genes (ISGs) is crucial to our understanding of the remarkable antiviral systems that evolved in nature and could enhance our preparedness for future pandemics.
Viruses maintain a complex relationship with their host cells, co-opting host factors for their replication while being targeted by cellular defense mechanisms. Such cellular defenses include the interferon (IFN) pathway, where the infected cell senses foreign molecules and secretes IFN to trigger an antiviral state in neighboring cells [ 1 ].
Results
Intrinsic PLSCR1 restricts SARS-CoV-2 independently of the IFN pathway To better characterize the function of PLSCR1 during SARS-CoV-2 infection, we generated and validated by western blot (WB) PLSCR1 KO bulk Huh-7.5 and A549-ACE2 lines (S5A Fig). As observed in the arrayed screen (S2B Fig), PLSCR1 KO cells were viable (S5B Fig). PLSCR1 depletion increased susceptibility to SARS-CoV-2 independently of IFN pretreatment (Fig 4A). Cell treatment with a JAK-STAT inhibitor, which effectively abrogated IFN signaling, confirmed that intrinsic PLSCR1 limits SARS-CoV-2 infection independently of the IFN signaling pathway (Fig 4A). SARS-CoV-2 susceptibility of PLSCR1 KO cells was reversed by the ectopic expression of PLSCR1 (Figs 4B and S5C). Interestingly, while PLSCR1 tagged with an N-terminal FLAG tag could rescue, PLSCR1 tagged with a C-terminal FLAG tag could not. The C-terminus of the protein is extracellular, and previous research suggests that this region is important for the protein’s scramblase activity and Ca2+ binding [117]. It is possible that the addition of this FLAG-tag impaired Ca2+ binding, affected PLSCR1’s localization at the plasma membrane, or otherwise disrupted the structure of this region, thereby abolishing PLSCR1’s antiviral ability. We cocultured PLSCR1 reconstituted cells and PLSCR1 KO cells in the same well and infected them with SARS-CoV-2. A higher proportion of PLSCR1 KO than PLSCR1 reconstituted cells were positive for SARS-CoV-2 indicating that PLSCR1 acts in a cell autonomous manner (Fig 4C). Overall, our data indicate that intrinsic PLSCR1 restricts SARS-CoV-2 in cell culture, even in the absence of IFN. Given that PLSCR1 mRNA is constitutively expressed in SARS-CoV-2 target cells (S5D Fig) [118], its intrinsic antiviral function may also be effective in vivo. PPT PowerPoint slide
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TIFF original image Download: Fig 4. PLSCR1 is a highly effective anti-SARS-CoV-2 effector ISG contributing to intrinsic immunity in the absence of IFN. (A) Cells were pretreated with a JAK-STAT inhibitor (InSolution 1 μM) for 2 hours, followed by IFN-ɑ2a (10 pM Huh-7.5 or 20 μM A549-ACE2) for 24 hours and were infected with SARS-CoV-2 for 24 hours followed by IF staining for viral N protein. Huh-7.5 infection using an MOI of 0.5 (titer determined by focus forming assay on Huh-7.5 WT cells). A549-ACE2 infection using an MOI of 0.01 (titer determined by focus forming assay on A549-ACE2 WT cells). The percentage of SARS-CoV-2-positive cells is plotted. n = 4 separate wells infected on the same day. (B) Cells were reconstituted with the indicated proteins by stable transduction with lentiviruses and then infected as in (A). n = 4 separate wells infected on the same day. (C) Cells were cocultured as indicated (50:50 mix) and then infected as in (A), and the % infection of each cell type was determined. n = 4 separate wells infected on the same day; ****, p ≤ 0.0001; two-tailed t test. The data underlying this Figure can be found in S1 Table. IF, immunofluorescence; IFN, interferon; ISG, IFN-stimulated gene; MOI, xxxx; PLSCR1, phospholipid scramblase 1; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; WT, wild type.
https://doi.org/10.1371/journal.pbio.3002767.g004
IFN signaling is unaffected by the loss of PLSCR1 in A549-ACE2 and Huh-7.5 cells PLSCR1 has been shown to potentiate ISG transcription in IFN-treated Hey1B cells [24]. We thus hypothesized PLSCR1 might enhance the type I IFN response in A549-ACE2 and Huh-7.5 cells. We investigated the PLSCR1’s role in the IFN response by infecting Huh-7.5 cells with chikungunya virus (CHIKV), which is unaffected by PLSCR1 KO without IFN (S6A Fig). PLSCR1 depletion did not functionally affect the antiviral effects of IFN treatment (S6B Fig). Furthermore, IFN treatment induced OAS1 and IFI6, 2 ISGs known to restrict SARS-CoV-2 [11,13,18,95,96,98,99], to a similar extent in both wild type (WT) and PLSCR1 KO cells, indicating that the IFN signaling pathway was unaffected by PLSCR1 depletion (S6C-S6J Fig). Finally, PLSCR1 depletion did not alter basal ISG transcription in the absence of IFN (S7 Fig and S14 and S15 Tables). These findings indicate that PLSCR1 limits SARS-CoV-2 infection independently of the IFN signaling pathway in A549-ACE2 and Huh-7.5 cells.
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