scholarly journals A hybrid soluble gp130/spike-nanobody fusion protein simultaneously blocks IL-6 trans-signaling and cellular infection with SARS-CoV2

2021 ◽  
Author(s):  
Julia Ettich ◽  
Julia Werner ◽  
Hendrik T. Weitz ◽  
Eva Mueller ◽  
Roland Schwarzer ◽  
...  
Keyword(s):  
Specific Inhibitor ◽  
Vero Cells ◽  
Organ Damage ◽  
Cell Entry ◽  
Spike Protein ◽  
Antibody Treatment ◽  
Glucocorticoid Treatment ◽  
Stat3 Phosphorylation ◽  
Dual Action ◽  
Viral Cell Entry

SARS-CoV2 infection can induce mild to life threatening symptoms. Especially individuals over 60 years of age or with underlying co-morbidities including heart or lung disease, and diabetes or immune compromised patients are at higher risk. Fatal multi-organ damage in COVID19 patients can be attributed to Interleukin (IL-)6 dominated cytokine storm. Consequently, IL-6R monoclonal antibody treatment for severe COVID19 cases has been approved for therapy. High concentrations of soluble IL-6R were found in COVID19 intensive care unit patients suggesting the involvement of IL-6 trans-signaling in disease pathology. Here, in analogy to bispecific antibodies (bsAbs), we developed the first bispecific IL-6 trans-signaling inhibitor c19s130Fc which blocks viral infection and IL-6 trans-signaling. c19s130Fc is a designer protein of the IL-6 trans-signaling inhibitor cs130 fused to a single domain nanobody directed against the receptor binding domain (RBD) of the SARS-CoV2 spike protein. c19s130Fc binds with high affinity to IL-6:sIL6R complexes as well as the spike protein of SARS-CoV2 as shown by surface plasmon resonance. Using cell-based assays, we demonstrate that c19s130Fc blocks IL-6 trans-signaling-induced proliferation and STAT3 phosphorylation of Ba/F3-gp130 cells as well as SARS-CoV2 infection and STAT3 phosphorylation in Vero cells. Taken together, c19s130Fc represents a new class of bispecific inhibitors consisting of a soluble cytokine receptor fused to anti-viral nanobodies and principally demonstrates the multi-functionalization of trans-signaling inhibitors. Importance The availability of effective SARS-CoV2 vaccines is a big step forward in managing the pandemic situation. In addition, therapeutic options e.g. monoclonal antibodies to prevent viral cell entry and anti-inflammatory therapies including glucocorticoid treatment are currently developed or in clinical use utilized to treat already infected patients. Here we report a novel dual-specific inhibitor to simultaneously target SARS-Cov2 infection and virus induced hyper-inflammation. This was achieved by fusing an inhibitor of viral cell entry with a molecule blocking IL-6, a key mediator of SARS-CoV2 induced hyper-inflammation. Through this dual action, this molecule may have the potential to efficiently ameliorate symptoms of COVID19 in infected individuals.

scholarly journals Differential Downregulation of ACE2 by the Spike Proteins of Severe Acute Respiratory Syndrome Coronavirus and Human Coronavirus NL63

2009 ◽  
Vol 84 (2) ◽  
pp. 1198-1205 ◽  
Author(s):  
Ilona Glowacka ◽  
Stephanie Bertram ◽  
Petra Herzog ◽  
Susanne Pfefferle ◽  
Imke Steffen ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Lung Injury ◽  
Vero Cells ◽  
Cell Entry ◽  
Spike Protein ◽  
Converting Enzyme ◽  
Human Coronavirus ◽  
Angiotensin Converting Enzyme 2 ◽  
Similar Activity ◽  
Human Coronaviruses

ABSTRACT The human coronaviruses (CoVs) severe acute respiratory syndrome (SARS)-CoV and NL63 employ angiotensin-converting enzyme 2 (ACE2) for cell entry. It was shown that recombinant SARS-CoV spike protein (SARS-S) downregulates ACE2 expression and thereby promotes lung injury. Whether NL63-S exerts a similar activity is yet unknown. We found that recombinant SARS-S bound to ACE2 and induced ACE2 shedding with higher efficiency than NL63-S. Shedding most likely accounted for the previously observed ACE2 downregulation but was dispensable for viral replication. Finally, SARS-CoV but not NL63 replicated efficiently in ACE2-positive Vero cells and reduced ACE2 expression, indicating robust receptor interference in the context of SARS-CoV but not NL63 infection.

scholarly journals A Furin Cleavage Site Inserted into the Spike Protein of SARS-CoV-2: A Structural Implication?

