An Oleanolic Acid Derivative Inhibits Hemagglutinin-Mediated Entry of Influenza A Virus

Influenza A viruses (IAV) have been a major public health threat worldwide, and options for antiviral therapy become increasingly limited with the emergence of drug-resisting virus strains. New and effective anti-IAV drugs, especially for highly pathogenic influenza, with different modes of action, are urgently needed. The influenza virus glycoprotein hemagglutinin (HA) plays critical roles in the early stage of virus infection, including receptor binding and membrane fusion, making it a potential target for the development of anti-influenza drugs. In this study, we show that OA-10, a newly synthesized triterpene out of 11 oleanane-type derivatives, exhibited significant antiviral activity against four different subtypes of IAV (H1N1, H5N1, H9N2 and H3N2) replications in A549 cell cultures with EC50 ranging from 6.7 to 19.6 μM and a negligible cytotoxicity (CC50 > 640 μM). It inhibited acid-induced hemolysis in a dose-dependent manner, with an IC50 of 26 µM, and had a weak inhibition on the adsorption of H5 HA to chicken erythrocytes at higher concentrations (≥40 µM). Surface plasmon resonance (SPR) analysis showed that OA-10 interacted with HA in a dose-dependent manner with the equilibrium dissociation constants (KD) of the interaction of 2.98 × 10−12 M. Computer-aided molecular docking analysis suggested that OA-10 might bind to the cavity in HA stem region which is known to undergo significant rearrangement during membrane fusion. Our results demonstrate that OA-10 inhibits H5N1 IAV replication mainly by blocking the conformational changes of HA2 subunit required for virus fusion with endosomal membrane. These findings suggest that OA-10 could serve as a lead for further development of novel virus entry inhibitors to prevent and treat IAV infections.

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Influenza Hemagglutinin (HA) Stem Region Mutations That Stabilize or Destabilize the Structure of Multiple HA Subtypes

Journal of Virology ◽

10.1128/jvi.00057-15 ◽

2015 ◽

Vol 89 (8) ◽

pp. 4504-4516 ◽

Cited By ~ 26

Author(s):

Lauren Byrd-Leotis ◽

Summer E. Galloway ◽

Evangeline Agbogu ◽

David A. Steinhauer

Keyword(s):

Membrane Fusion ◽

Receptor Binding ◽

Conformational Changes ◽

Influenza A ◽

Mutational Analysis ◽

Host Cells ◽

Influenza A Viruses ◽

Pathogenic Potential ◽

Additional Tool ◽

Improved Stability

ABSTRACTInfluenza A viruses enter host cells through endosomes, where acidification induces irreversible conformational changes of the viral hemagglutinin (HA) that drive the membrane fusion process. The prefusion conformation of the HA is metastable, and the pH of fusion can vary significantly among HA strains and subtypes. Furthermore, an accumulating body of evidence implicates HA stability properties as partial determinants of influenza host range, transmission phenotype, and pathogenic potential. Although previous studies have identified HA mutations that can affect HA stability, these have been limited to a small selection of HA strains and subtypes. Here we report a mutational analysis of HA stability utilizing a panel of expressed HAs representing a broad range of HA subtypes and strains, including avian representatives across the phylogenetic spectrum and several human strains. We focused on two highly conserved residues in the HA stem region: HA2 position 58, located at the membrane distal tip of the short helix of the hairpin loop structure, and HA2 position 112, located in the long helix in proximity to the fusion peptide. We demonstrate that a K58I mutation confers an acid-stable phenotype for nearly all HAs examined, whereas a D112G mutation consistently leads to elevated fusion pH. The results enhance our understanding of HA stability across multiple subtypes and provide an additional tool for risk assessment for circulating strains that may have other hallmarks of human adaptation. Furthermore, the K58I mutants, in particular, may be of interest for potential use in the development of vaccines with improved stability profiles.IMPORTANCEThe influenza A hemagglutinin glycoprotein (HA) mediates the receptor binding and membrane fusion functions that are essential for virus entry into host cells. While receptor binding has long been recognized for its role in host species specificity and transmission, membrane fusion and associated properties of HA stability have only recently been appreciated as potential determinants. We show here that mutations can be introduced at highly conserved positions to stabilize or destabilize the HA structure of multiple HA subtypes, expanding our knowledge base for this important phenotype. The practical implications of these findings extend to the field of vaccine design, since the HA mutations characterized here could potentially be utilized across a broad spectrum of influenza virus subtypes to improve the stability of vaccine strains or components.

