Would It Be Possible to Stabilize Prefusion SARS-COV-2 Spikes with Ligands?

2020 ◽  
Author(s):  
Julio Coll
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Membrane Fusion ◽  
Experimental Validation ◽  
Wild Type ◽  
Native Conformation ◽  
Enveloped Viruses ◽  
Computational Screening ◽  
Host Membrane ◽  
Nm Range

The<i> </i>infection by Severe Acute Respiratory Syndrome coronavirus (SARS)-CoV2 could be inhibited <i> in vitro </i>by mutations stabilizing their spike (S) native conformation in prefusion states, as reported by several authors. However, the possible S stabilization by binding-ligands, rather than by mutations, have not been computationally explored, nor it is known if that will be possible. Therefore, to first explore these possibilities, a binding target for predictive programs was focused to where the inhibiting mutations were described in the S coronavirus protein, in particular to the “spring-loaded switch-folding” (SLSF) segment of the S2 subunit, whose prefusion unfolding/refolding is required for viral/host membrane fusion. Similar SLSF prefusion mechanisms have been described in many other enveloped viruses. Results of a double computational screening of hundred of thousands of natural compounds for binding to wild-type SLSF conformer, predicted more leads in the low nM range for trimers than for monomers. Further ranked by the number of bound SLSF-conformers, some of the derived top-leads were predicted that may deserve experimental validation. Additionally, thousands of drugs were also included into the screening, resulting in a few top-lead drugs predicted to bind SLSF targets in the low nM range. All these potentially interacting S-ligands, similar structures and/or chemically improved designs, could be used to experimentally find out whether it will be possible to use them for inhibiting fusion and infection, offer new tools to investigate prefusion mechanism(s) and may contribute to therapeutic purposes.

scholarly journals Would It Be Possible to Stabilize Prefusion SARS-COV-2 Spikes with Ligands?

2020 ◽  
Author(s):  
Julio Coll
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Membrane Fusion ◽  
Experimental Validation ◽  
Wild Type ◽  
Native Conformation ◽  
Enveloped Viruses ◽  
Computational Screening ◽  
Host Membrane ◽  
Nm Range

The<i> </i>infection by Severe Acute Respiratory Syndrome coronavirus (SARS)-CoV2 could be inhibited <i> in vitro </i>by mutations stabilizing their spike (S) native conformation in prefusion states, as reported by several authors. However, the possible S stabilization by binding-ligands, rather than by mutations, have not been computationally explored, nor it is known if that will be possible. Therefore, to first explore these possibilities, a binding target for predictive programs was focused to where the inhibiting mutations were described in the S coronavirus protein, in particular to the “spring-loaded switch-folding” (SLSF) segment of the S2 subunit, whose prefusion unfolding/refolding is required for viral/host membrane fusion. Similar SLSF prefusion mechanisms have been described in many other enveloped viruses. Results of a double computational screening of hundred of thousands of natural compounds for binding to wild-type SLSF conformer, predicted more leads in the low nM range for trimers than for monomers. Further ranked by the number of bound SLSF-conformers, some of the derived top-leads were predicted that may deserve experimental validation. Additionally, thousands of drugs were also included into the screening, resulting in a few top-lead drugs predicted to bind SLSF targets in the low nM range. All these potentially interacting S-ligands, similar structures and/or chemically improved designs, could be used to experimentally find out whether it will be possible to use them for inhibiting fusion and infection, offer new tools to investigate prefusion mechanism(s) and may contribute to therapeutic purposes.

scholarly journals Would It Be Possible to Stabilize Prefusion SARS-COV-2 Spikes with Ligands?