2020 ◽  
Author(s):  
Wei Li
Keyword(s):  
Cleavage Site ◽  
Structural Data ◽  
Cell Entry ◽  
Spike Protein ◽  
Spike Glycoprotein ◽  
Furin Cleavage ◽  
Furin Cleavage Site ◽  
Structural Consequence ◽  
Viral Cell Entry

One notable features of the SARS-CoV-2 genome is that the spike protein of SARS-CoV-2 has a functional polybasic (furin) cleavage site (RRAR) at the S1–S2 boundary through the insertion of 12 nucleotides encoding PRRA. To date, the furin cleavage site (FCS) remains an experimentally uncharted territory both structurally and functionally. For instance, whether or not FCS is actually cleaved, before or after viral cell entry or exit, still remains to be experimentally investigated. With currently available structural data, this article presents a computational structural characterization of the FCS inserted into SARS-CoV-2 spike glycoprotein, and puts forward a set of structural hypothesis against the hypothesis of SARS-CoV-2 from purposeful manipulation: (1), the inserted FCS does not alter, neither stabilize nor de-stabilize, the overall structure of SARS-CoV-2 spike glycoprotein; (2), the net structural consequence of FCS is the insertion of a furin cleavage site into SARS-CoV-2 spike glycoprotein, whose S1 and S2 subunits will still be bonded together even if the FCS is actually cleaved by furin protease.

scholarly journals Blockade of TMPRSS2-mediated priming of SARS-CoV-2 by the N-terminal peptide of lactoferrin

2021 ◽  
Author(s):  
Anna Ohradanova-Repic ◽  
Laura Gebetsberger ◽  
Gabor Tajti ◽  
Gabriela Ondrovicova ◽  
Romana Prazenicova ◽  
...  
Keyword(s):  
Vero Cells ◽  
Proteolytic Processing ◽  
Cell Entry ◽  
Biologically Active ◽  
Spike Protein ◽  
Active Peptides ◽  
Biologically Active Peptides ◽  
Prevention And Treatment ◽  
Mediated Priming

In addition to vaccines, there is an urgent need for supplemental antiviral therapeutics to dampen the persistent COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). The transmembrane protease serine 2 (TMPRSS2), which is responsible for the proteolytic processing of the SARS-CoV-2 spike protein as virus priming for cell entry, appears as a rational therapeutic target for the clearance of SARS-CoV-2 infection. Accordingly, selective inhibitors of TMPRSS2 represent potential tools for prevention and treatment of COVID-19. Here, we tested the inhibitory capacities of the human milk glycoprotein lactoferrin and its N-terminal peptide pLF1, which we identified as inhibitors of plasminogen, a serine protease homologous to TMPRSS2. In vitro proteolysis assays revealed that, unlike full-length lactoferrin, pLF1 significantly inhibited the proteolytic activity of TMPRSS2. pLF1 inhibited both the proteolytic processing of the SARS-CoV-2 spike protein and the SARS-CoV-2 infection of simian Vero cells. Because lactoferrin is a natural product and several biologically active peptides, such as the N-terminally derived lactoferricins, are produced naturally by pepsin-mediated digestion, natural or synthetic peptides from lactoferrin represent well-achievable candidates for supporting prevention and treatment of COVID-19.

scholarly journals SARS-CoV-2 variants of concern have acquired mutations associated with an increased spike cleavage

