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1.
JACS Au ; 2(9): 2187-2202, 2022 Sep 26.
Article in English | MEDLINE | ID: covidwho-2050266

ABSTRACT

The COVID-19 pandemic caused by SARS-CoV-2 presents a global health emergency. Therapeutic options against SARS-CoV-2 are still very limited but urgently required. Molecular tweezers are supramolecular agents that destabilize the envelope of viruses resulting in a loss of viral infectivity. Here, we show that first-generation tweezers, CLR01 and CLR05, disrupt the SARS-CoV-2 envelope and abrogate viral infectivity. To increase the antiviral activity, a series of 34 advanced molecular tweezers were synthesized by insertion of aliphatic or aromatic ester groups on the phosphate moieties of the parent molecule CLR01. A structure-activity relationship study enabled the identification of tweezers with a markedly enhanced ability to destroy lipid bilayers and to suppress SARS-CoV-2 infection. Selected tweezer derivatives retain activity in airway mucus and inactivate the SARS-CoV-2 wildtype and variants of concern as well as respiratory syncytial, influenza, and measles viruses. Moreover, inhibitory activity of advanced tweezers against respiratory syncytial virus and SARS-CoV-2 was confirmed in mice. Thus, potentiated tweezers are broad-spectrum antiviral agents with great prospects for clinical development to combat highly pathogenic viruses.

2.
J Virol ; 96(11): e0059422, 2022 06 08.
Article in English | MEDLINE | ID: covidwho-1840553

ABSTRACT

It has recently been shown that an early SARS-CoV-2 isolate (NL-02-2020) hijacks interferon-induced transmembrane proteins (IFITMs) for efficient replication in human lung cells, cardiomyocytes, and gut organoids. To date, several "variants of concern" (VOCs) showing increased infectivity and resistance to neutralization have emerged and globally replaced the early viral strains. Here, we determined whether the five current SARS-CoV-2 VOCs (Alpha, Beta, Gamma, Delta, and Omicron) maintained the dependency on IFITM proteins for efficient replication. We found that depletion of IFITM2 strongly reduces viral RNA production by all VOCs in the human epithelial lung cancer cell line Calu-3. Silencing of IFITM1 had modest effects, while knockdown of IFITM3 resulted in an intermediate phenotype. Strikingly, depletion of IFITM2 generally reduced infectious virus production by more than 4 orders of magnitude. In addition, an antibody directed against the N terminus of IFITM2 inhibited SARS-CoV-2 VOC replication in induced pluripotent stem cell (iPSC)-derived alveolar epithelial type II cells, thought to represent major viral target cells in the lung. In conclusion, endogenously expressed IFITM proteins (especially IFITM2) are critical cofactors for efficient replication of genuine SARS-CoV-2 VOCs, including the currently dominant Omicron variant. IMPORTANCE Recent data indicate that SARS-CoV-2 requires endogenously expressed IFITM proteins for efficient infection. However, the results were obtained with an early SARS-CoV-2 isolate. Thus, it remained to be determined whether IFITMs are also important cofactors for infection of emerging SARS-CoV-2 VOCs that outcompeted the original strains in the meantime. This includes the Omicron VOC, which currently dominates the pandemic. Here, we show that depletion of endogenous IFITM2 expression almost entirely prevents productive infection of Alpha, Beta, Gamma, Delta, and Omicron SARS-CoV-2 VOCs in human lung cells. In addition, an antibody targeting the N terminus of IFITM2 inhibited SARS-CoV-2 VOC replication in iPSC-derived alveolar epithelial type II cells. Our results show that SARS-CoV-2 VOCs, including the currently dominant Omicron variant, are strongly dependent on IFITM2 for efficient replication, suggesting a key proviral role of IFITMs in viral transmission and pathogenicity.


Subject(s)
Lung , Membrane Proteins , SARS-CoV-2 , Virus Replication , COVID-19/virology , Cell Line, Tumor , Humans , Lung/virology , Membrane Proteins/genetics , Membrane Proteins/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Virus Internalization
3.
Am J Physiol Cell Physiol ; 322(4): C591-C604, 2022 04 01.
Article in English | MEDLINE | ID: covidwho-1701350