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Toxicity of chlordane at early developmental stage of zebrafish

10.1101/119248 ◽

2017 ◽

Cited By ~ 1

Author(s):

Jingxuan Xiong

Keyword(s):

Early Stage ◽

Real Life ◽

Potential Effect ◽

Early Developmental Stage ◽

Dependent Manner ◽

Lower Survival Rate ◽

Zebrafish Larvae ◽

Hatching Time ◽

Early Developmental ◽

Dose Dependent

AbstractChlordane is highly toxic organochlorine pesticides that have been widely used throughout the world for decades and posing adverse effects on the environment. Contents detected in tissue and blood samples have resulted in a raising concern for their potential effects on wildlife and humans. In this study, we investigate the potential effect of chlordane on the development of zebrafish embryos. Zebrafish larvae were treated with different concentrations (0, 25, 50, 100, 200 ng/L) of chlordane from 12 hours postfertilization (hpf). Different early stage parameters were observed at 1, 2, 3 and 4 day post-fertilization (dpf). Chlordane-exposed zebrafish larvae appeared significant lower survival rate, developmental and hatching time delay and decreased embryo productivity. The heartbeat rate and blood flow were decreased in a dose dependent manner. These results suggested that exposure to real life of chlordane led to direct morphological and phenotypic changes and effects systems related to development and reproduction even in short-term manner.

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Membrane-tethered mucin-like polypeptides sterically inhibit binding and slow fusion kinetics of influenza A virus

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1921962117 ◽

2020 ◽

Vol 117 (23) ◽

pp. 12643-12650 ◽

Cited By ~ 2

Author(s):

Corleone S. Delaveris ◽

Elizabeth R. Webster ◽

Steven M. Banik ◽

Steven G. Boxer ◽

Carolyn R. Bertozzi

Keyword(s):

Lipid Bilayers ◽

Influenza A ◽

Conformational Transition ◽

Molecular Model ◽

High Surface ◽

Protective Mechanism ◽

Dependent Manner ◽

Influenza A Viruses ◽

Virus Binding ◽

Kinetics Of

The mechanism(s) by which cell-tethered mucins modulate infection by influenza A viruses (IAVs) remain an open question. Mucins form both a protective barrier that can block virus binding and recruit IAVs to bind cells via the sialic acids of cell-tethered mucins. To elucidate the molecular role of mucins in flu pathogenesis, we constructed a synthetic glycocalyx to investigate membrane-tethered mucins in the context of IAV binding and fusion. We designed and synthesized lipid-tethered glycopolypeptide mimics of mucins and added them to lipid bilayers, allowing chemical control of length, glycosylation, and surface density of a model glycocalyx. We observed that the mucin mimics undergo a conformational change at high surface densities from a compact to an extended architecture. At high surface densities, asialo mucin mimics inhibited IAV binding to underlying glycolipid receptors, and this density correlated to the mucin mimic’s conformational transition. Using a single virus fusion assay, we observed that while fusion of virions bound to vesicles coated with sialylated mucin mimics was possible, the kinetics of fusion was slowed in a mucin density-dependent manner. These data provide a molecular model for a protective mechanism by mucins in IAV infection, and therefore this synthetic glycocalyx provides a useful reductionist model for studying the complex interface of host–pathogen interactions.