2021 ◽  
Author(s):  
Lorenzo, M.M. ◽  
Rafael Blasco ◽  
Julio Coll
Keyword(s):  
Membrane Fusion ◽  
Host Cells ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Native Conformation ◽  
Infectivity Assay ◽  
Computational Screening ◽  
Binding Score ◽  
Host Membrane

<p>Fusion to host cells and infection caused by Severe Acute Respiratory Syndrome coronavirus (SARS)-CoV2 was inhibited <i>in vitro</i> by PP mutations stabilizing prefusion states of their spike (S) protein native conformation, as reported by several authors. However, the possible stabilization of S by binding-ligands, rather than by mutations, have not been explored, nor it is yet known if it would be possible. In this work, the so called “spring-loaded switch-folding” (SLSF) expanding S amino acid residues 960-1010 was computationally targeted because SLSF surrounded the previously described PP mutations. The SLSF trimeric prefusion conformation consisted in 3x3 α-helices that require a transition to 3 longer α-helices before viral/host membrane fusion, similarly to what occurs in other enveloped viruses. Results of a double computational screening among hundred of thousands of natural compounds for binding to the wild-type isolated SLSF conformer predicted more leads for its trimers than for monomers. Further ranked by the number of SLSF-conformers bound, some of the predicted top-leads may deserve experimental validation. Additional screening among thousands of drugs identified Tinosorb, an star-shaped molecule, as the lowest binding-score lead to SLSF in the low nM range. However, despite its lower binding-score, 3-fold molecular symmetry and fitting the inner part of the SLSF α-helices, we were unable to experimentally show any specific inhibition of S-mediated membrane fusion using an VSV-pseudotyped infectivity assay, nor any virtual binding to S-SLSF using docking to whole native S trimers. Further exploring the star-shaped features may provide new molecular alternatives to cross-bind the α-helices of S-SLSF to hypothetically inhibit coronavirus fusion.<b></b></p>

scholarly journals Murine monoclonal antibodies against RBD of SARS-CoV-2 neutralize authentic wild type SARS-CoV-2 as well as B.1.1.7 and B.1.351 viruses and protect in vivo in a mouse model in a neutralization dependent manner

2021 ◽  
Author(s):  
Fatima Amanat ◽  
Shirin Strohmeier ◽  
Wen-Hsin Lee ◽  
Sandhya Bangaru ◽  
Andrew B Ward ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Severe Acute Respiratory Syndrome ◽  
Mouse Model ◽  
Neutralizing Antibodies ◽  
Dependent Manner ◽  
Receptor Binding Domain ◽  
Wild Type ◽  
Murine Monoclonal Antibodies

After first emerging in December 2019 in China, severe acute respiratory syndrome 2 (SARS-CoV-2) has since caused a pandemic leading to millions of infections and deaths worldwide. Vaccines have been developed and authorized but supply of these vaccines is currently limited. With new variants of the virus now emerging and spreading globally, it is essential to develop therapeutics that are broadly protective and bind conserved epitopes in the receptor binding domain (RBD) or the whole spike of SARS-CoV-2. In this study, we have generated mouse monoclonal antibodies (mAbs) against different epitopes on the RBD and assessed binding and neutralization against authentic SARS-CoV-2. We have demonstrated that antibodies with neutralizing activity, but not non-neutralizing antibodies, lower viral titers in the lungs when administered in a prophylactic setting in vivo in a mouse challenge model. In addition, most of the mAbs cross-neutralize the B.1.351 as well as the B.1.1.7 variants in vitro.

scholarly journals Exocytosis induction in Paramecium tetraurelia cells by exogenous phosphoprotein phosphatase in vivo and in vitro: possible involvement of calcineurin in exocytotic membrane fusion.

