SARS-CoV-2 variant Alpha has a spike-dependent replication advantage over the ancestral B.1 strain in human cells with low ACE2 expression.

Epidemiological data demonstrate that Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) Alpha and Delta are more transmissible, infectious, and pathogenic than previous variants. Phenotypic properties of VOC remain understudied. Here, we provide an extensive functional study of VOC Alpha replication and cell entry phenotypes assisted by reverse genetics, mutational mapping of spike in lentiviral pseudotypes, viral and cellular gene expression studies, and infectivity stability assays in an enhanced range of cell and epithelial culture models. In almost all models, VOC Alpha spread less or equally efficiently as ancestral (B.1) SARS-CoV-2. B.1. and VOC Alpha shared similar susceptibility to serum neutralization. Despite increased relative abundance of specific sgRNAs in the context of VOC Alpha infection, immune gene expression in infected cells did not differ between VOC Alpha and B.1. However, inferior spreading and entry efficiencies of VOC Alpha corresponded to lower abundance of proteolytically cleaved spike products presumably linked to the T716I mutation. In addition, we identified a bronchial cell line, NCI-H1299, which supported 24-fold increased growth of VOC Alpha and is to our knowledge the only cell line to recapitulate the fitness advantage of VOC Alpha compared to B.1. Interestingly, also VOC Delta showed a strong (595-fold) fitness advantage over B.1 in these cells. Comparative analysis of chimeric viruses expressing VOC Alpha spike in the backbone of B.1, and vice versa, showed that the specific replication phenotype of VOC Alpha in NCI-H1299 cells is largely determined by its spike protein. Despite undetectable ACE2 protein expression in NCI-H1299 cells, CRISPR/Cas9 knock-out and antibody-mediated blocking experiments revealed that multicycle spread of B.1 and VOC Alpha required ACE2 expression. Interestingly, entry of VOC Alpha, as opposed to B.1 virions, was largely unaffected by treatment with exogenous trypsin or saliva prior to infection, suggesting enhanced resistance of VOC Alpha spike to premature proteolytic cleavage in the extracellular environment of the human respiratory tract. This property may result in delayed degradation of VOC Alpha particle infectivity in conditions typical of mucosal fluids of the upper respiratory tract that may be recapitulated in NCI-H1299 cells closer than in highly ACE2-expressing cell lines and models. Our study highlights the importance of cell model evaluation and comparison for in-depth characterization of virus variant-specific phenotypes and uncovers a fine-tuned interrelationship between VOC Alpha- and host cell-specific determinants that may underlie the increased and prolonged virus shedding detected in patients infected with VOC Alpha.

Nonproductive exposure of PBMCs to SARS-CoV-2 induces cell-intrinsic innate immune responses.

Cell-intrinsic responses mounted in PBMCs during mild and severe COVID-19 differ quantitatively and qualitatively. Whether they are triggered by signals emitted by productively infected cells of the respiratory tract or result from physical interaction with virus particles remains unclear. Here, we analyzed susceptibility and expression profiles of PBMCs from healthy donors upon ex vivo exposure to SARS-CoV and SARS-CoV-2. In line with the absence of detectable ACE2 receptor expression, human PBMCs were refractory to productive infection. RT-PCR experiments and single-cell RNA sequencing revealed JAK/STAT-dependent induction of interferon-stimulated genes (ISGs) but not proinflammatory cytokines. This SARS-CoV-2-specific response was most pronounced in monocytes. SARS-CoV-2-RNA-positive monocytes displayed a lower ISG signature as compared to bystander cells of the identical culture. This suggests a preferential invasion of cells with a low ISG baseline profile or delivery of a SARS-CoV-2-specific sensing antagonist upon efficient particle internalization. Together, nonproductive physical interaction of PBMCs with SARS-CoV-2- and, to a much lesser extent, SARS-CoV particles stimulate JAK/STAT-dependent, monocyte-accentuated innate immune responses that resemble those detected in vivo in patients with mild COVID-19.

Corrigendum: Antiviral and Immunomodulatory Effects of Pelargonium sidoides DC. Root Extract EPs® 7630 in SARS-CoV-2-Infected Human Lung Cells.

[This corrects the article DOI: 10.3389/fphar.2021.757666.].

Antiviral and Immunomodulatory Effects of Pelargonium sidoides DC. Root Extract EPs® 7630 in SARS-CoV-2-Infected Human Lung Cells.

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-1ß 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.

SARS-CoV-2-mediated dysregulation of metabolism and autophagy uncovers host-targeting antivirals.

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.