Coronavirus entry is mediated by the spike protein that binds the receptor and mediates fusion after cleavage by host proteases. The proteases that mediate entry differ between cell lines, and it is ...currently unclear which proteases are relevant in vivo. A remarkable feature of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike is the presence of a multibasic cleavage site (MBCS), which is absent in the SARS-CoV spike. Here, we report that the SARS-CoV-2 spike MBCS increases infectivity on human airway organoids (hAOs). Compared with SARS-CoV, SARS-CoV-2 entered faster into Calu-3 cells and, more frequently, formed syncytia in hAOs. Moreover, the MBCS increased entry speed and plasma membrane serine protease usage relative to cathepsin-mediated endosomal entry. Blocking serine proteases, but not cathepsins, effectively inhibited SARS-CoV-2 entry and replication in hAOs. Our findings demonstrate that SARS-CoV-2 enters relevant airway cells using serine proteases, and suggest that the MBCS is an adaptation to this viral entry strategy.
SARS-CoV-2 productively infects human gut enterocytes Lamers, Mart M; Beumer, Joep; van der Vaart, Jelte ...
Science (American Association for the Advancement of Science),
07/2020, Letnik:
369, Številka:
6499
Journal Article
Recenzirano
Odprti dostop
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can cause coronavirus disease 2019 (COVID-19), an influenza-like disease that is primarily thought to infect the lungs with transmission ...through the respiratory route. However, clinical evidence suggests that the intestine may present another viral target organ. Indeed, the SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2) is highly expressed on differentiated enterocytes. In human small intestinal organoids (hSIOs), enterocytes were readily infected by SARS-CoV and SARS-CoV-2, as demonstrated by confocal and electron microscopy. Enterocytes produced infectious viral particles, whereas messenger RNA expression analysis of hSIOs revealed induction of a generic viral response program. Therefore, the intestinal epithelium supports SARS-CoV-2 replication, and hSIOs serve as an experimental model for coronavirus infection and biology.
The current pandemic coronavirus, severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2), was recently identified in patients with an acute respiratory syndrome, coronavirus disease 2019 ...(COVID-19). To compare its pathogenesis with that of previously emerging coronaviruses, we inoculated cynomolgus macaques with SARS-CoV-2 or Middle East respiratory syndrome (MERS)-CoV and compared the pathology and virology with historical reports of SARS-CoV infections. In SARS-CoV-2-infected macaques, virus was excreted from nose and throat in the absence of clinical signs and detected in type I and II pneumocytes in foci of diffuse alveolar damage and in ciliated epithelial cells of nasal, bronchial, and bronchiolar mucosae. In SARS-CoV infection, lung lesions were typically more severe, whereas they were milder in MERS-CoV infection, where virus was detected mainly in type II pneumocytes. These data show that SARS-CoV-2 causes COVID-19-like disease in macaques and provides a new model to test preventive and therapeutic strategies.
Abstract
Rapid identification of host genes essential for virus replication may expedite the generation of therapeutic interventions. Genetic screens are often performed in transformed cell lines ...that poorly represent viral target cells in vivo, leading to discoveries that may not be translated to the clinic. Intestinal organoids are increasingly used to model human disease and are amenable to genetic engineering. To discern which host factors are reliable anti-coronavirus therapeutic targets, we generate mutant clonal IOs for 19 host genes previously implicated in coronavirus biology. We verify ACE2 and DPP4 as entry receptors for SARS-CoV/SARS-CoV-2 and MERS-CoV respectively. SARS-CoV-2 replication in IOs does not require the endosomal Cathepsin B/L proteases, but specifically depends on the cell surface protease TMPRSS2. Other TMPRSS family members were not essential. The newly emerging coronavirus variant B.1.1.7, as well as SARS-CoV and MERS-CoV similarly depended on TMPRSS2. These findings underscore the relevance of non-transformed human models for coronavirus research, identify TMPRSS2 as an attractive pan-coronavirus therapeutic target, and demonstrate that an organoid knockout biobank is a valuable tool to investigate the biology of current and future emerging coronaviruses.
Virus propagation methods generally use transformed cell lines to grow viruses from clinical specimens, which may force viruses to rapidly adapt to cell culture conditions, a process facilitated by ...high viral mutation rates. Upon propagation in VeroE6 cells, SARS-CoV-2 may mutate or delete the multibasic cleavage site (MBCS) in the spike protein. Previously, we showed that the MBCS facilitates serine protease-mediated entry into human airway cells (Mykytyn et al., 2021). Here, we report that propagating SARS-CoV-2 on the human airway cell line Calu-3 - that expresses serine proteases - prevents cell culture adaptations in the MBCS and directly adjacent to the MBCS (S686G). Similar results were obtained using a human airway organoid-based culture system for SARS-CoV-2 propagation. Thus, in-depth knowledge on the biology of a virus can be used to establish methods to prevent cell culture adaptation.