1987 ◽  
Vol 105 (1) ◽  
pp. 181-189 ◽  
Author(s):  
M Momayezi ◽  
C J Lumpert ◽  
H Kersken ◽  
U Gras ◽  
H Plattner ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Membrane Fusion ◽  
Paramecium Tetraurelia ◽  
Wild Type ◽  
Current Finding ◽  
Phosphoprotein Phosphatase ◽  
Negative Results ◽  
Molecular Components

Since it had been previously shown that in Paramecium cells exocytosis involves the dephosphorylation of a 65-kD phosphoprotein (PP), we tried to induce exocytotic membrane fusion by exogenous phosphatases (alkaline phosphatase or calcineurin [CaN]). The occurrence of calmodulin (CaM) at preformed exocytosis sites (Momayezi, M., H. Kersken, U. Gras, J. Vilmart-Seuwen, and H. Plattner, 1986, J. Histochem. Cytochem., 34:1621-1638) and the current finding of the presence of the 65-kD PP and of a CaN-like protein in cell surface fragments ("cortices") isolated from Paramecium cells led us to also test the effect of antibodies (Ab) against CaM or CaN on exocytosis performance. Microinjected anti-CaN Ab strongly inhibit exocytosis. (Negative results with microinjected anti-CaM Ab can easily be explained by the abundance of CaM.) Alternatively, microinjection of a Ca2+-CaM-CaN complex triggers exocytosis. The same occurs with alkaline phosphatase. All these effects can also be mimicked in vitro with isolated cortices. In vitro exocytosis triggered by adding Ca2+-CaM-CaN or alkaline phosphatase is paralleled by dephosphorylation of the 65-kD PP. Exocytosis can also be inhibited in cortices by anti-CaM Ab or anti-CaN Ab. In wild-type cells, compounds that inhibit phosphatase activity, but none that inhibit kinases or proteases, are able to inhibit exocytosis. Exocytosis cannot be induced by phosphatase injection in a membrane-fusion-deficient mutant strain (nd9-28 degrees C) characterized by a defective organization of exocytosis sites (Beisson, J., M. Lefort-Tran, M. Pouphile, M. Rossignol, and B. Satir, 1976, J. Cell Biol., 69:126-143). We conclude that exocytotic membrane fusion requires an adequate assembly of molecular components to allow for the dephosphorylation of a 65-kD PP and that this step is crucial for the induction of exocytotic membrane fusion in Paramecium cells. In vivo this probably involves a Ca2+-CaM-stimulated CaN-like PP phosphatase.

scholarly journals ATP keeps exocytosis sites in a primed state but is not required for membrane fusion: an analysis with Paramecium cells in vivo and in vitro.

1986 ◽  
Vol 103 (4) ◽  
pp. 1279-1288 ◽  
Author(s):  
J Vilmart-Seuwen ◽  
H Kersken ◽  
R Stürzl ◽  
H Plattner
Keyword(s):  
Membrane Fusion ◽  
Time Course ◽  
Paramecium Tetraurelia ◽  
Wild Type ◽  
Primed State ◽  
Different Strains ◽  
Exogenous Trigger ◽  
Free Media

We have tried to specify a widespread hypothesis on the requirement of ATP for exocytosis (membrane fusion). With Paramecium tetraurelia cells, synchronously (approximately 1 s) exocytosing trichocysts, ATP pools have been measured in different strains, including wild type cells, "non-discharge" (nd), "trichless" (tl), and other mutations. The occurrence of a considerable and rapid ATP consumption also in nd and tl mutations as well as its time course (with a maximum 3-5 s after exocytosis) in exocytosis-competent strains does not match the actual extent of exocytosis performance. However, from in vivo as well as from in vitro experiments, we came to the conclusion that ATP might be required to keep the system in a primed state and its removal might facilitate membrane fusion. (For the study of exocytosis in vitro we have developed a new system, consisting of isolated cortices). In vivo as well as in vitro exocytosis is inhibited by increased levels of ATP or by a nonhydrolyzable ATP analogue. In vitro exocytosis is facilitated in ATP-free media. In vivo-microinjected ATP retards exocytosis in response to chemical triggers, whereas microinjected apyrase triggers exocytosis without exogenous trigger. Experiments with this system also largely exclude any overlaps with other processes that normally accompany exocytosis. Our data also explain why it was frequently assumed that ATP would be required for exocytosis. We conclude that membrane fusion during exocytosis does not require the presence of ATP; the occurrence of membrane fusion might involve the elimination of ATP from primed fusogenic sites; most of the ATP consumption measured in the course of exocytosis may be due to other effects, probably to recovery phenomena.

