ABSTRACT
Background: The bat coronavirus RaTG13 shares 96% sequence identity to SARS-CoV-2, the causative agent of the COVID-19 pandemic. However, the RaTG13 Spike (S) protein interacts only weakly with the human SCoV-2 receptor Angiotensin-converting Enzyme 2 (ACE2) and does not mediate efficient infection of human cells. Here, we examined which alterations are required to allow the RaTG13 S protein to use human ACE2 for efficient entry into human cells. Methods: Sequence alignments showed that SARS-CoV-2 almost invariantly encodes a positively charged amino acid at position 403 of its S protein, while RaTG13 has a neutral Threonine (T). REAX based computational modeling suggested that S R403 contributes to binding of human ACE2. Wild-type and T403R mutant RaTG13 S proteins were investigated for their ability to bind ACE2 and to mediate infection of pseudotyped VSV particles in human lung-and intestine-derived cell lines as well as hPSC-derived gut organoids. Replication-competent recombinant SCoV2 S R403T was produced and replication monitored. In addition, we mutated human ACE2 to map the interacting residue of S R403. Finally, sera of vaccinated individuals were analyzed for their neutralizing potential against various WT CoV and RaTG13 S as well as mutant S containing pseudoparticles. Results: Our results show that a single amino acid change of T403R allows the RaTG13 S to utilize human ACE2 for viral entry. Spike T403R enhanced infection of VSV-based RatG13 S pseudotypes in human lung and colon cells as well as gut-derived organoids. Vice versa R403T mutation reduced infectivity of SCoV2 S pseudotypes and recombinant SCoV2 replication. The enhancing effect of T403R in RaTG13 S depends on E37 in ACE2. RaTG13 T403R S-mediated infection was blocked by the fusion inhibitor EK-1 but not by the SCoV-2 antibody Casirivimab. SARS-CoV-2 and the T403R RaTG13 S were equally susceptible to neutralization by sera from individuals vaccinated against COVID-19. Conclusion: A positively charged amino acid at position 403 in the S protein of bat coronaviruses is critical for efficient utilization of human ACE2. Our results help to better assess the zoonotic potential of bat sarbecoviruses and suggest that COVID-19 vaccination will also protect against closely bat relatives of SARS-CoV-2 that may emerge in the future.
ABSTRACT
Background: We recently showed that genuine SARS-CoV-2 hijacks endogenously expressed interferon-induced transmembrane proteins, especially IFITM2, as entry cofactors for efficient infection (Prelli Bozzo, Nchioua et al., Nat. Com., 2021). This came as a surprise, since IFITMs have been reported to inhibit entry of numerous enveloped viruses, including SARS-CoV-2. However, most data were obtained using IFITM overexpression and pseudoparticle infection assays. In our initial study, we used a SARS-CoV-2 strain isolated in the Netherlands in February 2020 (NL-02-2020). Since then several "variants of concern" (VOCs) have emerged that show increased transmission fitness and evasion of vaccine-induced immunity. These VOCs contain various alterations in their Spike (S) proteins that may alter their dependency on entry cofactors. Here, we examined whether SARS-CoV-2 VOCs, including the currently dominating Delta variant, still depend on IFITMs for efficient infection and replication. Methods: To determine the role of IFITMs in infection of SARS-CoV-2 VOCs, we silenced IFITM1, 2, or 3 expression in Calu-3 cells using siRNAs and infected them with NL-02-2020 as well VOCs B.1.1.7, B.1.351, P.1 and B.1.617.2, also referred to as Alpha, Beta, Gamma and Delta variants, respectively. Viral entry and replication were quantified by qRT-PCR as well as TCID50 analysis. In addition, we determined the inhibitory effect of an α-IFITM2 antibody on VOC infection in iPSC-derived human alveolar epithelial type 2 (iAT2) cells. Results: Depletion of IFITM2 reduced viral RNA production from 31-(B.1.1.7) to 754-fold (P.1). In comparison, KD of IFITM1 generally had little effect, while silencing of IFITM3 resulted in 2-to 20-fold reduction of viral RNA yields by the four VOCs. An antibody directed against the N-terminus of IFITM2 inhibited SARS-CoV-2 VOC replication in iAT2 cells. Conclusion: Endogenously expressed IFITM proteins (especially IFITM2) are important cofactors for entry and replication of SARS-CoV-2 VOCs, including the Delta variant that currently dominates the COVID-19 pandemic.
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
Introduction: Viral infections may trigger diabetes. Clinical data suggest infection with the pandemic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causing coronavirus disease 2019 (COVID-19), may impact glucose homeostasis in patients. Notably, cases of new-onset diabetes upon SARS-CoV-2 infection have been reported. However, experimental evidence of pancreatic infection is still controversial. Aims & Methods: Here, we employ cadaveric human pancreatic islets, as well as pancreatic tissue from deceased COVID-19 patients to investigate the impact of SARS-CoV-2 on the pancreas. Results: We show that human β-cells express viral entry proteins ACE2 and TMPRSS2, making them susceptible to SARS-CoV-2 infection and replication. Our data further demonstrates that SARS-CoV-2 infects and replicates in ex vivo cultured human islets and infection. This infection is associated with morphological, transcriptional and functional changes, such as reduction of insulin-secretory granules in β-cells and impaired glucose-stimulated insulin secretion. In COVID-19 post-mortem examinations, we detected SARS-CoV-2 nucleocapsid protein in pancreatic exocrine cells, and in cells that stain positive for the β-cell marker NKX6.1 in all patients investigated. Conclusion: Taken together, our data define the human pancreas as a target of SARS-CoV-2 infection and suggest that β-cell infection might contribute to the metabolic dysregulation observed in patients with COVID- 19.
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.