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Otoferlin is a calcium sensor that directly regulates SNARE-mediated membrane fusion

The Journal of Cell Biology ◽

10.1083/jcb.201002089 ◽

2010 ◽

Vol 191 (1) ◽

pp. 187-197 ◽

Cited By ~ 80

Author(s):

Colin P. Johnson ◽

Edwin R. Chapman

Keyword(s):

Membrane Fusion ◽

Calcium Binding ◽

Dissociation Constants ◽

Dependent Manner ◽

Calcium Sensor ◽

Fusion Reactions ◽

Cochlear Hair Cells ◽

C2 Domains ◽

Synaptic Vesicle Exocytosis ◽

Vesicle Exocytosis

Otoferlin is a large multi–C2 domain protein proposed to act as a calcium sensor that regulates synaptic vesicle exocytosis in cochlear hair cells. Although mutations in otoferlin have been associated with deafness, its contribution to neurotransmitter release is unresolved. Using recombinant proteins, we demonstrate that five of the six C2 domains of otoferlin sense calcium with apparent dissociation constants that ranged from 13–25 µM; in the presence of membranes, these apparent affinities increase by up to sevenfold. Using a reconstituted membrane fusion assay, we found that five of the six C2 domains of otoferlin stimulate membrane fusion in a calcium-dependent manner. We also demonstrate that a calcium binding–deficient form of the C2C domain is incapable of stimulating membrane fusion, further underscoring the importance of calcium for the protein’s function. These results demonstrate for the first time that otoferlin is a calcium sensor that can directly regulate soluble N-ethyl-maleimide sensitive fusion protein attachment protein receptor–mediated membrane fusion reactions.

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Oral Supplementation with Omega3 Fatty Acids Inhibits NFkB Activation In Chronic Lymphocytic Leukemia (CLL) Cells.

Blood ◽

10.1182/blood.v116.21.4607.4607 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4607-4607

Author(s):

Oscar F. F Ballester ◽

Johannes Fahrmann ◽

Theodore Witte ◽

Gabriela Ballester ◽

W. Elaine Hardman

Keyword(s):

Conflicts Of Interest ◽

Early Stage ◽

Gradient Centrifugation ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Dependent Manner ◽

Red Cell ◽

Omega 3 ◽

Dose Dependent

Abstract Abstract 4607 Introduction: Nuclear factor kappa B (NFkB) is a critical transcription factor involved in the growth and survival of CLL cells. NFkB is recognized as an important target for the development of novel therapies for the treatment of various malignancies. In vitro and in experimental animal models, OMEGA-3 fatty acid (O3FA) supplementation has been shown to inhibit NFkB activity. Patients and Methods: Patients with early stage CLL (Rai stages 0-II) who required no therapy, where accrued to this phase I-II trial. O3FA supplements were given for a total of 12 months at doses ranging from 2250 mg (EPA plus DHA), escalated to 4500 mg and 6750 mg per day as tolerated. NFkB activity was measured in peripheral blood samples after separation of mononuclear cell by gradient centrifugation and expressed as luminescence units/μ g of protein. Baseline and multiple serial samples were obtained during the study period. In-vitro cytotoxicity assays to doxorubicin were conducted using standard LD50 methods. Compliance was monitored by analysis of red cell and lymphocyte membrane lipid composition by gas chromatography. Results: Fifteen patients have been accrued to the trial, 8 of them have currently completed the planned 12 months of the study period. No significant clinical changes in disease activity were noted. O3FA was well tolerated. Supplementation resulted in a dose-dependent increase of O3FA composition of red cell and lymphocyte membranes in a dose dependent manner. At baseline, CLL patients had NFkB above the range observed in normal controls (2.05 × 104 to 2.32 × 105 NFkB lum units/μ g). The median value in CLL patients at baseline was 11.60 × 106 NFkB lum units/μ g (range 0.9 × 105 to 23.12 × 106). Among 5 patients with the highest baseline levels of NFkB, a decrease in NFkB activity ranging from 0.02 to 0.19 of the baseline value, was noted at the 2 higher doses of O3FA supplementation. Similar results were seen in patients with relatively lower levels of baseline NFkB activity (0.9 × 105 to 2.96 × 106 lum units/μ g). In vitro, significant doxorubicin cytotoxicity (>50%) was noted in samples obtained during supplementation, at μ gM concentrations which produced no detectable cell kill in baseline samples. Conclusions: O3FA supplementation resulted in significant inhibition of NFkB activity in leukemic cells from patients with CLL. In-vitro, after O3FA supplementation CLL cells became more sensitive to doxorubicin. Preliminary analysis of whole genome micro arrays revealed significant down-regulation of multiple genes associated with O3FA supplementation. Disclosures: No relevant conflicts of interest to declare.

