scholarly journals FXa cleaves the SARS-CoV-2 spike protein and blocks cell entry to protect against infection with inferior effects in B.1.1.7 variant

2021 ◽  
Author(s):  
Jianhua Yu ◽  
Wenjuan Dong ◽  
Jing Wang ◽  
Lei Tian ◽  
Jianying Zhang ◽  
...  
Keyword(s):  
Viral Entry ◽  
Serine Proteases ◽  
Defense Mechanisms ◽  
Defense Mechanism ◽  
Spike Protein ◽  
Natural Defense ◽  
Host Defense Mechanism ◽  
Direct Inhibitors

The ongoing coronavirus disease 2019 (COVID-19) pandemic is caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Human natural defense mechanisms against SARS-CoV-2 are largely unknown. Serine proteases (SPs) including furin and TMPRSS2 cleave SARS-CoV-2 spike protein, facilitating viral entry. Here, we show that FXa, a SP for blood coagulation, is upregulated in COVID 19 patients compared to non-COVID-19 donors and exerts anti-viral activity. Mechanistically, FXa cleaves the SARS-CoV-2 spike protein, which prevents its binding to ACE2, and thus blocks viral entry. Furthermore, the variant B.1.1.7 with several mutations is dramatically resistant to the anti-viral effect of FXa compared to wild-type SARA-CoV-2 in vivo and in vitro. The anti-coagulant rivaroxaban directly inhibits FXa and facilitates viral entry, whereas the indirect inhibitor fondaparinux does not. In a lethal humanized hACE2 mouse model of SARS-CoV-2, FXa prolonged survival while combination with rivaroxaban but not fondaparinux abrogated this protection. These preclinical results identify a previously unknown SP function and associated anti-viral host defense mechanism and suggest caution in considering direct inhibitors for prevention or treatment of thrombotic complications in COVID-19 patients.

scholarly journals Autophagy-activating strategies to promote innate defense against mycobacteria

2019 ◽  
Vol 51 (12) ◽  
pp. 1-10 ◽  
Author(s):  
Yi Sak Kim ◽  
Prashanta Silwal ◽  
Soo Yeon Kim ◽  
Tamotsu Yoshimori ◽  
Eun-Kyeong Jo
Keyword(s):  
Host Defense ◽  
Defense Mechanism ◽  
Multidrug Resistant ◽  
Peroxisome Proliferator ◽  
Peroxisome Proliferator Activated Receptor ◽  
Innate Defense ◽  
Host Defense Mechanism ◽  
A Cell

AbstractMycobacterium tuberculosis (Mtb) is a major causal pathogen of human tuberculosis (TB), which is a serious health burden worldwide. The demand for the development of an innovative therapeutic strategy to treat TB is high due to drug-resistant forms of TB. Autophagy is a cell-autonomous host defense mechanism by which intracytoplasmic cargos can be delivered and then destroyed in lysosomes. Previous studies have reported that autophagy-activating agents and small molecules may be beneficial in restricting intracellular Mtb infection, even with multidrug-resistant Mtb strains. Recent studies have revealed the essential roles of host nuclear receptors (NRs) in the activation of the host defense through antibacterial autophagy against Mtb infection. In particular, we discuss the function of estrogen-related receptor (ERR) α and peroxisome proliferator-activated receptor (PPAR) α in autophagy regulation to improve host defenses against Mtb infection. Despite promising findings relating to the antitubercular effects of various agents, our understanding of the molecular mechanism by which autophagy-activating agents suppress intracellular Mtb in vitro and in vivo is lacking. An improved understanding of the antibacterial autophagic mechanisms in the innate host defense will eventually lead to the development of new therapeutic strategies for human TB.

scholarly journals Carbon monoxide in the treatment of sepsis

2015 ◽  
Vol 309 (12) ◽  
pp. L1387-L1393 ◽  
Author(s):  
Kiichi Nakahira ◽  
Augustine M. K. Choi
Keyword(s):  
Carbon Monoxide ◽  
Defense Mechanism ◽  
Inflammatory Responses ◽  
The Body ◽  
High Concentration ◽  
Novel Therapy ◽  
Autophagy Induction ◽  
Host Defense Mechanism

Carbon monoxide (CO), a low-molecular-weight gas, is endogenously produced in the body as a product of heme degradation catalyzed by heme oxygenase (HO) enzymes. As the beneficial roles of HO system have been elucidated in vitro and in vivo, CO itself has also been reported as a potent cytoprotective molecule. Whereas CO represents a toxic inhalation hazard at high concentration, low-dose exogenous CO treatment (∼250–500 parts per million) demonstrates protective functions including but not limited to the anti-inflammatory and antiapoptotic effects in preclinical models of human diseases. Of note, CO exposure confers protection in animal models of sepsis by inhibiting inflammatory responses and also enhancing bacterial phagocytosis in leukocytes. These unique functions of CO including both dampening inflammation and promoting host defense mechanism are mediated by multiple pathways such as autophagy induction or biosynthesis of specialized proresolving lipid mediators. We suggest that CO gas may represent a novel therapy for patients with sepsis.

