Autophagy is an essential cellular process affecting virus infections and other diseases and Beclin1 (BECN1) is one of its key regulators. Here, we identified S-phase kinase-associated protein 2 (SKP2) as E3 ligase that executes lysine-48-linked poly-ubiquitination of BECN1, thus promoting its proteasomal degradation. SKP2 activity is regulated by phosphorylation in a hetero-complex involving FKBP51, PHLPP, AKT1, and BECN1. Genetic or pharmacological inhibition of SKP2 decreases BECN1 ubiquitination, decreases BECN1 degradation and enhances autophagic flux. Middle East respiratory syndrome coronavirus (MERS-CoV) multiplication results in reduced BECN1 levels and blocks the fusion of autophagosomes and lysosomes. Inhibitors of SKP2 not only enhance autophagy but also reduce the replication of MERS-CoV up to 28,000-fold. The SKP2-BECN1 link constitutes a promising target for host-directed antiviral drugs and possibly other autophagy-sensitive conditions.

Detailed knowledge of the molecular biology of SARS-CoV-2 infection is crucial for understanding of viral replication, host responses and disease progression. Here, we report gene expression profiles of three SARS-CoV and SARS-CoV-2 infected human cell lines. SARS-CoV-2 elicited an approximately two-fold higher stimulation of the innate immune response compared to SARS-CoV in the human epithelial cell line Calu-3, including induction of miRNA-155. Single-cell RNA sequencing of infected cells showed that genes induced by virus infections were broadly upregulated, whereas interferon beta/lambda genes an pro-inflammatory cytokines such as IL-6 were expressed only in small subsets of infected cells. Temporal analysis suggested that transcriptional activities of interferon regulatory factors precede those of nuclear factor κB. Lastly, we identified heat shock protein 90 (HSP90) as a protein relevant for the infection. Inhibition of the HSP90 activity resulted in a reduction of viral replication and pro-inflammatory cytokine expression in primary human airway epithelial cells.

21Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses an acute threat to public health 22 and the world economy, especially because no approved specific drugs or vaccines are available. 23Pharmacological modulation of metabolism-dependent cellular pathways such as autophagy reduced 24 propagation of highly pathogenic Middle East respiratory syndrome (MERS)-CoV. 25Here we show that SARS-CoV-2 infection limits autophagy by interfering with multiple metabolic 26 pathways and that compound-driven interventions aimed at autophagy induction reduce SARS-CoV-2 27 propagation in vitro. In-depth analyses of autophagy signaling and metabolomics indicate that SARS-28 CoV-2 reduces glycolysis and protein translation by limiting activation of AMP-protein activated kinase 29 (AMPK) and mammalian target of rapamycin complex 1 (mTORC1). Infection also downregulates 30 autophagy-inducing spermidine, and facilitates AKT1/SKP2-dependent degradation of autophagy-31 initiating Beclin-1 (BECN1). Targeting of these pathways by exogenous administration of spermidine, 32AKT inhibitor MK-2206, and the Beclin-1 stabilizing, antihelminthic drug niclosamide inhibited SARS- 33CoV-2 propagation by 85, 88, and >99%, respectively. In sum, SARS-CoV-2 infection causally diminishes 34 autophagy. A clinically approved and well-tolerated autophagy-inducing compound shows potential 35 for evaluation as a treatment against SARS-CoV-2. 36 37 38 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint this version posted April 15, 2020. . https://doi.org/10.1101 3 The current pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses an 39 imminent threat to global health. As of 15 April 2020, 1,878,489 individuals were infected in >200 40 countries, with >119,000 fatalities (1). SARS-CoV-2 infections cause CoV-associated disease 19 (COVID-41 19), which can lead to severe atypical pneumonia in humans (2). Currently, there are no approved 42 therapeutics or vaccines available. The development and licensing of new FDA-approved drugs takes 43 years, which is problematic given the urgent need for effective therapies against novel, rapidly 44 emergent diseases like COVID-19. Antiviral drug screenings are commonly based on testing FDA-45 approved compound libraries against cellular and viral components (3). However, these undirected 46 approaches lack functional insights into how the drugs affect virus propagation. Risk evaluations for 47 drug repurposing and development of new therapeutics would benefit from rational drug design 48 founded on known SARS-CoV-2-host interactions. 49 Compound-based targeting of cellular proteins that are essential for the virus life cycle has led to the 50 discovery of broadly reactive drugs against a range of CoVs (3-6). As virus propagation strongly 51 depends on energy and catabolic substrates of host cells, drug target identification should consider 52 the metabolism of infected cells (3)....