2021 ◽  
Author(s):  
Alba Escalera ◽  
Ana S. Gonzalez-Reiche ◽  
Sadaf Aslam ◽  
Ignacio Mena ◽  
Rebecca L. Pearl ◽  
...  
Keyword(s):  
Cell Entry ◽  
Host Susceptibility ◽  
Spike Protein ◽  
Protein Cleavage ◽  
S Protein ◽  
The Impact ◽  
Viral Cell Entry

For efficient cell entry and membrane fusion, SARS-CoV-2 spike (S) protein needs to be cleaved at two different sites, S1/S2 and S2 by different cellular proteases such as furin and TMPRSS2. Polymorphisms in the S protein can affect cleavage, viral transmission, and pathogenesis. Here, we investigated the role of arising S polymorphisms in vitro and in vivo to understand the emergence of SARS-CoV-2 variants. First, we showed that the S:655Y is selected after in vivo replication in the mink model. This mutation is present in the Gamma Variant Of Concern (VOC) but it also occurred sporadically in early SARS-CoV-2 human isolates. To better understand the impact of this polymorphism, we analyzed the in vitro properties of a panel of SARS-CoV-2 isolates containing S:655Y in different lineage backgrounds. Results demonstrated that this mutation enhances viral replication and spike protein cleavage. Viral competition experiments using hamsters infected with WA1 and WA1-655Y isolates showed that the variant with 655Y became dominant in both direct infected and direct contact animals. Finally, we investigated the cleavage efficiency and fusogenic properties of the spike protein of selected VOCs containing different mutations in their spike proteins. Results showed that all VOCs have evolved to acquire an increased spike cleavage and fusogenic capacity despite having different sets of mutations in the S protein. Our study demonstrates that the S:655Y is an important adaptative mutation that increases viral cell entry, transmission, and host susceptibility. Moreover, SARS-COV-2 VOCs showed a convergent evolution that promotes the S protein processing.

scholarly journals Peptides derived from the SARS-CoV-2 receptor binding motif bind to ACE2 but do not block ACE2-mediated host cell entry or pro-inflammatory cytokine induction

PLoS ONE ◽  
2021 ◽  
Vol 16 (11) ◽  
pp. e0260283
Author(s):  
Amit Mahindra ◽  
Gonzalo Tejeda ◽  
Mario Rossi ◽  
Omar Janha ◽  
Imogen Herbert ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Direct Interaction ◽  
Human Cell Line ◽  
Cell Entry ◽  
Host Cells ◽  
Spike Protein ◽  
Binding Motif ◽  
Viral Attachment ◽  
Contact Residues ◽  
Viral Cell Entry

SARS-CoV-2 viral attachment and entry into host cells is mediated by a direct interaction between viral spike glycoproteins and membrane bound angiotensin-converting enzyme 2 (ACE2). The receptor binding motif (RBM), located within the S1 subunit of the spike protein, incorporates the majority of known ACE2 contact residues responsible for high affinity binding and associated virulence. Observation of existing crystal structures of the SARS-CoV-2 receptor binding domain (SRBD)–ACE2 interface, combined with peptide array screening, allowed us to define a series of linear native RBM-derived peptides that were selected as potential antiviral decoy sequences with the aim of directly binding ACE2 and attenuating viral cell entry. RBM1 (16mer): S443KVGGNYNYLYRLFRK458, RBM2A (25mer): E484GFNCYFPLQSYGFQPTNGVGYQPY508, RBM2B (20mer): F456NCYFPLQSYGFQPTNGVGY505 and RBM2A-Sc (25mer): NYGLQGSPFGYQETPYPFCNFVQYG. Data from fluorescence polarisation experiments suggested direct binding between RBM peptides and ACE2, with binding affinities ranging from the high nM to low μM range (Kd = 0.207–1.206 μM). However, the RBM peptides demonstrated only modest effects in preventing SRBD internalisation and showed no antiviral activity in a spike protein trimer neutralisation assay. The RBM peptides also failed to suppress S1-protein mediated inflammation in an endogenously expressing ACE2 human cell line. We conclude that linear native RBM-derived peptides are unable to outcompete viral spike protein for binding to ACE2 and therefore represent a suboptimal approach to inhibiting SARS-CoV-2 viral cell entry. These findings reinforce the notion that larger biologics (such as soluble ACE2, ‘miniproteins’, nanobodies and antibodies) are likely better suited as SARS-CoV-2 cell-entry inhibitors than short-sequence linear peptides.