scholarly journals Intestinal P Glycoprotein Acts as a Natural Defense Mechanism against Listeria monocytogenes

2004 ◽  
Vol 72 (7) ◽  
pp. 3849-3854 ◽  
Author(s):  
Brien L. Neudeck ◽  
Jennifer M. Loeb ◽  
Nancy G. Faith ◽  
Charles J. Czuprynski
Keyword(s):  
Listeria Monocytogenes ◽  
Defense Mechanism ◽  
Transepithelial Transport ◽  
Bacterial Invasion ◽  
Wild Type ◽  
In Vivo Experiments ◽  
Natural Defense ◽  
P Glycoprotein

ABSTRACT Mechanisms by which the intestinal epithelium resists invasion by food-borne pathogens such as Listeria monocytogenes are an evolving area of research. Intestinal P glycoprotein is well known to limit the absorption of xenobiotics and is believed to act as a cytotoxic defense mechanism. The aim of this study was to determine if intestinal P glycoprotein is involved in host defense against L. monocytogenes. Caco-2 cells and a P-glycoprotein-overexpressing subclone (Caco-2/MDR) were employed in addition to mdr1a−/− mice and wild-type controls. In vitro invasion assays and in vivo experiments were employed to measure bacterial invasion and dissemination. In addition, L. monocytogenes proteins were labeled with [35S]methionine, and the transepithelial transport across Caco-2 monolayers was characterized in both directions. Overexpression of P glycoprotein in Caco-2/MDR cells led to increased resistance to L. monocytogenes invasion, whereas P-glycoprotein inhibition led to increased invasion. Flux of [35S]methionine-labeled L. monocytogenes proteins was significantly greater in the basolateral-to-apical direction than in the apical-to-basolateral direction, indicating dependence on an apically located efflux transporter. Moreover, inhibiting P glycoprotein reduced the basolateral-to-apical flux of the proteins. Early dissemination of L. monocytogenes from the gastrointestinal tract was significantly greater in the mdr1a−/− mice than in wild-type controls. Expression and function of intestinal P glycoprotein is an important determinant in resistance to early invasion of L. monocytogenes.

scholarly journals In-vivo Protection from SARS-CoV-2 infection by ATN-161 in k18-hACE2 transgenic mice

2021 ◽  
Author(s):  
Amruta Narayanappa ◽  
Elizabeth B Engler-Chiurazzi ◽  
Isabel C Murray-Brown ◽  
Timothy E Gressett ◽  
Ifechukwude J Biose ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Viral Entry ◽  
Therapeutic Potential ◽  
Host Cells ◽  
Spike Protein ◽  
Cell Infection ◽  
Integrin Alpha5beta1 ◽  
Chemokine Ligand

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an infectious disease that has spread worldwide. Current treatments are limited in both availability and efficacy, such that improving our understanding of the factors that facilitate infection is urgently needed to more effectively treat infected individuals and to curb the pandemic. We and others have previously demonstrated the significance of interactions between the SARS-CoV-2 spike protein, integrin alpha5beta1 and human ACE2 to facilitate viral entry into host cells in vitro. We previously found that inhibition of integrin alpha5beta1 by the clinically validated small peptide ATN-161 inhibits these spike protein interactions and cell infection in vitro. In continuation with our previous findings, here we have further evaluated the therapeutic potential of ATN-161 on SARS-CoV-2 infection in k18-hACE2 transgenic (SARS-CoV-2 susceptible) mice in vivo. We discovered that treatment with single- or repeated intravenous doses of ATN-161 (1 mg/kg) within 48 hours after intranasal inoculation with SARS-CoV-2 lead to a reduction of lung viral load, viral immunofluorescence and improved lung histology in a majority of mice 72 hours post-infection. Furthermore, ATN-161 reduced SARS-CoV-2-induced increased expression of lung integrin alpha 5 and alpha v (an alpha 5-related integrin that has also been implicated in SARS-CoV-2 interactions) as well as the C-X-C motif chemokine ligand 10 (Cxcl10), further supporting the potential involvement of these integrins, and the anti-inflammatory potential of ATN-161, respectively, in SARS-CoV-2 infection. To the best of our knowledge, this is the first study demonstrating the potential therapeutic efficacy of targeting integrin alpha5beta1 in SARS-CoV-2 infection in vivo and supports the development of ATN-161 as a novel SARS-CoV-2 therapy.

scholarly journals TMPRSS2 structure-phylogeny repositions Avoralstat for SARS-CoV-2 prophylaxis in mice

