scholarly journals SARS-CoV-2 variants with mutations at the S1/S2 cleavage site are generated in vitro during propagation in TMPRSS2-deficient cells

2021 ◽  
Vol 17 (1) ◽  
pp. e1009233 ◽  
Author(s):  
Michihito Sasaki ◽  
Kentaro Uemura ◽  
Akihiko Sato ◽  
Shinsuke Toba ◽  
Takao Sanaki ◽  
...  
Keyword(s):  
Cleavage Site ◽  
De Novo ◽  
Gene Mutations ◽  
Critical Factor ◽  
Cell Receptor ◽  
Cell Tropism ◽  
Protein Cleavage ◽  
S Protein ◽  
S Gene

The spike (S) protein of Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) binds to a host cell receptor which facilitates viral entry. A polybasic motif detected at the cleavage site of the S protein has been shown to broaden the cell tropism and transmissibility of the virus. Here we examine the properties of SARS-CoV-2 variants with mutations at the S protein cleavage site that undergo inefficient proteolytic cleavage. Virus variants with S gene mutations generated smaller plaques and exhibited a more limited range of cell tropism compared to the wild-type strain. These alterations were shown to result from their inability to utilize the entry pathway involving direct fusion mediated by the host type II transmembrane serine protease, TMPRSS2. Notably, viruses with S gene mutations emerged rapidly and became the dominant SARS-CoV-2 variants in TMPRSS2-deficient cells including Vero cells. Our study demonstrated that the S protein polybasic cleavage motif is a critical factor underlying SARS-CoV-2 entry and cell tropism. As such, researchers should be alert to the possibility of de novo S gene mutations emerging in tissue-culture propagated virus strains.

scholarly journals SARS-CoV-2 variants with mutations at the S1/S2 cleavage site are generated in vitro during propagation in TMPRSS2-deficient cells

2020 ◽  
Author(s):  
Michihito Sasaki ◽  
Kentaro Uemura ◽  
Akihiko Sato ◽  
Shinsuke Toba ◽  
Takao Sanaki ◽  
...  
Keyword(s):  
Cleavage Site ◽  
De Novo ◽  
Gene Mutations ◽  
Critical Factor ◽  
Cell Receptor ◽  
Cell Tropism ◽  
Protein Cleavage ◽  
S Protein ◽  
S Gene

AbstractThe spike (S) protein of Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) binds to a host cell receptor which facilitates viral entry. A polybasic motif detected at the cleavage site of the S protein has been shown to broaden the cell tropism and transmissibility of the virus. Here we examine the properties of SARS-CoV-2 variants with mutations at the S protein cleavage site that undergo inefficient proteolytic cleavage. Virus variants with S gene mutations generated smaller plaques and exhibited a more limited range of cell tropism compared to the wild-type strain. These alterations were shown to result from their inability to utilize the entry pathway involving direct fusion mediated by the host type II transmembrane serine protease, TMPRSS2. Notably, viruses with S gene mutations emerged rapidly and became the dominant SARS-CoV-2 variants in TMPRSS2-deficient cells including Vero cells. Our study demonstrated that the S protein polybasic cleavage motif is a critical factor underlying SARS-CoV-2 entry and cell tropism. As such, researchers should be alert to the possibility of de novo S gene mutations emerging in tissue-culture propagated virus strains.

scholarly journals Implications of Spike-glycoprotein processing at S1/S2 by Furin, at S2′ by Furin and/or TMPRSS2 and shedding of ACE2: cell-to-cell fusion, cell entry and infectivity of SARS-CoV-2

2021 ◽  
Author(s):  
Rachid Essalmani ◽  
Jaspreet Jain ◽  
Delia Susan-Resiga ◽  
Ursula Andreo ◽  
Alexandra Evagelidis ◽  
...  
Keyword(s):  
Cell Fusion ◽  
Viral Entry ◽  
Hela Cells ◽  
Protein Trafficking ◽  
Cleavage Site ◽  
Cell Receptor ◽  
Hek293 Cells ◽  
Furin Cleavage ◽  
S Protein

