Identification and Targeting of Regulators of SARS-CoV-2-Host Interactions in the Airway Epithelium.

Research Square (Research Square), Journal Year: 2024, Volume and Issue: unknown

Published: Oct. 28, 2024

Background In spite of the wealth literature available, mechanistic determinants SARS-CoV-2-mediated host-cell hijacking that results in massive infection human airway epithelium are still poorly understood. While ciliated cells have been identified as a major target and sink SARS-CoV-2 during COVID-19 pathogenesis, contribution other epithelial components specific host factors hijacked maintain their pro-infective cell state remains unclear. Limitations part due to overreliance on single-cell gene expression profiling which may not reflect protein activation status analyses biased toward downstream effects rather than actual infection. These ultimately hampered progress understanding mechanisms implemented by different types identification compounds effectively counteract these factors. Methods Here we used organotypic culture system known model cellular diversity epithelium, network-based platform identify master regulator (MR) proteins facilitate reprogramming key at stages The analysis was coupled large-scale drug perturbation screen cultures using library FDA-approved drugs able invert SARS-CoV-2-induced activities cells. Results top MR differentially activated (NCOR, HDAC1), secretory (KAT2B), or basal/ciliated (MED21/MED7) suggested distinct mechanisms. Notably, crucial proviral required for (USP33, CUL5, SNX27 PBRM1) collectively all 3 main revealed potential mechanism viral propagation common both basal luminal compartments. assay 11 entire signature types, with 9 targeting recognized Conclusions Leveraging perturbational profiles primary represents relevant mechanism-based investigation disease pathogenesis discovery conditions affecting epithelium.

Language: Английский

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Generation of a SARS-CoV-2 reverse genetics system and novel human lung cell lines that exhibit high virus-induced cytopathology

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2023, Volume and Issue: unknown

Published: March 9, 2023

Abstract The global COVID-19 pandemic continues with an increasing number of cases worldwide and the emergence new SARS-CoV-2 variants. In our study, we have developed novel tools applications for screening antivirals, identifying virus-host dependencies, characterizing viral Using reverse genetics, rescued Wuhan1 (D614G variant) wild type (WTFL) reporter virus (NLucFL) using molecular BAC clones. replication kinetics, plaque morphology titers were comparable between clones a clinical isolate (VIDO-01 strain), thus providing confidence that viruses can be used as effective tools. Furthermore, NLucFL exhibited robust luciferase values over time course infection was to develop rapid antiviral assay remdesivir proof-of-principle. addition, tool study lung-relevant interactions, established human lung cell lines support high virus-induced cytopathology. Six (NCI-H23, A549, NCI-H1703, NCI-H520, NCI-H226, HCC827) HEK293T cells, transduced stably express ACE2 tested their ability infection. A549 B1 A2 more than 70% death line NCI-H23 A3 showed about ∼99% post-infection. These are ideal assays relying on live-dead selection currently being in CRISPR knockout activation screens lab. Importance We genetics system generate well nanoluciferase-expressing clone SARS-CoV-2. allows transient throughput by detection assays. genetic mutant phenotypes variant mutations. Additionally, unique supporting will aid studying environment based cytopathology induced some lines, useful rely selection. Our aims enhance contribute current available methods, lines.

Language: Английский

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The importance of activated TMPRSS2 in the proviral role of small extracellular vesicles in SARS‐CoV‐2 infection

Journal of Extracellular Vesicles, Journal Year: 2022, Volume and Issue: 11(12)

Published: Dec. 1, 2022

The importance of activated TMPRSS in the proviral role small extracellular vesicles

Language: Английский

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Human Cytomegalovirus Degrades DMXL1 to Inhibit Autophagy, Lysosomal Acidification and Viral Assembly

Published: Jan. 1, 2023

Human cytomegalovirus (HCMV) is an important human pathogen and a master regulator of host intrinsic, innate, adaptive immunity. HCMV hijacks intracellular compartments to assemble new virions, lysosomes are essential for this process. We combined comprehensive proteomic analysis proteins targeted degradation by with database involved in vacuolar acidification. Dmx-like protein 1 (DMXL1) was the only that acidifies vacuoles yet degraded HCMV.Systematic comparison viral deletion mutants revealed uncharacterised 7kDa US33A necessary sufficient DMXL1 degradation. Functional experiments using cells stably expressing US33A, or infected adenovirus vectors recombinant deleted demonstrated degrades inhibit lysosomal acidification autophagic cargo Formation assembly compartment, which known require lysosomes, occurred significantly later US33A-expressing virus, reduced replication. These data thus identify entirely strategy cellular remodelling. recruits E3 ubiquitin ligase Kip1 ubiquitination-promoting complex (KPC) acts akin proteolysis-targeting chimera (PROTAC) degrade DMXL1. The potential exists employ novel therapies infection rheumatic conditions, inhibition lysosome can attenuate disease.

Language: Английский

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Whole Genome CRISPR Screening Strategy to Identify Genes Contributing to SARS-CoV-2 Spike and VSV-G Mediated Entry.

Authorea (Authorea), Journal Year: 2023, Volume and Issue: unknown

Published: Aug. 12, 2023

Background Understanding the cellular host factors that promote and inhibit viral entry is important for identifying countermeasures. CRISPR whole genome screens can be used to rapidly discover contribute or impair entry. However, when using live viruses lethality selection, these identify an overwhelming number of genes without specificity stage infection cycle. New screening methods are needed machinery contributing specific steps infection. Here, we developed a screen counter strategy based on pseudoviral platform allowed identification SARS-CoV-2 spike vesicular stomatitis virus glycoprotein VSV-G mediated Methods To focus onto step, non-lytic fluorescent reporters in combination with comparative distinguish affecting reporter from those unique envelope-mediated Screening same lentiviral pseudovirus entry-specific relative associated retro-transcription, integration, expression pseudovirus. Second, Cre-Gag fusion protein was bypass retro-transcription integration by directly activating floxed GFP upon reduce gene hits increase Results Our approach correctly identified receptors ACE2 LDLR, respectively distinguished retroviral Moreover, CRE-Gag fusion/flox increased genes. Validation few demonstrates this distinguishes envelope-specific expression. Conclusion Overall, provides new influencing confounding complexity live-viral which produce long lists all aspects pathogenesis replication. This pathway increasing

Language: Английский

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