2021 ◽  
Author(s):  
Young Joo Sun ◽  
Gabriel Velez ◽  
Dylan Parsons ◽  
Kun Li ◽  
Miguel Ortiz ◽  
...  
Keyword(s):  
Viral Entry ◽  
Serine Proteases ◽  
Tertiary Structure ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Cell Infection ◽  
Effective Inhibition ◽  
Transmembrane Serine Protease

Drugs targeting host proteins can act prophylactically to reduce viral burden early in disease and limit morbidity, even with antivirals and vaccination. Transmembrane serine protease 2 (TMPRSS2) is a human protease required for SARS-CoV-2 viral entry and may represent such a target. We hypothesized drugs selected from proteins related by their tertiary structure, rather than their primary structure, were likely to interact with TMPRSS2. We created a structure-based phylogenetic computational tool 3DPhyloFold to systematically identify structurally similar serine proteases with known therapeutic inhibitors and demonstrated effective inhibition of SARS-CoV-2 infection in vitro and in vivo. Several candidate compounds, Avoralstat, PCI-27483, Antipain, and Soybean-Trypsin-Inhibitor, inhibited TMPRSS2 in biochemical and cell infection assays. Avoralstat, a clinically tested Kallikrein-related B1 inhibitor, inhibited SARS-CoV-2 entry and replication in human airway epithelial cells. In an in vivo proof of principle, Avoralstat significantly reduced lung tissue titers and mitigated weight-loss when administered prophylactically to SARS-CoV-2 susceptible mice indicating its potential to be repositioned for COVID-19 prophylaxis in humans.

scholarly journals The sequence at Spike S1/S2 site enables cleavage by furin and phospho-regulation in SARS-CoV2 but not in SARS-CoV1 or MERS-CoV

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Mihkel Örd ◽  
Ilona Faustova ◽  
Mart Loog
Keyword(s):  
Viral Entry ◽  
Spike Protein ◽  
Target Cells ◽  
The Novel ◽  
Novel Coronavirus ◽  
Cleavage Motif ◽  
Left Middle ◽  
Surprising Finding

Abstract The Spike protein of the novel coronavirus SARS-CoV2 contains an insertion 680SPRRAR↓SV687 forming a cleavage motif RxxR for furin-like enzymes at the boundary of S1/S2 subunits. Cleavage at S1/S2 is important for efficient viral entry into target cells. The insertion is absent in other CoV-s of the same clade, including SARS-CoV1 that caused the 2003 outbreak. However, an analogous cleavage motif was present at S1/S2 of the Spike protein of the more distant Middle East Respiratory Syndrome coronavirus MERS-CoV. We show that a crucial third arginine at the left middle position, comprising a motif RRxR is required for furin recognition in vitro, while the general motif RxxR in common with MERS-CoV is not sufficient for cleavage. Further, we describe a surprising finding that the two serines at the edges of the insert SPRRAR↓SV can be efficiently phosphorylated by proline-directed and basophilic protein kinases. Both phosphorylations switch off furin’s ability to cleave the site. Although phospho-regulation of secreted proteins is still poorly understood, further studies, supported by a recent report of ten in vivo phosphorylated sites in the Spike protein of SARS-CoV2, could potentially uncover important novel regulatory mechanisms for SARS-CoV2.

scholarly journals Susceptibilities of human ACE2 genetic variants in coronavirus infection

2021 ◽  
Author(s):  
Wenlin Ren ◽  
Yunkai Zhu ◽  
Jun Lan ◽  
Hedi Chen ◽  
Yuyan Wang ◽  
...  
Keyword(s):  
Viral Entry ◽  
Integrated Approach ◽  
Spike Protein ◽  
Nucleotide Polymorphisms ◽  
Coding Region ◽  
Angiotensin Converting Enzyme 2 ◽  
Coronavirus Infection ◽  
Ace2 Gene

The COVID-19 pandemic, caused by SARS-CoV-2, has resulted in more than 1603 million cases worldwide and 3.4 million deaths (as of May 2021), with varying incidences and death rates among regions/ethnicities. Human genetic variation can affect disease progression and outcome, but little is known about genetic risk factors for SARS-CoV-2 infection. The coronaviruses SARS-CoV, SARS-CoV-2 and HCoV-NL63 all utilize the human protein angiotensin-converting enzyme 2 (ACE2) as the receptor to enter cells. We hypothesized that the genetic variability in ACE2 may contribute to the variable clinical outcomes of COVID-19. To test this hypothesis, we first conducted an in silico investigation of single-nucleotide polymorphisms (SNPs) in the coding region of ACE2 gene. We then applied an integrated approach of genetics, biochemistry and virology to explore the capacity of select ACE2 variants to bind coronavirus spike protein and mediate viral entry. We identified the ACE2 D355N variant that restricts the spike protein-ACE2 interaction and consequently limits infection both in vitro and in vivo. In conclusion, ACE2 polymorphisms could modulate susceptibility to SARS-CoV-2, which may lead to variable disease severity.