The Spike (S)-protein of SARS-CoV-2 binds host-cell receptor ACE2 and requires proteolytic "priming" at PRRAR685↓ into S1 and S2 (cleavage at S1/S2), and "fusion-activation" at KPSKR815↓ (cleavage at S2′) for viral entry. Both cleavages occur at Furin-like motifs suggesting that proprotein convertases might promote virus entry. In vitro Furin cleaved peptides mimicking the S1/S2 cleavage site more efficiently than S2′, whereas TMPRSS2 cleaved at both sites. In HeLa cells endogenous Furin-like enzymes cleave mainly at S1/S2 during intracellular protein trafficking, as confirmed by mutagenesis. We also mapped the S2′ cleavage site by proteomics and further showed that S2′-processing by Furin, while limited, was strongly enhanced in the presence of ACE2. In contrast, the S2′ KRRKR815↓ mutant (μS2′) was considerably better cleaved by Furin, whereas individual/double KR815AA mutants are retained in the endoplasmic reticulum (ER). Pharmacological inhibitors of convertases (Boston Pharmaceuticals - BOS-inhibitors) effectively blocked endogenous S-protein processing in HeLa cells. However, under co-expression the S-protein was prematurely cleaved by TMPRSS2 into ER-retained, non-O-glycosylated S2 and S2′ products. Quantitative analysis of cell-to-cell fusion and Spike processing using Hela cells revealed the key importance of the Furin sites for syncytia formation and unveiled the enhanced fusogenic potential of the α- and δ-variants of the S-protein of SARS-CoV-2. Our fusion assay indicated that TMPRSS2 enhances S2′ formation, especially in the absence of Furin cleavage, as well as ACE2 shedding. Furthermore, we provide evidence using pseudoparticles that while entry by a "pH-dependent" endocytosis pathway in HEK293 cells did not require Furin processing at S1/S2, a "pH-independent" viral entry in lung-derived Calu-3 cells was sensitive to inhibitors of Furin and TMPRSS2. Consistently, in Calu-3 cells BOS-inhibitors or Camostat potently reduce infectious viral titer and cytopathic effects and this outcome was enhanced when both compounds were combined. Overall, our results show that Furin and TMPRSS2 play synergistic roles in generating fusion-competent S-protein, and promote viral entry, supporting the combination of Furin and TMPRSS2 inhibitors as potent antivirals against SARS-CoV-2.

scholarly journals Newcastle Disease Virus Establishes Persistent Infection in Tumor Cells In Vitro: Contribution of the Cleavage Site of Fusion Protein and Second Sialic Acid Binding Site of Hemagglutinin-Neuraminidase

2017 ◽  
Vol 91 (16) ◽  
Author(s):  
Udaya S. Rangaswamy ◽  
Weijia Wang ◽  
Xing Cheng ◽  
Patrick McTamney ◽  
Danielle Carroll ◽  
...  
Keyword(s):  
Newcastle Disease Virus ◽  
Disease Virus ◽  
Receptor Binding ◽  
Newcastle Disease ◽  
Persistent Infection ◽  
Cleavage Site ◽  
Protein Cleavage ◽  
F Protein ◽  
Sialic Acid Binding

ABSTRACT Newcastle disease virus (NDV) is an oncolytic virus being developed for the treatment of cancer. Following infection of a human ovarian cancer cell line (OVCAR3) with a recombinant low-pathogenic NDV, persistent infection was established in a subset of tumor cells. Persistently infected (PI) cells exhibited resistance to superinfection with NDV and established an antiviral state, as demonstrated by upregulation of interferon and interferon-induced genes such as myxoma resistance gene 1 (Mx1) and retinoic acid-inducing gene-I (RIG-I). Viruses released from PI cells induced higher cell-to-cell fusion than the parental virus following infection in two tumor cell lines tested, HT1080 and HeLa, and remained attenuated in chickens. Two mutations, one in the fusion (F) protein cleavage site, F117S (F117S), and another in hemagglutinin-neuraminidase (HN), G169R (HN169R), located in the second sialic acid binding region, were responsible for the hyperfusogenic phenotype. F117S improves F protein cleavage efficiency, facilitating cell-to-cell fusion, while HN169R possesses a multifaceted role in contributing to higher fusion, reduced receptor binding, and lower neuraminidase activity, which together result in increased fusion and reduced viral replication. Thus, establishment of persistent infection in vitro involves viral genetic changes that facilitate efficient viral spread from cell to cell as a potential mechanism to escape host antiviral responses. The results of our study also demonstrate a critical role in the viral life cycle for the second receptor binding region of the HN protein, which is conserved in several paramyxoviruses. IMPORTANCE Oncolytic Newcastle disease virus (NDV) could establish persistent infection in a tumor cell line, resulting in a steady antiviral state reflected by constitutively expressed interferon. Viruses isolated from persistently infected cells are highly fusogenic, and this phenotype has been mapped to two mutations, one each in the fusion (F) and hemagglutinin-neuraminidase (HN) proteins. The F117S mutation in the F protein cleavage site improved F protein cleavage efficiency while the HN169R mutation located at the second receptor binding site of the HN protein contributed to a complex phenotype consisting of a modest increase in fusion and cell killing, lower neuraminidase activity, and reduced viral growth. This study highlights the intricate nature of these two mutations in the glycoproteins of NDV in the establishment of persistent infection. The data also shed light on the critical balance between the F and HN proteins required for efficient NDV infection and their role in avian pathogenicity.