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Towards Al3+-Induced Manganese-Containing Superoxide Dismutase Inactivation and Conformational Changes: An Integrating Study with Docking Simulations

Enzyme Research ◽

10.4061/2011/307464 ◽

2011 ◽

Vol 2011 ◽

pp. 1-7

Author(s):

Jiang-Liu Yang ◽

Shang-Jun Yin ◽

Yue-Xiu Si ◽

Zhi-Rong Lü ◽

Xiangrong Shao ◽

...

Keyword(s):

Superoxide Dismutase ◽

Conformational Changes ◽

Tertiary Structure ◽

Kinetic Studies ◽

Computational Prediction ◽

Enzyme Inactivation ◽

Dependent Manner ◽

Inhibitory Effects ◽

Docking Simulations ◽

Dose Dependent

Superoxide dismutase (SOD, EC 1.15.1.1) plays an important antioxidant defense role in skins exposed to oxygen. We studied the inhibitory effects of Al3+ on the activity and conformation of manganese-containing SOD (Mn-SOD). Mn-SOD was significantly inactivated by Al3+ in a dose-dependent manner. The kinetic studies showed that Al3+ inactivated Mn-SOD follows the first-order reaction. Al3+ increased the degree of secondary structure of Mn-SOD and also disrupted the tertiary structure of Mn-SOD, which directly resulted in enzyme inactivation. We further simulated the docking between Mn-SOD and Al3+ (binding energy for Dock 6.3: −14.07 kcal/mol) and suggested that ASP152 and GLU157 residues were predicted to interact with Al3+, which are not located in the Mn-contained active site. Our results provide insight into the inactivation of Mn-SOD during unfolding in the presence of Al3+ and allow us to describe a ligand binding via inhibition kinetics combined with the computational prediction.

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Strain-dependent effects of PB1-F2 of triple-reassortant H3N2 influenza viruses in swine

Journal of General Virology ◽

10.1099/vir.0.045005-0 ◽

2012 ◽

Vol 93 (10) ◽

pp. 2204-2214 ◽

Cited By ~ 20

Author(s):

Lindomar Pena ◽

Amy L. Vincent ◽

Crystal L. Loving ◽

Jamie N. Henningson ◽

Kelly M. Lager ◽

...

Keyword(s):

