Survival-based CRISPR genetic screens across a panel of permissive cell lines identify common and cell-specific SARS-CoV-2 host factors

Chan, Katherine; Farias, Adrian Granda; Lee, Hunsang; Guvenc, Furkan; Mero, Patricia; Brown, Kevin R.; Ward, Henry N.; Billmann, Maximilian; Aulakh, Kamaldeep; Astori, Audrey; Haider, Shahan; Marcon, Edyta; Braunschweig, Ulrich; Pu, Shuye; Habsid, Andrea; Tong, Amy; Christie-Holmes, Natasha; Budylowski, Patrick; Ghalami, Ayoob; Mubareka, Samira; Maguire, Finlay; Banerjee, Arinjay; Mossman, Karen; Greenblatt, Jack; Gray-Owen, Scott D.; Raught, Brian; Blencowe, Benjamin J.; Taipale, Mikko; Myers, Chad L.; Moffat, Jason

doi:10.1016/j.heliyon.2022.e12744

Cited by 7 publications

(4 citation statements)

References 78 publications

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“…In Tab 3: GOF-enriched screen analysis, for antiviral genes, we used FDR ≤ 0.05. Chan, et al [74]: in Multimedia component 6, for Vero E6 (T16); UM-UC-4 (T23); HEK293+A+T (T12); HuH-7 (T15) and Calu-3 (T43), we considered gene as hits at FDR < 0.1 (as in Figure 4A). We listed the genes as proviral if differential ≥ 0 or antiviral if differential ≤ 0.…”

Section: Methodsmentioning

confidence: 99%

A Genome-Wide Arrayed CRISPR Screen Reveals PLSCR1 as an Intrinsic Barrier to SARS-CoV-2 Entry

Le Pen,

Paniccia,

Bauer

et al. 2024

Preprint

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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 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 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 over-expression, 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. Our study contributes to understanding the association between PLSCR1 variants and severe COVID-19 cases reported in a recent GWAS.

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Section: Methodsmentioning

confidence: 99%

A Genome-Wide Arrayed CRISPR Screen Reveals PLSCR1 as an Intrinsic Barrier to SARS-CoV-2 Entry

Le Pen,

Paniccia,

Bauer

et al. 2024

Preprint

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“…293T human embryonic kidney and Vero E6 transmembrane serine protease 2 (TMPRSS2) [ 30 , 31 ] were maintained at 37 °C under 5% CO 2 in Dulbecco’s modified Eagle’s medium (DMEM) (Wisent) containing 5% fetal bovine serum (FBS) (VWR) and 100 μg/mL of penicillin-streptomycin (Wisent). CEM.NKr CCR5+ cells (NIH AIDS reagent program) were maintained at 37 °C under 5% CO 2 in Roswell Park Memorial Institute (RPMI) 1640 medium (GIBCO) containing 10% FBS and 100 μg/mL of penicillin-streptomycin.…”

Section: Methodsmentioning

confidence: 99%

Humoral Responses Elicited by SARS-CoV-2 mRNA Vaccine in People Living with HIV

Marchitto,

Chatterjee,

Ding

et al. 2023

Viruses

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While mRNA SARS-CoV-2 vaccination elicits strong humoral responses in the general population, humoral responses in people living with HIV (PLWH) remain to be clarified. Here, we conducted a longitudinal study of vaccine immunogenicity elicited after two and three doses of mRNA SARS-CoV-2 vaccine in PLWH stratified by their CD4 count. We measured the capacity of the antibodies elicited by vaccination to bind the Spike glycoprotein of different variants of concern (VOCs). We also evaluated the Fc-mediated effector functions of these antibodies by measuring their ability to eliminate CEM.NKr cells stably expressing SARS-CoV-2 Spikes. Finally, we measured the relative capacity of the antibodies to neutralize authentic SARS-CoV-2 virus after the third dose of mRNA vaccine. We found that after two doses of SARS-CoV-2 mRNA vaccine, PLWH with a CD4 count < 250/mm3 had lower levels of anti-RBD IgG antibodies compared to PLWH with a CD4 count > 250/mm3 (p < 0.05). A third dose increased these levels and importantly, no major differences were observed in their capacity to mediate Fc-effector functions and neutralize authentic SARS-CoV-2. Overall, our work demonstrates the importance of mRNA vaccine boosting in immuno-compromised individuals presenting low levels of CD4.

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“…We evolved this virus in four human cell lines: A549-ACE2 (A549-A), A549-ACE2-TMPRSS2 (A549-AT), Huh-7, and IGROV-1 (Figure 1A). These are well-characterized cell lines [39][40][41][42][43] originating from different tissues and show large differences in the mRNA expression levels of key factors related to spike function such as ACE2, TMPRSS2, furin, and cathepsin L, as determined by RT-qPCR (Figure 1B). For instance, ACE2 mRNA levels spanned three orders of magnitude, with Huh-7 expressing the lowest and A549-AT the highest values.…”

Section: Adaptation Of the Sars-cov-2 Spike To Different Human Cell T...mentioning

confidence: 99%

Cell type-specific adaptation of the SARS-CoV-2 spike

Carrascosa-Sàez,

Marqués,

Geller

et al. 2023

Preprint

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SARS-CoV-2 can infect various human tissues and cell types, principally via interaction with its cognate receptor ACE2. However, how the virus evolves in different cellular environments is poorly understood. Here, we used experimental evolution to study the adaptation of the SARS-CoV-2 spike to four human cell lines expressing different levels of key entry factors. After 20 passages, cell type-specific phenotypic changes were observed. Selected spike mutations were identified and functionally characterized in terms of entry efficiency, ACE2 affinity, spike processing, TMPRSS2 usage, entry pathway and syncytia formation. We found that the effects of these mutations varied across cell types. Interestingly, two spike mutations (L48S and A372T) that emerged in cells expressing low ACE2 levels increased receptor affinity, syncytia induction, and entry efficiency under low-ACE2 conditions. Our results demonstrate specific adaptation of the SARS-CoV-2 spike to different cell types and have implications for understanding SARS-CoV-2 tissue tropism and evolution.

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Survival-based CRISPR genetic screens across a panel of permissive cell lines identify common and cell-specific SARS-CoV-2 host factors

Cited by 7 publications

References 78 publications

A Genome-Wide Arrayed CRISPR Screen Reveals PLSCR1 as an Intrinsic Barrier to SARS-CoV-2 Entry

A Genome-Wide Arrayed CRISPR Screen Reveals PLSCR1 as an Intrinsic Barrier to SARS-CoV-2 Entry

Humoral Responses Elicited by SARS-CoV-2 mRNA Vaccine in People Living with HIV

Cell type-specific adaptation of the SARS-CoV-2 spike

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