scholarly journals Functional Relevance of the N-Terminal Domain of Pseudorabies Virus Envelope Glycoprotein H and Its Interaction with Glycoprotein L

2017 ◽  
Vol 91 (9) ◽  
Author(s):  
Melina Vallbracht ◽  
Sascha Rehwaldt ◽  
Barbara G. Klupp ◽  
Thomas C. Mettenleiter ◽  
Walter Fuchs
Keyword(s):  
Fusion Protein ◽  
Membrane Fusion ◽  
Receptor Binding ◽  
Pseudorabies Virus ◽  
Envelope Glycoproteins ◽  
Virus Envelope ◽  
Enveloped Viruses ◽  
Gh Gene ◽  
Functional Relevance

ABSTRACT Several envelope glycoproteins are involved in herpesvirus entry into cells, direct cell-to-cell spread, and induction of cell fusion. The membrane fusion protein glycoprotein B (gB) and the presumably gB-activating heterodimer gH/gL are essential for these processes and conserved throughout the Herpesviridae. However, after extended cell culture passage of gL-negative mutants of the alphaherpesvirus pseudorabies virus (PrV), phenotypic revertants could be isolated which had acquired spontaneous mutations affecting the gL-interacting N-terminal part of the gH ectodomain (gDH and gHB4.1) (B. G. Klupp and T. C. Mettenleiter, J Virol 73:3014–3022, 1999; C. Schröter, M. Vallbracht, J. Altenschmidt, S. Kargoll, W. Fuchs, B. G. Klupp, and T. C. Mettenleiter, J Virol 90:2264–2272, 2016). To investigate the functional relevance of this part of gH in more detail, we introduced an in-frame deletion of 66 codons at the 5′ end of the plasmid-cloned gH gene (gH32/98). The N-terminal signal peptide was retained, and the deletion did not affect expression or processing of gH but abrogated its function in in vitro fusion assays. Insertion of the engineered gH gene into the PrV genome resulted in a defective mutant (pPrV-gH32/98K), which was incapable of entry and spread. Interestingly, in vitro activity of mutated gH32/98 was restored when it was coexpressed with hyperfusogenic gBB4.1, obtained from a passaged gL deletion mutant of PrV. Moreover, the entry and spread defects of pPrV-gH32/98K were compensated by the mutations in gBB4.1 in cis, as well as in trans, independent of gL. Thus, PrV gL and the gL-interacting domain of gH are not strictly required for function. IMPORTANCE Membrane fusion is crucial for infectious entry and spread of enveloped viruses. While many enveloped viruses require only one or two proteins for receptor binding and membrane fusion, herpesvirus infection depends on several envelope glycoproteins. Besides subfamily-specific receptor binding proteins, the core fusion machinery consists of the conserved fusion protein gB and the gH/gL complex. The role of the latter is unclear, but it is hypothesized to interact with gB for fusion activation. Using isogenic virus recombinants, we demonstrate here that gL and the gL-binding domain of PrV gH are not strictly required for membrane fusion during virus entry and spread when concomitantly mutations in gB are present which increase its fusogenicity. Thus, our results strongly support the notion of a functional gB-gH interaction during the fusion process.

scholarly journals The degree of polymerization and sulfation patterns in heparan sulfate are critical determinants of cytomegalovirus entry into host cells

2021 ◽  
Vol 17 (8) ◽  
pp. e1009803
Author(s):  
Dipanwita Mitra ◽  
Mohammad H. Hasan ◽  
John T. Bates ◽  
Michael A. Bierdeman ◽  
Dallas R. Ederer ◽  
...  
Keyword(s):  
Cell Surface ◽  
Heparan Sulfate ◽  
Virus Entry ◽  
Degree Of Polymerization ◽  
Host Cells ◽  
Virus Infectivity ◽  
Wild Type ◽  
Enveloped Viruses ◽  
Host Membrane ◽  
Mutant Cells