scholarly journals The Integrin Binding Peptide, ATN-161, as a Novel Therapy for SARS-CoV-2 Infection

2020 ◽  
Author(s):  
Brandon Beddingfield ◽  
Naoki Iwanaga ◽  
Prem Chapagain ◽  
Wenshu Zheng ◽  
Chad J. Roy ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Protein Interaction ◽  
Cell Entry ◽  
Spike Protein ◽  
Novel Therapy ◽  
Binding Peptide ◽  
Viral Cell Entry ◽  
Α5β1 Integrin

SUMMARYMany efforts to design and screen therapeutics for severe acute respiratory syndrome coronavirus (SARS-CoV-2) have focused on inhibiting viral cell entry by disrupting ACE2 binding with the SARS-CoV-2 spike protein. This work focuses on inhibiting SARS-CoV-2 entry through a hypothesized α5β1 integrin-based mechanism, and indicates that inhibiting the spike protein interaction with α5β1 integrin (+/− ACE2), and the interaction between α5β1 integrin and ACE2 using a molecule ATN-161 represents a promising approach to treat COVID-19.

scholarly journals Studies in humanized mice and convalescent humans yield a SARS-CoV-2 antibody cocktail

Science ◽  
2020 ◽  
Vol 369 (6506) ◽  
pp. 1010-1014 ◽  
Author(s):  
Johanna Hansen ◽  
Alina Baum ◽  
Kristen E. Pascal ◽  
Vincenzo Russo ◽  
Stephanie Giordano ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Selective Pressure ◽  
Three Dimensional ◽  
Spike Protein ◽  
Humanized Mice ◽  
Dimensional Structure ◽  
Antibody Treatment ◽  
Virus Escape ◽  
Escape Mutants ◽  
Antibody Cocktail

Neutralizing antibodies have become an important tool in treating infectious diseases. Recently, two separate approaches yielded successful antibody treatments for Ebola—one from genetically humanized mice and the other from a human survivor. Here, we describe parallel efforts using both humanized mice and convalescent patients to generate antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein, which yielded a large collection of fully human antibodies that were characterized for binding, neutralization, and three-dimensional structure. On the basis of these criteria, we selected pairs of highly potent individual antibodies that simultaneously bind the receptor binding domain of the spike protein, thereby providing ideal partners for a therapeutic antibody cocktail that aims to decrease the potential for virus escape mutants that might arise in response to selective pressure from a single-antibody treatment.

scholarly journals Marek's Disease Virus Disables the ATR-Chk1 Pathway by Activating STAT3

2019 ◽  
Vol 93 (9) ◽  
Author(s):  
Xue Lian ◽  
Chenyi Bao ◽  
Xueqi Li ◽  
Xunhai Zhang ◽  
Hongjun Chen ◽  
...  
Keyword(s):  
Dna Damage ◽  
Viral Replication ◽  
Disease Virus ◽  
Virus Replication ◽  
Specific Inhibitor ◽  
Marek's Disease Virus ◽  
Marek's Disease ◽  
Marek’S Disease Virus ◽  
Marek’S Disease ◽  
Stat3 Phosphorylation