Potential inhibitors for blocking the interaction of the coronavirus SARS-CoV-2 spike protein and its host cell receptor ACE2

2021 ◽  
Author(s):  
Changzhi Li ◽  
Hongjuan Zhou ◽  
Lingling Guo ◽  
Dehuan Xie ◽  
Huiping He ◽  
...  
Keyword(s):  
Cell Receptor ◽  
Economic Stability ◽  
Time Resolved ◽  
S Protein ◽  
Host Cell Receptor ◽  
Compound Libraries ◽  
Screening Platform ◽  
Potential Inhibitors ◽  
Validation Experiments

The outbreak of SARS-CoV-2 continues to pose a serious threat to human health and social and economic stability. In this study, we established an anti-coronavirus drug screening platform based on the Homogeneous Time Resolved Fluorescence (HTRF) technology and the interaction between the coronavirus S protein and its host receptor ACE2. This platform is a rapid, sensitive, specific, and high throughput system. With this platform, we screened two compound libraries of 2,864 molecules and identified three potential anti-coronavirus compounds: tannic acid (TA), TS-1276 (anthraquinone), and TS-984 (9-Methoxycanthin-6-one). Our in vitro validation experiments indicated that TS-984 strongly inhibits the interaction of the coronavirus S-protein and the human cell ACE2 receptor. This data suggests that TS-984 is a potent blocker of the interaction between the S-protein and ACE2, which might have the potential to be developed into an effective anti-coronavirus drug.

In-Vitro Fluorescence Microscopy Studies Show Retention of Spike-Protein (SARS-Cov-2) on Cell Membrane in the Presence of Amodiaquin Dihydrochloride Dihydrate Drug

2021 ◽  
Author(s):  
Partha Pratim Mondal ◽  
Subhra Mandal
Keyword(s):  
Cell Membrane ◽  
Large Fraction ◽  
Degradation Process ◽  
Cell Receptor ◽  
Endocytic Pathway ◽  
Receptor Protein ◽  
Potential Candidate ◽  
S Protein ◽  
Cellular Environment

The ability of S-glycoprotein (S-protein) in SARS-Cov-2 to bind to the host cell receptor protein (angiotensinconverting enzyme 2 (ACE2)) leading to its entry in cellular system determines its contagious index and global spread. Three available drugs (Riboflavin, Amodiaquin dihydrochloride dihydrate (ADD) and Remidesivir) were investigated to understand the kinetics of S-protein and its entry inside a cellular environment. Optical microscopy and fluorescence-based assays on 293T cells (transfected with ACE2 plasmid) were used as the preamble for assessing the behaviour of S-protein in the presence of these drugs for the first 12 hours post S-protein - ACE2 binding. Preliminary results suggest relatively long retention of S-protein on the cell membrane in the presence of ADD drug. Evident from the %-overlap and colocalization of S-protein with endosome studies, a large fraction of S-protein entering the cell escape endosomal degradation process, suggesting S-protein takes non-endocytic mediated entry in the presence of ADD, whereas in the presence of Riboflavin, S-protein carry out normal endocytic pathway, comparable to control (no drug) group. Therefore, present study indicates ADD potentially affects S-protein’s entry mechanism (endocytic pathway) in addition to its reported target action mechanism. Hence, ADD substantially interfere with S-protein cellular entrance mechanism. However, further detailed studies at molecular scale will clarify our understanding of exact intermediate molecular processes. The present study (based on limited data) reveal ADD could be potential candidate to manage Covid-19 functions through yet unknown molecular mechanism.

scholarly journals Duplication of the IL2RA locus causes excessive IL-2 signaling and may predispose to very early onset colitis

2021 ◽  
Author(s):  
Maria E. Joosse ◽  
Fabienne Charbit-Henrion ◽  
Remy Boisgard ◽  
Rolien C. Raatgeep ◽  
Dicky J. Lindenbergh-Kortleve ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Early Onset ◽  
Intestinal Inflammation ◽  
De Novo ◽  
Cell Receptor ◽  
Effector Memory ◽  
Colonic Disease ◽  
Cd25 Expression

