Project description:Reveal differentially regulated genes and cellular pathways within allergic and non-allergic asthmatic children compared to healthy controls Peripheral blood mononuclear cells (PBMCs) were obtained from allergic asthmatics (n=14), non-allergic asthmatics (n=8) and healthy controls (n=14) and kept with anti-CD3/CD28 (CD328), LpA or without stimulation (M).
Project description:VAF347 is a low molecular weight compound which inhibits allergic lung inflammation in vivo. This effect is likely due to a block of dendritic cell (DC) function to generate pro-inflammatory T-helper (Th) cells since VAF347 inhibits IL-6, CD86 and HLA-DR expression by human monocyte derived DC, three relevant molecules for Th-cell generation. Here we demonstrate that VAF347 interacts with the aryl hydrocarbon receptor (AhR) protein resulting in activation of the AhR signaling pathway. Functional AhR is responsible for the biological activity of VAF347 since, i) other AhR agonists display an identical activity profile in vitro, ii) gene silencing of wild type AhR expression or forced over-expression of a trans-dominant negative AhR ablates VAF347 activity to inhibit cytokine induced IL-6 expression in a human monocytic cell line and iii) AhR deficient mice are resistant to the compoundâs ability to block allergic lung inflammation in vivo. These data identify the AhR protein as key molecular target of VAF347 and its essential role for mediating the anti-inflammatory effects of the compound in vitro and in vivo. Experiment Overall Design: Immature monocyte-derived DC were activated with anti-CD40 antibodies for 8 hours in the absence or presence of VAF347. Two donors (D1,D2) were used, resulting in four data sets: D1_ctrl, D2_ctrl (ctrl=no treatment), D1_VAF347, D2_VAF347.
Project description:SARS-CoV-2 virus mimics host mRNA by capping its viral RNA to promote replication and evade host immune sensing. SARS-CoV-2 NSP14 is the N7-guanosine methyltransferase (N7-MTase) responsible for RNA cap-0 formation. Targeting NSP14 for antiviral drug development is an under-explored but promising strategy. Here we conducted a high-throughput screening on natural products library derived from Chinese herbal medicine to discover Emodin as a SARS-CoV-2 NSP14 inhibitor. Exploring Emodin derivatives, Questin was identified with potent cellular inhibitory activity (EC50=249 nM) against SARS-CoV-2, which inhibits NSP14 in an RNA cap competitive manner, making it one the most potent anti-coronaviral natural products. Mechanistically, besides catalyzing viral RNA capping, NSP14 by itself could remodel host transcriptome such as enriching CREBBP, a key host factor in cellular cyclic AMP response pathway, to promote viral infection. As a result, targeting NSP14 by Questin significantly impairs viral Replication & Translation step and reverses host transcriptome remodeled by NSP14. We next validated Questin as a promising lead with significantly improved toxicity upon acute exposure in zebrafish larvae. Taken together, our study not only demonstrates Questin as a potent drug lead for clinical antiviral application, but also highlights multiple antiviral potentials of NSP14 as therapeutic target.
Project description:SARS-CoV-2, the causative agent of COVID-19, manipulates host gene expression through multiple mechanisms, including disruption of RNA processing. Here, we identify a novel function of the viral nonstructural protein 14 (NSP14) in inducing N7-methylguanosine (m7G) modification in the internal sequences of host mRNA. We demonstrate that NSP14 catalyzes the conversion of guanosine triphosphate (GTP) to m7GTP, which is subsequently incorporated into mRNA by RNA polymerase II, resulting in widespread internal m7G modification. This activity is dependent on NSP14's N7-methyltransferase (N7-MTase) domain and is enhanced by interaction with NSP10. Internal m7G modification by NSP14 predominantly occurs in the nucleus and is conserved across alpha-, beta- and gamma-coronaviruses. Mechanistically, we show that this RNA modification disrupts normal splicing by promoting intron retention and generating novel splice junctions. Importantly, inhibition of m7G modification, through pharmacological targeting of NSP14 or RNA polymerase II, impairs SARS-CoV-2 replication, indicating that the virus hijacks host transcriptomic machinery to support infection. Our findings reveal a previously unrecognized epitranscriptomic mechanism by which coronaviruses reprogram host gene expression and suggest that NSP14-induced m7G modification is a potential therapeutic target.