Virus Replication ◽

Influenza A ◽

Reverse Genetics ◽

Influenza Viruses ◽

Dependent Manner ◽

Lung Pathology ◽

Influenza A Viruses ◽

Virus Shedding ◽

The Impact ◽

Strain Dependent

The PB1-F2 protein of the influenza A viruses (IAVs) can act as a virulence factor in mice. Its contribution to the virulence of IAV in swine, however, remains largely unexplored. In this study, we chose two genetically related H3N2 triple-reassortant IAVs to assess the impact of PB1-F2 in virus replication and virulence in pigs. Using reverse genetics, we disrupted the PB1-F2 ORF of A/swine/Wisconsin/14094/99 (H3N2) (Sw/99) and A/turkey/Ohio/313053/04 (H3N2) (Ty/04). Removing the PB1-F2 ORF led to increased expression of PB1-N40 in a strain-dependent manner. Ablation of the PB1-F2 ORF (or incorporation of the N66S mutation in the PB1-F2 ORF, Sw/99 N66S) affected the replication in porcine alveolar macrophages of only the Sw/99 KO (PB1-F2 knockout) and Sw/99 N66S variants. The Ty/04 KO strain showed decreased virus replication in swine respiratory explants, whereas no such effect was observed in Sw/99 KO, compared with the wild-type (WT) counterparts. In pigs, PB1-F2 did not affect virus shedding or viral load in the lungs for any of these strains. Upon necropsy, PB1-F2 had no effect on the lung pathology caused by Sw/99 variants. Interestingly, the Ty/04 KO-infected pigs showed significantly increased lung pathology at 3 days post-infection compared with pigs infected with the Ty/04 WT strain. In addition, the pulmonary levels of interleukin (IL)-6, IL-8 and gamma interferon were regulated differentially by the expression of PB1-F2. Taken together, these results indicate that PB1-F2 modulates virus replication, virulence and innate immune responses in pigs in a strain-dependent fashion.

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Membrane-Tethered Mucin-like Polypeptides Sterically Inhibit Binding and Slow Fusion Kinetics of Influenza A Virus

10.26434/chemrxiv.11312567 ◽

2019 ◽

Cited By ~ 1

Author(s):

Corleone Delaveris ◽

Elizabeth Webster ◽

Steven Banik ◽

Steven Boxer ◽

Carolyn Bertozzi

Keyword(s):

Lipid Bilayers ◽

Influenza A ◽

Conformational Transition ◽

Molecular Model ◽

High Surface ◽

Protective Mechanism ◽

Dependent Manner ◽

Influenza A Viruses ◽

Virus Binding ◽

Kinetics Of

<div> <div> <div> <p>The mechanism(s) by which cell-tethered mucins modulate infection by Influenza A viruses (IAVs) remains an open question. Mucins form both a protective barrier that can block virus binding and recruit IAVs to bind cells via the sialic acids of cell-tethered mucins. To elucidate the molecular role of mucins in flu pathogenesis, we constructed a synthetic glycocalyx to investigate membrane-tethered mucins in the context of IAV binding and fusion. We designed and synthesized lipid-tethered glycopolypeptide mimics of mucins and added them to lipid bilayers, allowing chemical control of length, glycosylation, and surface density of a model glycocalyx. We observed that the mucin mimics undergo a conformational change at high surface densities from a compact to an extended architecture. At high surface densities asialo mucin mimics inhibited IAV binding to underlying glycolipid receptors and this density correlated to the mucin mimic’s conformational transition. Using a single virus fusion assay, we observed that while fusion of virions bound to vesicles coated with sialylated mucin mimics was possible, the kinetics of fusion were slowed in a mucin density-dependent manner. These data provide a molecular model for a protective mechanism by mucins in IAV infection, and therefore this synthetic glycocalyx provides a useful reductionist model for studying the complex interface of host-pathogen interactions. </p> </div> </div> </div>

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In-Vivo Toxicity Studies and In-Vitro Inactivation of SARS-CoV-2 by Povidone-iodine In-situ Gel Forming Formulations

10.1101/2020.05.18.103184 ◽

2020 ◽

Cited By ~ 2

Author(s):

Bo Liang ◽

Xudong Yuan ◽

Gang Wei ◽

Wei Wang ◽

Ming Zhang ◽

...