AbstractSingle genetic mutations predispose to very early onset inflammatory bowel disease (VEO-IBD). Here, we identify a de novo duplication of the 10p15.1 chromosomal region, including the IL2RA locus, in a 2-year-old girl with treatment-resistant pancolitis that was brought into remission by colectomy. Strikingly, after colectomy while the patient was in clinical remission and without medication, the peripheral blood CD4:CD8 ratio was constitutively high and CD25 expression was increased on circulating effector memory, Foxp3+, and Foxp3neg CD4+ T cells compared to healthy controls. This high CD25 expression increased IL-2 signaling, potentiating CD4+ T-cell-derived IFNγ secretion after T-cell receptor (TCR) stimulation. Restoring CD25 expression using the JAK1/3-inhibitor tofacitinib controlled TCR-induced IFNγ secretion in vitro. As diseased colonic tissue, but not the unaffected duodenum, contained mainly CD4+ T cells with a prominent IFNγ-signature, we hypothesize that local microbial stimulation may have initiated colonic disease. Overall, we identify that duplication of the IL2RA locus can associate with VEO-IBD and suggest that increased IL-2 signaling predisposes to colonic intestinal inflammation.

scholarly journals Natural isolate and recombinant SARS-CoV-2 rapidly evolve in vitro to higher infectivity through more efficient binding to heparan sulfate and reduced S1/S2 cleavage

2021 ◽  
Author(s):  
Nikita Shiliaev ◽  
Tetyana Lukash ◽  
Oksana Palchevska ◽  
David K Crossman ◽  
Todd J. Green ◽  
...  
Keyword(s):  
Heparan Sulfate ◽  
Cleavage Site ◽  
Heparin Binding ◽  
Natural Isolate ◽  
Furin Cleavage ◽  
S Protein ◽  
Viral Spread ◽  
Furin Cleavage Site ◽  
Viral Particles

One of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virulence factors is the ability to interact with high affinity to the ACE2 receptor, which mediates viral entry into cells. The results of our study demonstrate that within a few passages in cell culture, both the natural isolate of SARS-CoV-2 and the recombinant, cDNA-derived variant acquire an additional ability to bind to heparan sulfate (HS). This promotes a primary attachment of viral particles to cells before their further interactions with the ACE2. Interaction with HS is acquired through multiple mechanisms. These include i) accumulation of point mutations in the N-terminal domain (NTD) of the S protein, which increase the positive charge of the surface of this domain, ii) insertions into NTD of heterologous peptides, containing positively charged amino acids, and iii) mutation of the first amino acid downstream of the furin cleavage site. This last mutation affects S protein processing, transforms the unprocessed furin cleavage site into the heparin-binding peptide and makes viruses less capable of syncytia formation. These viral adaptations result in higher affinity of viral particles to heparin sepharose, dramatic increase in plaque sizes, more efficient viral spread, higher infectious titers and two orders of magnitude lower GE:PFU ratios. The detected adaptations also suggest an active role of NTD in virus attachment and entry. As in the case of other RNA+ viruses, evolution to HS binding may result in virus attenuation in vivo.

scholarly journals The AKT–mTOR axis regulates de novo differentiation of CD4+Foxp3+ cells

2008 ◽  
Vol 205 (3) ◽  
pp. 565-574 ◽  
Author(s):  
Sokol Haxhinasto ◽  
Diane Mathis ◽  
Christophe Benoist
Keyword(s):  
Signaling Pathways ◽  
De Novo ◽  
Mammalian Target Of Rapamycin ◽  
Foxp3 Expression ◽  
Cell Receptor ◽  
Immunological Tolerance ◽  
Dependent Manner ◽  
Transcriptional Signature

CD4+Foxp3+ regulatory T (T reg) cells play an essential role in maintaining immunological tolerance via their suppressive function on conventional CD4+ T (Tconv) cells. Repertoire studies suggest that distinct T cell receptor signaling pathways lead to T reg differentiation, but the signals that regulate T reg specification are largely unknown. We identify AKT as a strong repressor of entry into the T reg phenotype in vitro and in vivo. A constitutively active allele of AKT substantially diminished TGF-β–induced Foxp3 expression in a kinase-dependent manner and via a rapamycin-sensitive pathway, implicating the AKT–mammalian target of rapamycin axis. The observed impairment in Foxp3 induction was part of a broad dampening of the typical T reg transcriptional signature. Expression of active AKT at a stage before Foxp3 turn on during normal T reg differentiation in the thymus selectively impaired differentiation of CD4+Foxp3+ cells without any alteration in the positive selection of Tconv. Activated AKT, in contrast, did not affect established Foxp3 expression in T reg cells. These results place AKT at a nexus of signaling pathways whose proper activation has a strong and broad impact on the onset of T reg specification.

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