ABSTRACT Oncogenic virus replication often leads to genomic instability, causing DNA damage and inducing the DNA damage response (DDR) pathway. The DDR pathway is a cellular pathway that senses DNA damage and regulates the cell cycle to maintain genomic stability. Therefore, the DDR pathway is critical for the viral lifecycle and tumorigenesis. Marek’s disease virus (MDV), an alphaherpesvirus that causes lymphoma in chickens, has been shown to induce DNA damage in infected cells. However, the interaction between MDV and the host DDR is unclear. In this study, we observed that MDV infection causes DNA strand breakage in chicken fibroblast (CEF) cells along with an increase in the DNA damage markers p53 and p21. Interestingly, we showed that phosphorylation of STAT3 was increased during MDV infection, concomitantly with a decrease of Chk1 phosphorylation. In addition, we found that MDV infection was enhanced by VE-821, an ATR-specific inhibitor, but attenuated by hydroxyurea, an ATR activator. Moreover, inhibition of STAT3 phosphorylation by Stattic eliminates the ability of MDV to inhibit Chk1 phosphorylation. Finally, we showed that MDV replication was decreased by Stattic treatment. Taken together, these results suggest that MDV disables the ATR-Chk1 pathway through STAT3 activation to benefit its replication. IMPORTANCE MDV is used as a biomedical model to study virus-induced lymphoma due to the similar genomic structures and physiological characteristics of MDV and human herpesviruses. Upon infection, MDV induces DNA damage, which may activate the DDR pathway. The DDR pathway has a dual impact on viruses because it manipulates repair and recombination factors to facilitate viral replication and also initiates antiviral action by regulating other signaling pathways. Many DNA viruses evolve to manipulate the DDR pathway to promote virus replication. In this study, we identified a mechanism used by MDV to inhibit ATR-Chk1 pathways. ATR is a cellular kinase that responds to broken single-stranded DNA, which has been less studied in MDV infection. Our results suggest that MDV infection activates STAT3 to disable the ATR-Chk1 pathway, which is conducive to viral replication. This finding provides new insight into the role of STAT3 in interrupting the ATR-Chk1 pathway during MDV replication.

Inhibition of canine distemper virus replication by blocking pyrimidine nucleotide synthesis with A77 1726, the active metabolite of the anti-inflammatory drug leflunomide

2021 ◽  
Author(s):  
Yao Li ◽  
Li Yi ◽  
Sipeng Cheng ◽  
Yongshan Wang ◽  
Jiongjiong Wang ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Active Metabolite ◽  
Canine Distemper Virus ◽  
De Novo ◽  
Specific Inhibitor ◽  
Vero Cells ◽  
Canine Distemper ◽  
Nucleotide Synthesis ◽  
Pyrimidine Nucleotide ◽  
Rate Limiting

Canine distemper virus (CDV) is the aetiological agent that causes canine distemper (CD). Currently, no antiviral drugs have been approved for CD treatment. A77 1726 is the active metabolite of the anti-rheumatoid arthritis (RA) drug leflunomide. It inhibits the activity of Janus kinases (JAKs) and dihydroorotate dehydrogenase (DHO-DHase), a rate-limiting enzyme in de novo pyrimidine nucleotide synthesis. A77 1726 also inhibits the activity of p70 S6 kinase (S6K1), a serine/threonine kinase that phosphorylates and activates carbamoyl-phosphate synthetase (CAD), a second rate-limiting enzyme in the de novo pathway of pyrimidine nucleotide synthesis. Our present study focuses on the ability of A77 1726 to inhibit CDV replication and its underlying mechanisms. Here we report that A77 1726 decreased the levels of the N and M proteins of CDV and lowered the virus titres in the conditioned media of CDV-infected Vero cells. CDV replication was not inhibited by Ruxolitinib (Rux), a JAK-specific inhibitor, but by brequinar sodium (BQR), a DHO-DHase-specific inhibitor, and PF-4708671, an S6K1-specific inhibitor. Addition of exogenous uridine, which restores intracellular pyrimidine nucleotide levels, blocked the antiviral activity of A77 1726, BQR and PF-4708671. A77 1726 and PF-4708671 inhibited the activity of S6K1 in CDV-infected Vero cells, as evidenced by the decreased levels of CAD and S6 phosphorylation. S6K1 knockdown suppressed CDV replication and enhanced the antiviral activity of A77 1726. These observations collectively suggest that the antiviral activity of A77 1726 against CDV is mediated by targeting pyrimidine nucleotide synthesis via inhibiting DHO-DHase activity and S6K1-mediated CAD activation.

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