Keyword(s):

Povidone Iodine ◽

Early Stage ◽

Etiologic Agent ◽

Dependent Manner ◽

Forming Technology ◽

Long Acting ◽

Dose Dependent

AbstractTo curb the spread of SARS-CoV-2, the etiologic agent of the COVID-19 pandemic, we characterize the virucidal activity of long-acting Povidone Iodine (PVP-I) compositions developed using an in-situ gel forming technology. The PVP-I gel forming nasal spray (IVIEW-1503) and PVP-I gel forming ophthalmic eye drop (IVIEW-1201) rapidly inactivated SARS-CoV-2, inhibiting the viral infection of VERO76 cells. No toxicity was observed for the PVP-I formulations. Significant inactivation was noted with preincubation of the virus with these PVP-I formulations at the lowest concentrations tested. It has been demonstrated that both PVP-I formulations can inactivate SARS-CoV-2 virus efficiently in both a dose-dependent and a time-dependent manner. These results suggest IVIEW-1503 and IVIEW-1201 could be potential agents to reduce or prevent the transmission of the virus through the nasal cavity and the eye, respectively. Further studies are needed to clinically evaluate these formulations in early-stage COVID-19 patients.

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pH Optimum of Hemagglutinin-Mediated Membrane Fusion Determines Sensitivity of Influenza A Viruses to the Interferon-Induced Antiviral State and IFITMs

Journal of Virology ◽

10.1128/jvi.00246-17 ◽

2017 ◽

Vol 91 (11) ◽

Cited By ~ 29

Author(s):

Thomas Gerlach ◽

Luca Hensen ◽

Tatyana Matrosovich ◽

Janina Bergmann ◽

Michael Winkler ◽

...

Keyword(s):

Epithelial Cells ◽

Membrane Fusion ◽

Influenza A ◽

Target Cells ◽

Viral Fusion ◽

Influenza A Viruses ◽

Ph Optimum ◽

High Ph ◽

Innate Defense ◽

Antiviral State

ABSTRACT The replication and pathogenicity of influenza A viruses (IAVs) critically depend on their ability to tolerate the antiviral interferon (IFN) response. To determine a potential role for the IAV hemagglutinin (HA) in viral sensitivity to IFN, we studied the restriction of IAV infection in IFN-β-treated human epithelial cells by using 2:6 recombinant IAVs that shared six gene segments of A/Puerto Rico/8/1934 virus (PR8) and contained HAs and neuraminidases of representative avian, human, and zoonotic H5N1 and H7N9 viruses. In A549 and Calu-3 cells, viruses displaying a higher pH optimum of HA-mediated membrane fusion, H5N1-PR8 and H7N9-PR8, were less sensitive to the IFN-induced antiviral state than their counterparts with HAs from duck and human viruses, which fused at a lower pH. The association between a high pH optimum of fusion and reduced IFN sensitivity was confirmed by using HA point mutants of A/Hong Kong/1/1968-PR8 that differed solely by their fusion properties. Furthermore, similar effects of the viral fusion pH on IFN sensitivity were observed in experiments with (i) primary human type II alveolar epithelial cells and differentiated cultures of human airway epithelial cells, (ii) nonrecombinant zoonotic and pandemic IAVs, and (iii) preparations of IFN-α and IFN-λ1. A higher pH of membrane fusion and reduced sensitivity to IFN correlated with lower restriction of the viruses in MDCK cells stably expressing the IFN-inducible transmembrane proteins IFITM2 and IFITM3, which are known to inhibit viral fusion. Our results reveal that the pH optimum of HA-driven membrane fusion of IAVs is a determinant of their sensitivity to IFN and IFITM proteins. IMPORTANCE The IFN system constitutes an important innate defense against viral infection. Substantial information is available on how IAVs avoid detection by sensors of the IFN system and disable IFN signaling pathways. Much less is known about the ability of IAVs to tolerate the antiviral activity of IFN-induced cellular proteins. The IFN-induced proteins of the IFITM family block IAV entry into target cells and can restrict viral spread and pathogenicity. Here we show for the first time that the sensitivity of IAVs to the IFN-induced antiviral state and IFITM2 and IFITM3 proteins depends on the pH value at which the viral HA undergoes a conformational transition and mediates membrane fusion. Our data imply that the high pH optimum of membrane fusion typical of zoonotic IAVs of gallinaceous poultry, such as H5N1 and H7N9, may contribute to their enhanced virulence in humans.

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