ABSTRACT

Primary airway epithelial cells (pAECs) cultivated at air-liquid interface (ALI) conditions are widely used as surrogates for human in vivo epithelia. To extend the proliferative capacity and to enable serially passaging of pAECs, conditional reprogramming (cr) has been employed in recent years. However, ALI epithelia derived from cr cells often display functional changes with increasing passages. This highlights the need for thorough validation of the ALI cultures for the respective application. In our study, we evaluated the use of serially passaged cr nasal epithelial cells (crNECs) as a model to study SARS-CoV-2 infection and effects on ion and water transport. NECs were obtained from healthy individuals and cultivated as ALI epithelia derived from passages 1, 2, 3, and 5. We compared epithelial differentiation, ion and water transport, and infection with SARS-CoV-2 between passages. Our results show that epithelia maintained major differentiation characteristics and physiological ion and water transport properties through all passages. However, the frequency of ciliated cells, short circuit currents reflecting epithelial Na+ channel (ENaC) and cystic fibrosis transmembrane conductance regulator (CFTR) activity and expression of aquaporin 3 and 5 decreased gradually over passages. crNECs also expressed SARS-CoV-2 receptors angiotensin converting enzyme 2 (ACE2) and transmembrane serin2 protease 2 (TMPRSS2) across all passages and allowed SARS-CoV-2 replication in all passages. In summary, we provide evidence that passaged crNECs provide an appropriate model to study SARS-CoV-2 infection and also epithelial transport function when considering some limitations that we defined herein.


Subject(s)
COVID-19 , Cell Differentiation , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Epithelial Cells/metabolism , Humans , Infant, Newborn , SARS-CoV-2
4.
EuropePMC; 2020.
Preprint in English | EuropePMC | ID: ppcovidwho-317546

ABSTRACT

Preexisting diabetes increases the risk of a severe course of the pandemic coronavirus disease 2019 (COVID-19). Vice versa, exacerbations of a preexisting diabetes as well as new-onset diabetes have been reported upon SARS-CoV-2 infection. Thus, there is an imperative need to clarify whether human pancreatic endocrine cells organized within an islet of Langerhans are permissive for and affected by SARS-CoV-2 infection, and to elucidate the mechanisms underlying the development of diabetes upon COVID-19. Here, we (i) defined ACE2 and TMPRSS2 expression patterns in human pancreatic endocrine and exocrine cell types, (ii) employed human pancreatic islet cultures to demonstrate susceptibility to SARS-CoV-2 infection and to viral replication in β-cells, (iii) showed that SARS-CoV-2 attenuates glucose-stimulated insulin secretion, and (iv) tested remdesivir as eventually effective to prevent β-cell failure. In addition, we (v) visualized viral particles replicating in endocrine pancreatic cells and define their subcellular localization patterns via transmission electron microscopy, and finally (vi) present examples of cell type specific pancreatic infection patterns of COVID-19 deceased patients. Overall, our data demonstrate that SARS-CoV-2 can infect both the exocrine and endocrine compartments of the pancreas and can perturb β-cell integrity, which might lead to an increased risk for diabetes.

5.
Cells ; 11(1)2021 12 24.
Article in English | MEDLINE | ID: covidwho-1580995

ABSTRACT

The lamellar body (LB) of the alveolar type II (ATII) cell is a lysosome-related organelle (LRO) that contains surfactant, a complex mix of mainly lipids and specific surfactant proteins. The major function of surfactant in the lung is the reduction of surface tension and stabilization of alveoli during respiration. Its lack or deficiency may cause various forms of respiratory distress syndrome (RDS). Surfactant is also part of the innate immune system in the lung, defending the organism against air-borne pathogens. The limiting (organelle) membrane that encloses the LB contains various transporters that are in part responsible for translocating lipids and other organic material into the LB. On the other hand, this membrane contains ion transporters and channels that maintain a specific internal ion composition including the acidic pH of about 5. Furthermore, P2X4 receptors, ligand gated ion channels of the danger signal ATP, are expressed in the limiting LB membrane. They play a role in boosting surfactant secretion and fluid clearance. In this review, we discuss the functions of these transporting pathways of the LB, including possible roles in disease and as therapeutic targets, including viral infections such as SARS-CoV-2.