Several enveloped viruses, including herpesviruses attach to host cells by initially interacting with cell surface heparan sulfate (HS) proteoglycans followed by specific coreceptor engagement which culminates in virus-host membrane fusion and virus entry. Interfering with HS-herpesvirus interactions has long been known to result in significant reduction in virus infectivity indicating that HS play important roles in initiating virus entry. In this study, we provide a series of evidence to prove that specific sulfations as well as the degree of polymerization (dp) of HS govern human cytomegalovirus (CMV) binding and infection. First, purified CMV extracellular virions preferentially bind to sulfated longer chain HS on a glycoarray compared to a variety of unsulfated glycosaminoglycans including unsulfated shorter chain HS. Second, the fraction of glycosaminoglycans (GAG) displaying higher dp and sulfation has a larger impact on CMV titers compared to other fractions. Third, cell lines deficient in specific glucosaminyl sulfotransferases produce significantly reduced CMV titers compared to wild-type cells and virus entry is compromised in these mutant cells. Finally, purified glycoprotein B shows strong binding to heparin, and desulfated heparin analogs compete poorly with heparin for gB binding. Taken together, these results highlight the significance of HS chain length and sulfation patterns in CMV attachment and infectivity.

scholarly journals Residues in the Heptad Repeat A Region of the Fusion Protein Modulate the Virulence of Sendai Virus in Mice

2009 ◽  
Vol 84 (2) ◽  
pp. 810-821 ◽  
Author(s):  
Laura E. Luque ◽  
Olga A. Bridges ◽  
John N. Mason ◽  
Kelli L. Boyd ◽  
Allen Portner ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Sendai Virus ◽  
Syncytium Formation ◽  
Heptad Repeat ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
F Protein

ABSTRACT While the molecular basis of fusion (F) protein refolding during membrane fusion has been studied extensively in vitro, little is known about the biological significance of membrane fusion activity in parainfluenza virus replication and pathogenesis in vivo. Two recombinant Sendai viruses, F-L179V and F-K180Q, were generated that contain F protein mutations in the heptad repeat A region of the ectodomain, a region of the protein known to regulate F protein activation. In vitro, the F-L179V virus caused increased syncytium formation (cell-cell membrane fusion) yet had a rate of replication and levels of F protein expression and cleavage similar to wild-type virus. The F-K180Q virus had a reduced replication rate along with reduced levels of F protein expression, cleavage, and fusogenicity. In DBA/2 mice, the hyperfusogenic F-L179V virus induced greater morbidity and mortality than wild-type virus, while the attenuated F-K180Q virus was much less pathogenic. During the first week of infection, virus replication and inflammation in the lungs were similar for wild-type and F-L179V viruses. After approximately 1 week of infection, the clearance of F-L179V virus was delayed, and more extensive interstitial inflammation and necrosis were observed in the lungs, affecting entire lobes of the lungs and having significantly greater numbers of syncytial cell masses in alveolar spaces on day 10. On the other hand, the slower-growing F-K180Q virus caused much less extensive inflammation than wild-type virus, presumably due to its reduced replication rate, and did not cause observable syncytium formation in the lungs. Overall, the results show that residues in the heptad repeat A region of the F protein modulate the virulence of Sendai virus in mice by influencing both the spread and clearance of the virus and the extent and severity of inflammation. An understanding of how the F protein contributes to infection and inflammation in vivo may assist in the development of antiviral therapies against respiratory paramyxoviruses.

scholarly journals The pH of Activation of the Hemagglutinin Protein Regulates H5N1 Influenza Virus Pathogenicity and Transmissibility in Ducks

