ABSTRACT
Background: The innate immune system is a powerful anti-viral defense mechanism, which includes the interferon (IFN) system and autophagy. Thus, successful pathogens like SARS-CoV-2 need to counteract or evade these defenses to establish an infection. However, due to its ongoing, worldwide spread in the human population SARS-CoV-2 is evolving and in the meantime four variants with selection advantages (variants of concern) emerged. Methods: Using expression constructs for 29 SARS-CoV-2 proteins we evaluated the impact of individual viral proteins on induction of cytokines (IFNA4, IFNB1, IRF3-signalling, NF-κB-signaling) and cytokine signaling (IFNα2, IFNβ, IFNγ, IFNa;1, IL-1α, TNFα) in luciferase reporter assays, validated by endogenous transcription factor phosphorylation analysis. We assessed the influence of SARS-CoV-2 proteins on autophagy using a flow cytometry-based system. Underlying molecular mechanisms were investigated on an endogenous level using Western blot, confocal fluorescence microscopy, and flow cytometry. In addition, we examined the susceptibility of SARS-CoV-2 including all variants of concern towards type-I,-II, and-III interferons. Results: To understand how SARS-CoV-2 efficiently manipulates the host's innate immune defenses, we systematically analyzed the impact of SARS-CoV-2 encoded proteins on induction of various IFNs and pro-inflammatory cytokines, IFN signaling, and autophagy. Our results reveal the range of innate immune antagonists encoded by SARS-CoV-2 and we characterized selected molecular mechanisms employed by Nsp1 and Nsp14 to downregulate the IFN system or ORF3a and ORF7a to prevent autophagic degradation. Interestingly, our assays show that variants of concern of SARS-CoV-2 remain sensitive to type-II interferon signaling but show increased resistance towards type-I and/or type-III interferons. Conclusion: SARS-CoV-2 has evolved to counteract innate immunity using several synergistic approaches but remains relatively sensitive to type-II and-III interferons. However, emerged variants of concern remain sensitive overall but are less susceptible towards IFNα2/β and IFNa;1 than early SARS-CoV-2 isolates.
ABSTRACT
Background: The coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), mainly affects the lung, but may also result in extrapulmonary manifestations such as lesions in kidneys, heart, brain, gastrointestinal and endocrine organs. Clinical data suggest that a SARS-CoV-2 infection disturbs glucose homeostasis, and cases of new-onset diabetes mellitus after SARS-CoV-2 infection have been reported. However, experimental evidence that SARS-CoV-2 can infect pancreatic tissue is lacking. We here explored whether pancreatic tissue is susceptible to SARS-CoV-2 infection. Methods: We analyzed healthy human pancreas tissue and cells for ACE2 and TMPRSS2 expression by immunohistochemistry. We exposed human Langerhans islets to SARS-CoV-2 ex vivo and determined viral infection by staining for SARS-CoV-2 spike and nucleoprotein. Viral replication was monitored by detection of released viral RNA by qPCR and infectious titers by TCID50 titration. In addition, infection and the impact of SARS-CoV-2 on cell morphology was examined by electron microscopy. Consequential changes in cell functionality were analyzed by determining insulin secretion and performing transcriptomics. Finally, we performed immunohistochemistry staining of pancreatic sections of four COVID-19 deceased individuals for the presence of SARS-CoV-2 nucleoprotein. Results: Our results show that SARS-CoV-2 infects cells of the human exocrine and endocrine pancreas ex vivo and in vivo. We demonstrate that human β-cells express ACE2 and TMPRSS2, and support SARS-CoV-2 replication. The infection was associated with morphological, transcriptional and functional changes, including reduced numbers of insulin secretory granules in β-cells, upregulation of antiviral gene expression, and impaired glucose-stimulated insulin secretion. Finally, all four analyzed full body autopsies of COVID-19 patients showed SARSCoV-2 nucleoprotein in pancreatic cells, including those that stain positive for the β-cell marker NKX6.1. Conclusion: Our data demonstrate that the human pancreas is a target of SARS-CoV-2 ex vivo and in vivo and suggest that β-cell infection may contribute to pancreatic dysregulation observed in COVID-19 patients.
ABSTRACT
Background: Interferon-induced transmembrane proteins (IFITMs 1, 2 and 3) are a family of interferon (IFN) stimulated genes (ISGs) well-known to inhibit entry of numerous enveloped viruses including the severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1). However, the mechanism(s) underlying the antiviral activity of IFITM proteins are not fully understood and most evidence comes from single-round pseudoparticle infection assays of cells artificially overexpressing IFITM proteins. Here, we examined whether and how endogenous IFITM proteins may affect infection by the novel pandemic coronavirus SARS-CoV-2. Methods: SARS-CoV-2 Spike (S) and ACE2 mediated pseudoparticle entry in HEK293T cells overexpressing IFITMs was quantified using luciferase reporter assays. To determine the role of IFITMs under more physiological conditions, we silenced IFITM protein expression in Calu-3 cells and in primary airway epithelial cells (SAEC) using siRNAs and infected them with genuine SARS-CoV-2. Viral entry and replication were quantified by qRT-PCR as well as plaque assays. To clarify whether IFITMs represent suitable therapeutic targets, we analyzed whether antibodies against IFITM proteins inhibit SARS-CoV-2 infection of lung cells. Results: Our results show that overexpression of IFITM protein blocks ACE2 and SARS-CoV-2 Spike mediated pseudoparticle infection. In striking contrast, however, endogenous IFITM expression was required for efficient entry and replication of genuine SARS-CoV-2 in Calu-3 lung cells and primary lung cells both in the presence and absence of interferons. Efficient endogenous expression of IFITM1 and IFITm2 enhanced SARS-CoV-2 replication in Calu-3 and SAEC by several orders of magnitude. In addition, antibodies directed against IFITM proteins inhibited SARS-CoV-2 replication in lung cells. Conclusion: IFITM proteins are cofactors for efficient SARS-CoV-2 infection of human lung cells and represent novel, unexpected targets for the treatment of COVID-19.