Subject(s)
COVID-19/metabolism , Ion Channels/metabolism , Lung/metabolism , Membrane Transport Proteins/metabolism , Pulmonary Surfactants/metabolism , COVID-19/virology , Humans , Lung/virology , Organelles/metabolism , Organelles/virology , Pulmonary Alveoli/metabolism , Pulmonary Alveoli/virology , SARS-CoV-2/physiology
6.
EuropePMC; 2021.
Preprint in English | EuropePMC | ID: ppcovidwho-296869

ABSTRACT

It has recently been shown that an early SARS-CoV-2 isolate (NL-02-2020) hijacks interferon-induced transmembrane proteins (IFITMs) for efficient replication in human cells. To date, several "Variants of Concern" (VOCs) showing increased infectivity and resistance to neutralization have emerged and globally replaced the early viral strains. Here, we determined whether the four SARS-CoV-2 VOCs (Alpha, Beta, Gamma and Delta) maintained the dependency on IFITM proteins for efficient replication. We found that depletion of IFITM2 strongly reduces viral RNA production by all four VOCs in the human epithelial lung cancer cell line Calu-3. Silencing of IFITM1 had little effect, while knock-down of IFITM3 resulted in an intermediate phenotype. Strikingly, depletion of IFITM2 generally reduced infectious virus production by more than four orders of magnitude. In addition, an antibody directed against the N-terminus of IFITM2 inhibited SARS-CoV-2 VOC replication in iPSC-derived alveolar epithelial type II cells thought to represent major viral target cells in the lung. In conclusion, endogenously expressed IFITM proteins (especially IFITM2) are critical cofactors for efficient replication of genuine SARS-CoV-2 VOCs, including the currently dominating Delta variant.

7.
EuropePMC; 2021.
Preprint in English | EuropePMC | ID: ppcovidwho-293046

ABSTRACT

We have recently shown that a SARS-CoV-2 strain isolated in the Netherlands in February 2020 (NL-02-2020) hijacks interferon-induced transmembrane proteins, especially IFITM2, as entry cofactors for efficient infection. Here, we examined whether SARS-CoV-2 'variants of concern' (VOCs), including the currently dominating delta variant, maintained the dependency on IFITMs for efficient replication. Depletion of IFITM2 reduced viral RNA production from 31- (B.1.1.7) to 755-fold (P.1). In comparison, silencing of IFITM1 had little effect, while knock-down of IFITM3 resulted in an intermediate phenotype. Strikingly, silencing of IFITM2 generally reduced infectious virus production in Calu-3 cells to near background levels. An antibody directed against the N-terminus of IFITM2 inhibited SARS-CoV-2 VOC replication in iPSC-derived alveolar epithelial type II cells. In conclusion, endogenously expressed IFITM proteins (especially IFITM2) are important cofactors for replication of genuine SARS-CoV-2 VOCs, including the Delta variant.

8.
EuropePMC; 2021.
Preprint in English | EuropePMC | ID: ppcovidwho-293045

ABSTRACT

Interferons are a major part of the anti-viral innate defense system. Successful pathogens, including the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), need to overcome these defenses to establish an infection. Early induction of interferons (IFNs) protects against severe coronavirus disease 2019 (COVID-19). In line with this, SARS-CoV-2 is inhibited by IFNs in vitro, and IFN-based therapies against COVID-19 are investigated in clinical trials. However, SARS-CoV-2 continues to adapt to the human population resulting in the emergence of variants characterized by increased transmission fitness and/or decreased sensitivity to preventive or therapeutic measures. It has been suggested that the efficient spread of these so-called "Variants of Concern" (VOCs) may also involve reduced sensitivity to IFNs. Here, we examined whether the four current VOCs (Alpha, Beta, Gamma and Delta) differ in replication efficiency or IFN sensitivity from an early isolate of SARS-CoV-2. All viruses replicated in a human lung cell line and in iPSC-derived alveolar type II cells (iAT2). The Delta variant showed accelerated replication kinetics and higher infectious virus production compared to the early 2020 isolate. Replication of all SARS-CoV-2 VOCs was reduced in the presence of exogenous type I, II and III IFNs. On average, the Alpha variant was the least susceptible to IFNs and the Alpha, Beta and Gamma variants show increased resistance against type III IFN. Although the Delta variant has outcompeted all other variants in humans it remained as sensitive to IFNs as an early 2020 SARS-CoV-2 isolate. This suggests that increased replication fitness rather than IFN resistance may be a reason for its dominance. Our results may help to understand changes in innate immune susceptibility of VOCs, and inform clinical trials exploring IFN-based COVID-19 therapies.