The rapid emergence of SARS-CoV-2, the causative agent of COVID-19, and its dissemination globally has caused an unprecedented strain on public health. Animal models are urgently being developed for ...SARS-CoV-2 to aid rational design of vaccines and therapeutics. Immunohistochemistry and in situ hybridisation techniques that facilitate reliable and reproducible detection of SARS-CoV and SARS-CoV-2 viral products in formalin-fixed paraffin-embedded (FFPE) specimens would be of great utility. A selection of commercial antibodies generated against SARS-CoV spike protein and nucleoprotein, double stranded RNA, and RNA probe for spike genes were evaluated for the ability to detect FFPE infected cells. We also tested both heat- and enzymatic-mediated virus antigen retrieval methods to determine the optimal virus antigen recovery as well as identifying alternative retrieval methods to enable flexibility of IHC methods. In addition to using native virus infected cells as positive control material, the evaluation of non-infected cells expressing coronavirus (SARS, MERS) spike as a biosecure alternative to assays involving live virus was undertaken. Optimized protocols were successfully applied to experimental animal-derived tissues. The diverse techniques for virus detection and control material generation demonstrated in this study can be applied to investigations of coronavirus pathogenesis and therapeutic research in animal models.
Middle East respiratory syndrome (MERS) cases continue to be reported, predominantly in Saudi Arabia and occasionally other countries. Although dromedaries are the main reservoir, other animal ...species might be susceptible to MERS coronavirus (MERS-CoV) infection and potentially serve as reservoirs. To determine whether other animals are potential reservoirs, we inoculated MERS-CoV into llamas, pigs, sheep, and horses and collected nasal and rectal swab samples at various times. The presence of MERS-CoV in the nose of pigs and llamas was confirmed by PCR, titration of infectious virus, immunohistochemistry, and in situ hybridization; seroconversion was detected in animals of both species. Conversely, in sheep and horses, virus-specific antibodies did not develop and no evidence of viral replication in the upper respiratory tract was found. These results prove the susceptibility of llamas and pigs to MERS-CoV infection. Thus, the possibility of MERS-CoV circulation in animals other than dromedaries, such as llamas and pigs, is not negligible.
Susceptibility of rabbits to SARS-CoV-2 Mykytyn, Anna Z.; Lamers, Mart M.; Okba, Nisreen M. A. ...
Emerging microbes & infections,
01/2021, Letnik:
10, Številka:
1
Journal Article
Recenzirano
Odprti dostop
Transmission of severe acute respiratory coronavirus-2 (SARS-CoV-2) between livestock and humans is a potential public health concern. We demonstrate the susceptibility of rabbits to SARS-CoV-2, ...which excrete infectious virus from the nose and throat upon experimental inoculation. Therefore, investigations on the presence of SARS-CoV-2 in farmed rabbits should be considered.
SARS-CoV-2 can enter cells after its spike protein is cleaved by either type II transmembrane serine proteases (TTSPs), like TMPRSS2, or cathepsins. It is now widely accepted that the Omicron variant ...uses TMPRSS2 less efficiently and instead enters cells via cathepsins, but these findings have yet to be verified in more relevant cell models. Although we could confirm efficient cathepsin-mediated entry for Omicron in a monkey kidney cell line, experiments with protease inhibitors showed that Omicron (BA.1 and XBB1.5) did not use cathepsins for entry into human airway organoids and instead utilized TTSPs. Likewise, CRISPR-edited intestinal organoids showed that entry of Omicron BA.1 relied on the expression of the serine protease TMPRSS2 but not cathepsin L or B. Together, these data force us to rethink the concept that Omicron has adapted to cathepsin-mediated entry and indicate that TTSP inhibitors should not be dismissed as prophylactic or therapeutic antiviral strategy against SARS-CoV-2. IMPORTANCE Coronavirus entry relies on host proteases that activate the viral fusion protein, spike. These proteases determine the viral entry route, tropism, host range, and can be attractive drug targets. Whereas earlier studies using cell lines suggested that the Omicron variant of SARS-CoV-2 has changed its protease usage, from cell surface type II transmembrane serine proteases (TTSPs) to endosomal cathepsins, we report that this is not the case in human airway and intestinal organoid models, suggesting that host TTSP inhibition is still a viable prophylactic or therapeutic antiviral strategy against current SARS-CoV-2 variants and highlighting the importance of relevant human
cell models.
PDF