2009 ◽  
Vol 84 (3) ◽  
pp. 1527-1535 ◽  
Author(s):  
Mark L. Reed ◽  
Olga A. Bridges ◽  
Patrick Seiler ◽  
Jeong-Ki Kim ◽  
Hui-Ling Yen ◽  
...  
Keyword(s):  
Weight Loss ◽  
Influenza Virus ◽  
Membrane Fusion ◽  
Influenza Viruses ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
H5n1 Influenza ◽  
Ha Protein

ABSTRACT While the molecular mechanism of membrane fusion by the influenza virus hemagglutinin (HA) protein has been studied extensively in vitro, the role of acid-dependent HA protein activation in virus replication, pathogenesis, and transmission in vivo has not been characterized. To investigate the biological significance of the pH of activation of the HA protein, we compared the properties of four recombinant viruses with altered HA protein acid stability to those of wild-type influenza virus A/chicken/Vietnam/C58/04 (H5N1) in vitro and in mallards. Membrane fusion by wild-type virus was activated at pH 5.9. Wild-type virus had a calculated environmental persistence of 62 days and caused extensive morbidity, mortality, shedding, and transmission in mallards. An N114K mutation that increased the pH of HA activation by 0.5 unit resulted in decreased replication, genetic stability, and environmental stability. Changes of +0.4 and −0.5 unit in the pH of activation by Y23H and K58I mutations, respectively, reduced weight loss, mortality, shedding, and transmission in mallards. An H24Q mutation that decreased the pH of activation by 0.3 unit resulted in weight loss, mortality, clinical symptoms, and shedding similar to those of the wild type. However, the HA-H241Q virus was shed more extensively into drinking water and persisted longer in the environment. The pH of activation of the H5 HA protein plays a key role in the propagation of H5N1 influenza viruses in ducks and may be a novel molecular factor in the ecology of influenza viruses. The data also demonstrate that H5N1 neuraminidase activity increases the pH of activation of the HA protein in vitro.

scholarly journals Important Role for the Transmembrane Domain of Severe Acute Respiratory Syndrome Coronavirus Spike Protein during Entry

2006 ◽  
Vol 80 (3) ◽  
pp. 1302-1310 ◽  
Author(s):  
Rene Broer ◽  
Bertrand Boson ◽  
Willy Spaan ◽  
François-Loïc Cosset ◽  
Jeroen Corver
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Membrane Fusion ◽  
Cell Fusion ◽  
Transmembrane Domain ◽  
Cytoplasmic Tail ◽  
Transmembrane Domains ◽  
Spike Protein ◽  
Fusion Activity ◽  
Wild Type ◽  
Cell Cell

ABSTRACT The spike protein (S) of severe acute respiratory syndrome coronavirus (SARS-CoV) is responsible for receptor binding and membrane fusion. It contains a highly conserved transmembrane domain that consists of three parts: an N-terminal tryptophan-rich domain, a central domain, and a cysteine-rich C-terminal domain. The cytoplasmic tail of S has previously been shown to be required for assembly. Here, the roles of the transmembrane and cytoplasmic domains of S in the infectivity and membrane fusion activity of SARS-CoV have been studied. SARS-CoV S-pseudotyped retrovirus (SARSpp) was used to measure S-mediated infectivity. In addition, the cell-cell fusion activity of S was monitored by a Renilla luciferase-based cell-cell fusion assay. Svsv-cyt, an S chimera with a cytoplasmic tail derived from vesicular stomatitis virus G protein (VSV-G), and Smhv-tmdcyt, an S chimera with the cytoplasmic and transmembrane domains of mouse hepatitis virus, displayed wild-type-like activity in both assays. Svsv-tmdcyt, a chimera with the cytoplasmic and transmembrane domains of VSV-G, was impaired in the SARSpp and cell-cell fusion assays, showing 3 to 25% activity compared to the wild type, depending on the assay and the cells used. Examination of the oligomeric state of the chimeric S proteins in SARSpp revealed that Svsv-tmdcyt trimers were less stable than wild-type S trimers, possibly explaining the lowered fusogenicity and infectivity.

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