9.
Nat Commun ; 12(1): 1726, 2021 03 19.
Article in English | MEDLINE | ID: covidwho-1142436

ABSTRACT

SARS-CoV-2 is a respiratory pathogen and primarily infects the airway epithelium. As our knowledge about innate immune factors of the respiratory tract against SARS-CoV-2 is limited, we generated and screened a peptide/protein library derived from bronchoalveolar lavage for inhibitors of SARS-CoV-2 spike-driven entry. Analysis of antiviral fractions revealed the presence of α1-antitrypsin (α1AT), a highly abundant circulating serine protease inhibitor. Here, we report that α1AT inhibits SARS-CoV-2 entry at physiological concentrations and suppresses viral replication in cell lines and primary cells including human airway epithelial cultures. We further demonstrate that α1AT binds and inactivates the serine protease TMPRSS2, which enzymatically primes the SARS-CoV-2 spike protein for membrane fusion. Thus, the acute phase protein α1AT is an inhibitor of TMPRSS2 and SARS-CoV-2 entry, and may play an important role in the innate immune defense against the novel coronavirus. Our findings suggest that repurposing of α1AT-containing drugs has prospects for the therapy of COVID-19.


Subject(s)
COVID-19/drug therapy , SARS-CoV-2/drug effects , Serine Endopeptidases/metabolism , Serine Proteinase Inhibitors/pharmacology , alpha 1-Antitrypsin/pharmacology , Antibodies, Viral/blood , Antiviral Agents/pharmacology , COVID-19/blood , Caco-2 Cells , Humans , Immunoglobulin G/blood , Molecular Docking Simulation , Spike Glycoprotein, Coronavirus/metabolism , Virus Internalization/drug effects , Virus Replication/drug effects
10.
Am J Physiol Lung Cell Mol Physiol ; 320(5): L750-L756, 2021 05 01.
Article in English | MEDLINE | ID: covidwho-1076012

ABSTRACT

Pharmaceutical interventions are urgently needed to prevent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and transmission. As SARS-CoV-2 infects and spreads via the nasopharyngeal airways, we analyzed the antiviral effect of selected nasal and oral sprays on virus infection in vitro. Two nose sprays showed virucidal activity but were cytotoxic precluding further analysis in cell culture. One nasal and one mouth spray suppressed SARS-CoV-2 infection of TMPRSS2-expressing Vero E6 cells and primary differentiated human airway epithelial cultures. The antiviral activity in both sprays could be attributed to polyanionic ι- and κ-carrageenans. Thus, application of carrageenan-containing nasal and mouth sprays may reduce the risk of acquiring SARS-CoV-2 infection and may limit viral spread, warranting further clinical evaluation.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , COVID-19/prevention & control , Carrageenan/pharmacology , SARS-CoV-2/drug effects , Adult , Animals , Cell Line , Chlorocebus aethiops , Epithelial Cells/drug effects , Epithelial Cells/virology , Female , Humans , Male , Middle Aged , Nasal Sprays , Oral Sprays , Serine Endopeptidases/metabolism , Vero Cells
11.
Nat Metab ; 3(2): 149-165, 2021 02.
Article in English | MEDLINE | ID: covidwho-1065968

ABSTRACT

Infection-related diabetes can arise as a result of virus-associated ß-cell destruction. Clinical data suggest that the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causing the coronavirus disease 2019 (COVID-19), impairs glucose homoeostasis, but experimental evidence that SARS-CoV-2 can infect pancreatic tissue has been lacking. In the present study, we show that SARS-CoV-2 infects cells of the human exocrine and endocrine pancreas ex vivo and in vivo. We demonstrate that human ß-cells express viral entry proteins, and SARS-CoV-2 infects and replicates in cultured human islets. Infection is associated with morphological, transcriptional and functional changes, including reduced numbers of insulin-secretory granules in ß-cells and impaired glucose-stimulated insulin secretion. In COVID-19 full-body postmortem examinations, we detected SARS-CoV-2 nucleocapsid protein in pancreatic exocrine cells, and in cells that stain positive for the ß-cell marker NKX6.1 and are in close proximity to the islets of Langerhans in all four patients investigated. Our data identify the human pancreas as a target of SARS-CoV-2 infection and suggest that ß-cell infection could contribute to the metabolic dysregulation observed in patients with COVID-19.


Subject(s)
Islets of Langerhans/virology , SARS-CoV-2/growth & development , Aged , Aged, 80 and over , Angiotensin-Converting Enzyme 2/biosynthesis , Angiotensin-Converting Enzyme 2/genetics , COVID-19/physiopathology , Cells, Cultured , Diabetes Mellitus , Female , Humans , Islets of Langerhans/cytology , Islets of Langerhans/physiopathology , Male , Pancreas, Exocrine/cytology , Pancreas, Exocrine/physiopathology , Pancreas, Exocrine/virology , Pancreatic Diseases/etiology , Pancreatic Diseases/virology , Serine Endopeptidases/biosynthesis , Serine Endopeptidases/genetics , Virus Internalization , Virus Replication
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