scholarly journals Biochemical evidence of furin specificity and potential for phospho-regulation at Spike protein S1/S2 cleavage site in SARS-CoV2 but not in SARS-CoV1 or MERS-CoV

2020 ◽  
Author(s):  
Mihkel Örd ◽  
Ilona Faustova ◽  
Mart Loog
Keyword(s):  
Viral Entry ◽  
Spike Protein ◽  
Target Cells ◽  
The Novel ◽  
Novel Coronavirus ◽  
Cleavage Motif ◽  
Left Middle ◽  
Surprising Finding

AbstractThe Spike protein of the novel coronavirus SARS-CoV2 contains an insertion 680SPRRAR↓SV687 forming a cleavage motif RxxR for furin-like enzymes at the boundary of S1/S2 subunits. Cleavage at S1/S2 is important for efficient viral entry into target cells. The insertion is absent in other CoV-s of the same clade, including SARS-CoV1 that caused the 2003 outbreak. However, an analogous insertion was present in the Spike protein of the more distant Middle East Respiratory Syndrome coronavirus MERS-CoV. We show that a crucial third arginine at the left middle position, comprising a motif RRxR is required for furin recognition in vitro, while the general motif RxxR in common with MERS-CoV is not sufficient for cleavage. Further, we describe a surprising finding that the two serines at the edges of the insert SPRRAR↓SV can be efficiently phosphorylated by proline-directed and basophilic protein kinases. Both phosphorylations switch off furin’s ability to cleave the site. Although phosphoregulation of secreted proteins is still poorly understood, further studies, supported by a recent report of ten in vivo phosphorylated sites in the Spike protein of SARS-CoV2, could potentially uncover important novel regulatory mechanisms for SARS-CoV2.

Spike Protein of SARS-CoV-2 Activates Macrophages and Contributes to Induction of Acute Lung Inflammations in Mice

2020 ◽  
Author(s):  
Xiaoling Cao ◽  
Yan Tian ◽  
Vi Nguyen ◽  
Yuping Zhang ◽  
Chao Gao ◽  
...  
Keyword(s):  
Viral Entry ◽  
Inflammatory Responses ◽  
Host Cells ◽  
Spike Protein ◽  
Raw264.7 Cells ◽  
Inflammatory Factors ◽  
Mrna Levels ◽  
Viral Burden

Background: Coronavirus disease 2019 (COVID-19) patients exhibit multiple organ malfunctions with a primary manifestation of acute and diffuse lung injuries. The Spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is crucial to mediate viral entry into host cells; however, whether it can be cellularly pathogenic and contribute to pulmonary hyper-inflammations in COVID-19 is not well known. Methods and Findings: In this study, we developed a Spike protein-pseudotyped (Spp) lentivirus with the proper tropism of SARS-CoV-2 Spike protein on the surface and tracked down the fate of Spp in wild type C57BL/6J mice receiving intravenous injection of the virus. A lentivirus with vesicular stomatitis virus glycoprotein (VSV-G) was used as the control. Two hours post-infection (hpi), Spp showed more than 27-75 times more viral burden in the lungs than other organs; it also exhibited about 3-5 times more viral burden than VSV-G lentivirus in the lungs, liver, kidney and spleen. Acute pneumonia was evident in animals 24 hpi. Spp lentivirus was mainly found in LDLR+ macrophages and pneumocytes in the lungs, but not in MARC1+ macrophages. IL6, IL10, CD80 and PPAR-γ were quickly upregulated in response to infection of Spp lentivirus in the lungs in vivo as well as in macrophage-like RAW264.7 cells in vitro. We further confirmed that forced expression of the Spike protein in RAW264.7 cells could significantly increase the mRNA levels of the same panel of inflammatory factors. Conclusions: Our results demonstrate that the Spike protein of SARS-CoV-2 alone can induce cellular pathology, e.g. activating macrophages and contributing to induction of acute inflammatory responses.

scholarly journals SARS-CoV-2 entry into human airway organoids is serine protease-mediated and facilitated by the multibasic cleavage site

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Anna Z Mykytyn ◽  
Tim I Breugem ◽  
Samra Riesebosch ◽  
Debby Schipper ◽  
Petra B van den Doel ◽  
...  
Keyword(s):  
Serine Protease ◽  
Viral Entry ◽  
Cleavage Site ◽  
Serine Proteases ◽  
Spike Protein ◽  
Remarkable Feature ◽  
Human Airway ◽  
Entry Strategy ◽  
Entry Speed

Coronavirus entry is mediated by the spike protein which 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 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.

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