Briefly, 5?l of the aqueous metabolite extract were injected in full loop mode using an overfill factor of 3, onto a Dionex IonPac AS11-HC column (2?mm 250?mm, 4 m particle size, Thermo Scientific) equipped with a Dionex IonPac AG11-HC guard column (2?mm 50?mm, 4 m, Thermo Scientific). autophagy limits SARS-CoV-2 propagation. In detail, SARS-CoV-2-infected cells show accumulation of key metabolites, activation of autophagy inhibitors (AKT1, SKP2) and reduction of proteins responsible for autophagy initiation (AMPK, TSC2, ULK1), membrane nucleation, and phagophore formation (BECN1, VPS34, ATG14), SGI-7079 as well as autophagosome-lysosome fusion (BECN1, ATG14 oligomers). Consequently, phagophore-incorporated autophagy markers LC3B-II and P62 accumulate, which we SGI-7079 confirm in a hamster model and lung samples of COVID-19 patients. Single-nucleus and single-cell sequencing of patient-derived?lung and mucosal samples SGI-7079 show differential transcriptional regulation of autophagy and immune genes depending on cell SGI-7079 type, disease duration, and SARS-CoV-2 replication levels. Targeting of autophagic pathways by exogenous administration of the polyamines spermidine and spermine, the selective AKT1 inhibitor MK-2206, and the BECN1-stabilizing anthelmintic drug niclosamide inhibit SARS-CoV-2 propagation in vitro with IC50 values of 136.7, 7.67, 0.11, and 0.13?M, respectively. Autophagy-inducing compounds reduce SARS-CoV-2 propagation in primary human lung cells and intestinal organoids emphasizing their potential as treatment options against COVID-19. expression in SARS-CoV-2-infected cells (Supplementary Fig.?5c). is a known IFN-stimulated gene18 and its upregulation was comparable to other prototypic innate immune genes (and encoding key autophagy markers LC3B and P62 remained unaffected whereas and showed that low pH autophagolysosomes (AL, red) were reduced compared to autophagosomes (AP, green + red = yellow) in virus-infected cells. Microscopic read-out was done by a scientist blind to the experimental conditions. For mock (and remained overall unchanged throughout the course of infection (Supplementary Fig.?8) suggesting that increased P62 and LC3B-II levels were not caused by transcriptional upregulation, but rather accumulated by a SARS-CoV-2-induced blockage of autophagy. Furthermore, we compared postmortem lung samples of COVID-19 patients (and mRNA levels or again by a reduced autophagic flux and protein accumulation (Fig.?3b, Supplementary Table?6). To distinguish between these possible explanations, we performed sNuc-seq-based transcriptional analyses for seven deceased individuals with severe COVID-19, of which three deceased early ( 14 days) and four late ( 14 days) post onset of symptoms (Supplementary Table?7). Cell types were defined as previously described38 (Supplementary Fig.?9aCi). CDKN2A The analyses focused on autophagy-affected genes (to monitor activation, autophagy-affecting polyamine-regulating and IFN-stimulated genes (ISGs) and in multiple cell types (Supplementary Fig.?9j) especially secretory, ciliated and AT2 cells, as the latter two are the predominant target cells for SARS-CoV-2 infection39 (Fig.?3c). In secretory cells (Fig.?3c, left), most target genes were comparably expressed between control and early-deceased COVID-19 patients but downregulated in cells from late-deceased individuals. In the main SARS-CoV-2 target cells (ciliated, AT2), we observed a similar pattern between early- and late-deceased patients. However, mRNA levels for IFN-stimulated and autophagy genes were much more increased compared to secretory cells and were highly comparable to a cellular subpopulation in which high viral RNA levels were detected (Fig.?3c, SARS2high, Supplementary Fig.?9d). mRNA levels SGI-7079 were similar to those of all other ISGs, further supporting a link between IFN response and polyamine-dependent autophagy interventions (see Fig.?1c). As early- compared to late-deceased patients showed higher virus RNA levels ( 105 virus RNA copies per 10,000 cells, Supplementary Table?7), we suspected that SARS-CoV-2 replication levels might influence transcriptional regulation. To further explore this, we re-analyzed single-cell sequencing (scSeq) data sets from mucosal brushes of hospitalized COVID-19 patients38 experiencing low to moderate ( 105 GE/ml, was upregulated in secretory cells and comparable or even reduced in putatively SARS-CoV-2-infected ciliated cells (Fig.?3d). Congruently, and similar to our in vitro metabolomics data (see Fig.?1), polyamine-regulating.
Briefly, 5?l of the aqueous metabolite extract were injected in full loop mode using an overfill factor of 3, onto a Dionex IonPac AS11-HC column (2?mm 250?mm, 4 m particle size, Thermo Scientific) equipped with a Dionex IonPac AG11-HC guard column (2?mm 50?mm, 4 m, Thermo Scientific)