Viruses manipulate cellular metabolism and macromolecule recycling processes like autophagy. Dysregulated metabolism might lead to excessive inflammatory and autoimmune responses as observed in severe and long COVID-19 patients. Here we show that SARS-CoV-2 modulates cellular metabolism and reduces autophagy. Accordingly, compound-driven induction of 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), as well as autophagosome-lysosome fusion (BECN1, ATG14 oligomers). Consequently, phagophore-incorporated autophagy markers LC3B-II and P62 accumulate, which we 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 show differential transcriptional regulation of autophagy and immune genes depending on cell 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 μM, 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.

Treatment options for COVID-19 are currently limited. Drugs reducing both viral loads and SARS-CoV-2-induced inflammatory responses would be ideal candidates for COVID-19 therapeutics. Previous in vitro and clinical studies suggest that the proprietary Pelargonium sidoides DC. root extract EPs 7630 has antiviral and immunomodulatory properties, limiting symptom severity and disease duration of infections with several upper respiratory viruses. Here we assessed if EPs 7630 affects SARS-CoV-2 propagation and the innate immune response in the human lung cell line Calu-3. In direct comparison to other highly pathogenic CoV (SARS-CoV, MERS-CoV), SARS-CoV-2 growth was most efficiently inhibited at a non-toxic concentration with an IC50 of 1.61 μg/ml. Particularly, the cellular entry step of SARS-CoV-2 was significantly reduced by EPs 7630 pretreatment (10–100 μg/ml) as shown by spike protein-carrying pseudovirus particles and infectious SARS-CoV-2. Using sequential ultrafiltration, EPs 7630 was separated into fractions containing either prodelphinidins of different oligomerization degrees or small molecule constituents like benzopyranones and purine derivatives. Prodelphinidins with a low oligomerization degree and small molecule constituents were most efficient in inhibiting SARS-CoV-2 entry already at 10 μg/ml and had comparable effects on immune gene regulation as EPs 7630. Downregulation of multiple pro-inflammatory genes (CCL5, IL6, IL1B) was accompanied by upregulation of anti-inflammatory TNFAIP3 at 48 h post-infection. At high concentrations (100 μg/ml) moderately oligomerized prodelphinidins reduced SARS-CoV-2 propagation most efficiently and exhibited pronounced immune gene modulation. Assessment of cytokine secretion in EPs 7630-treated and SARS-CoV-2-coinfected Calu-3 cells showed that pro-inflammatory cytokines IL-1β and IL-6 were elevated whereas multiple other COVID-19-associated cytokines (IL-8, IL-13, TNF-α), chemokines (CXCL9, CXCL10), and growth factors (PDGF, VEGF-A, CD40L) were significantly reduced by EPs 7630. SARS-CoV-2 entry inhibition and the differential immunomodulatory functions of EPs 7630 against SARS-CoV-2 encourage further in vivo studies.

Highlights d Bat mumps virus can readily replicate in human cells d Host cell entry factors of bat mumps virus are identical to those of human viruses d Bat mumps virus can evade innate immune responses in human and in bat cells d Neurotoxic properties of bat mumps virus are comparable to human field isolates

BackgroundSmall mammals such as bats and rodents have been increasingly recognized as reservoirs of novel potentially zoonotic pathogens. However, few in vitro model systems to date allow assessment of zoonotic viruses in a relevant host context. The cotton rat (Sigmodon hispidus) is a New World rodent species that has a long-standing history as an experimental animal model due to its unique susceptibility to human viruses. Furthermore, wild cotton rats are associated with a large variety of known or potentially zoonotic pathogens.MethodsA method for the isolation and culture of airway epithelial cell lines recently developed for bats was applied for the generation of rodent airway and renal epithelial cell lines from the cotton rat. Continuous cell lines were characterized for their epithelial properties as well as for their interferon competence. Susceptibility to members of zoonotic Bunya-, Rhabdo-, and Flaviviridae, in particular Rift Valley fever virus (RVFV), vesicular stomatitis virus (VSV), West Nile virus (WNV), and tick-borne encephalitis virus (TBEV) was tested. Furthermore, novel arthropod-derived viruses belonging to the families Bunya-, Rhabdo-, and Mesoniviridae were tested.ResultsWe successfully established airway and kidney epithelial cell lines from the cotton rat, and characterized their epithelial properties. Cells were shown to be interferon-competent. Viral infection assays showed high-titre viral replication of RVFV, VSV, WNV, and TBEV, as well as production of infectious virus particles. No viral replication was observed for novel arthropod-derived members of the Bunya-, Rhabdo-, and Mesoniviridae families in these cell lines.ConclusionIn the current study, we showed that newly established cell lines from the cotton rat can serve as host-specific in vitro models for viral infection experiments. These cell lines may also serve as novel tools for virus isolation, as well as for the investigation of virus-host interactions in a relevant host species.

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