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Patrick C Wilson - One of the best experts on this subject based on the ideXlab platform.

  • hemagglutinin stalk reactive antibodies interfere with influenza virus neuraminidase activity by steric hindrance
    Journal of Virology, 2018
    Co-Authors: Yaoqing Chen, Linda Yuling Lan, Min Huang, Carole Henry, Patrick C Wilson
    Abstract:

    Hemagglutinin (HA) stalk-reactive antibodies are the basis of several current "one-shot" universal influenza vaccine efforts because they protect against a wide spectrum of influenza virus strains. The appreciated mechanism of protection by HA stalk-reactive antibodies is to inhibit HA stalk reconfiguration, blocking viral fusion and entry. This study shows that HA stalk-reactive antibodies also inhibit neuraminidase (NA) enzymatic activity, prohibiting viral egress. NA inhibition (NI) was evident for an attached substrate but not for unattached small-molecule cleavage of sialic acid. This finding suggests that the antibodies inhibit NA enzymatic activity through steric hindrance, thus limiting NA access to sialic acids when adjacent to HA on whole virions. Consistently, F(ab')2 fragments that occupied reduced area without loss of avidity or disrupted HA/NA interactions showed significantly reduced NI activity. Notably, HA stalk-binding antibodies lacking NI activity were unable to neutralize viral infection via microneutralization assays. This work suggests that NI activity is an important component of protection mediated by HA stalk-reactive antibodies.IMPORTANCE This study reports a new mechanism of protection mediated by influenza hemagglutinin stalk-reactive antibodies, i.e., inhibition of neuraminidase activity by steric hindrance, blocking access of neuraminidase to sialic acids when it abuts hemagglutinin on whole virions.

  • hemagglutinin stalk reactive antibodies interfere with influenza virus neuraminidase activity by steric hindrance
    bioRxiv, 2018
    Co-Authors: Yaoqing Chen, Linda Yuling Lan, Min Huang, Carole Henry, Patrick C Wilson
    Abstract:

    Abstract: Hemagglutinin (HA) stalk-reactive antibodies are the basis of several current “one-shot” universal influenza vaccine efforts because they protect against a wide spectrum of influenza virus strains. The appreciated mechanism of protection by HA-stalk antibodies is to inhibit HA stalk reconfiguration, blocking viral fusion and entry. This study shows that HA stalk-reactive antibodies also inhibit neuraminidase (NA) enzymatic activity, prohibiting viral egress. NA inhibition (NI) is evident for an attached substrate but not for unattached small molecule cleavage of sialic acid. This suggests that the antibodies inhibit NA enzymatic activity through steric hindrance, thus limiting NA access to sialic acids when adjacent to HA on whole virions. Consistently, F(ab’)2 fragments that occupy reduced area without loss of avidity or disrupted HA/NA interactions show significantly reduced NI activity. Notably, HA stalk binding antibodies lacking NI activity were unable to neutralize viral infection via microneutralization assays. This work suggests that NI activity is an important component of HA-stalk antibody mediated protection. Summary This study reports a new mechanism of protection that is mediated by influenza hemagglutinin-stalk reactive antibodies: inhibition of neuraminidase activity by steric hindrance, blocking access of neuraminidase to sialic acids when it is abutted next to hemagglutinin on whole virions.

Melkote S. Shaila - One of the best experts on this subject based on the ideXlab platform.

  • Expression of biologically active Hemagglutinin-Neuraminidase protein of Peste des petits ruminants virus in transgenic pigeonpea [Cajanus cajan (L) Millsp.]
    Plant Science, 2004
    Co-Authors: Venkatesh Prasad, Melkote S. Shaila, V. V. Satyavathi, Sanjaya, K.m Valli, Abha Khandelwal, G. Lakshmi Sita
    Abstract:

    Hemagglutinin-Neuraminidase (HN) gene of Peste des petits ruminants virus (PPRV) has been expressed in pigeonpea for the development of an edible vaccine for Peste des Petits ruminant (PPR). PPRV, causes PPR disease in sheep and goats with high mortality rate. The two surface glycoproteins of PPRV Hemagglutinin-Neuraminidase and fusion protein (F) confer protective immunity. We report the successful generation of transgenic pigeonpea (Cajanus cajan (L.) Millsp.) plants and expression of HN protein having biological activity. A 2 fragment containing the coding region of the HN gene from an Indian isolate was cloned into the binary vector pBI121 and mobilized into Agrobacterium tumefaciens strain GV3 101. Cotyledonary nodes from germinated seeds of pigeonpea were used for transformation. The presence of transgenes, NPTII and HN in the plants was confirmed by PCR. The expression of HN protein in the transgenic lines was further confirmed by Western blot analysis using polyclonal monospecific antibody to HN and more importantly plant-derived HN protein was shown to be biologically active as demonstrated by neuraminidase activity. Transgenic plants were fertile and PCR of $T_1$ plants confirmed the inheritance of the transgene.

  • The Hemagglutinin-Neuraminidase protein of peste des petits ruminants virus is biologically active when transiently expressed in mammalian cells.
    Virus research, 2001
    Co-Authors: Shaguna Seth, Melkote S. Shaila
    Abstract:

    The genes coding for the surface glycoproteins hemagglutinin–neuraminidase (HN) of the peste des petits ruminants virus (PPRV) and hemagglutinin (H) of rinderpest virus (RPV) were cloned in a cytomagalovirus promoter driven expression vector and expressed transiently in mammalian cells. The protein expression was apparent 24 h after transfection and the expressed proteins were detected at the cell surface. The transiently expressed PPRV HN protein was found to be biologically active in possessing hemadsorption and neuraminidase activities. On the other hand, RPV H protein exhibited neuraminidase activity but was deficient in hemadsorption activity. The substrate specificity of the neuraminidase activity of these two proteins differed distinctly. The presence of neuraminidase activity in both PPRV HN and RPV H proteins is unusual among members of the morbillivirus genus.

Yaoqing Chen - One of the best experts on this subject based on the ideXlab platform.

  • hemagglutinin stalk reactive antibodies interfere with influenza virus neuraminidase activity by steric hindrance
    Journal of Virology, 2018
    Co-Authors: Yaoqing Chen, Linda Yuling Lan, Min Huang, Carole Henry, Patrick C Wilson
    Abstract:

    Hemagglutinin (HA) stalk-reactive antibodies are the basis of several current "one-shot" universal influenza vaccine efforts because they protect against a wide spectrum of influenza virus strains. The appreciated mechanism of protection by HA stalk-reactive antibodies is to inhibit HA stalk reconfiguration, blocking viral fusion and entry. This study shows that HA stalk-reactive antibodies also inhibit neuraminidase (NA) enzymatic activity, prohibiting viral egress. NA inhibition (NI) was evident for an attached substrate but not for unattached small-molecule cleavage of sialic acid. This finding suggests that the antibodies inhibit NA enzymatic activity through steric hindrance, thus limiting NA access to sialic acids when adjacent to HA on whole virions. Consistently, F(ab')2 fragments that occupied reduced area without loss of avidity or disrupted HA/NA interactions showed significantly reduced NI activity. Notably, HA stalk-binding antibodies lacking NI activity were unable to neutralize viral infection via microneutralization assays. This work suggests that NI activity is an important component of protection mediated by HA stalk-reactive antibodies.IMPORTANCE This study reports a new mechanism of protection mediated by influenza hemagglutinin stalk-reactive antibodies, i.e., inhibition of neuraminidase activity by steric hindrance, blocking access of neuraminidase to sialic acids when it abuts hemagglutinin on whole virions.

  • hemagglutinin stalk reactive antibodies interfere with influenza virus neuraminidase activity by steric hindrance
    bioRxiv, 2018
    Co-Authors: Yaoqing Chen, Linda Yuling Lan, Min Huang, Carole Henry, Patrick C Wilson
    Abstract:

    Abstract: Hemagglutinin (HA) stalk-reactive antibodies are the basis of several current “one-shot” universal influenza vaccine efforts because they protect against a wide spectrum of influenza virus strains. The appreciated mechanism of protection by HA-stalk antibodies is to inhibit HA stalk reconfiguration, blocking viral fusion and entry. This study shows that HA stalk-reactive antibodies also inhibit neuraminidase (NA) enzymatic activity, prohibiting viral egress. NA inhibition (NI) is evident for an attached substrate but not for unattached small molecule cleavage of sialic acid. This suggests that the antibodies inhibit NA enzymatic activity through steric hindrance, thus limiting NA access to sialic acids when adjacent to HA on whole virions. Consistently, F(ab’)2 fragments that occupy reduced area without loss of avidity or disrupted HA/NA interactions show significantly reduced NI activity. Notably, HA stalk binding antibodies lacking NI activity were unable to neutralize viral infection via microneutralization assays. This work suggests that NI activity is an important component of HA-stalk antibody mediated protection. Summary This study reports a new mechanism of protection that is mediated by influenza hemagglutinin-stalk reactive antibodies: inhibition of neuraminidase activity by steric hindrance, blocking access of neuraminidase to sialic acids when it is abutted next to hemagglutinin on whole virions.

Franco Dallocchio - One of the best experts on this subject based on the ideXlab platform.

  • Anomeric specificity and protein-substrate interactions support the 3D model for the Hemagglutinin-Neuraminidase from sendai virus.
    Biochemical and biophysical research communications, 1999
    Co-Authors: Tiziana Bellini, Claudia Pasti, Maria Cristina Manfrinato, Maurizio Tomasi, Franco Dallocchio
    Abstract:

    The 3D structure of paramyxovirus Hemagglutinin-Neuraminidase has not yet been resolved; however, a theoretical model has been built by using influenza virus and bacterial neuraminidases as template [V. C. Epa (1997) Proteins Struct. Funct. Gen. 29, 264-281]. Two common features of the catalytic mechanism of the neuraminidases of known 3D structure are the anomeric specificity and the involvement of a tyrosine residue in the stabilization of the transition state. These key features have been investigated on the water-soluble ectodomain of the Hemagglutinin-Neuraminidase from Sendai virus (cHN). The anomeric specificity of the hydrolysis of the substrate by cHN has been investigated by NMR spectroscopy. The immediate product of the reaction was the alpha-anomer, meaning that cHN belongs between glycohydrolases retaining anomeric configuration like influenza virus neuraminidase. Measurements of the UV difference spectrum upon binding of the substrate analogue 2,3-dehydro 2-deossi N-acetyl neuraminic acid indicate the ionization of a tyrosine residue and decreased polarity in the environment of a tryptophan residue. Functional significance of the spectral data was derived from the known structure of influenza neuraminidase, where a tyrosinate ion is involved in the stabilization of the transition-state carbonium ion, and a tryptophan residue is involved in the binding of the acetyl moiety of the substrate. The data give experimental support to the 3D model of paramyxovirus neuraminidase.

  • Inhibition of Sendai virus hemagglutinin neuraminidase by the fusion protein.
    Biochemical and biophysical research communications, 1994
    Co-Authors: Franco Dallocchio, Maurizio Tomasi, Tiziana Bellini
    Abstract:

    Abstract The Sendai virus envelope contains two glycoproteins: the fusion (F) protein and the Hemagglutinin-Neuraminidase (HN). Inactivation of F causes the loss of fusogenic activity and an increase of the neuraminidase activity of HN. After inactivation of F, HN can be inhibited by fetuin or asialofetuin, as already observed on the water-soluble, C-terminal fragment of HN (Dallocchio, F., Bellini, T., Martuscelli, G., Baiocchi, M., and Tomasi, M. (1991) Biochem. Int. 25, 663-668). Disruption of viral envelopes by detergents does not affect the neuraminidase activity of virions contaning inactive F, while it causes an increase of the neuraminidase activity in native virions. Reconstitution of HN into liposomes is accompanied by a decrease of enzymatic activity, due to the random inside-outside distribution of the protein. However, the decrease of the neuraminidase activity is higher in liposomes contianig both HN and F. These data suggest that F inhibits the neuraminidase activity of HN.

  • Selective modification of Sendai virus hemagglutinin neuraminidase by pyridoxal 5'-phosphate: evidence for an allosteric modulation of neuraminidase activity.
    Biochimica et biophysica acta, 1993
    Co-Authors: Tiziana Bellini, Maurizio Tomasi, Franco Dallocchio
    Abstract:

    Abstract Incubation of Sendai virus with pyridoxal 5′-phosphate (PLP) causes inhibition of hemolytic activity, a slight reduction of hemagglutinating activity, and an increase in neuraminidase activity. The effects on hemagglutination and neuraminidase are prevented by the presence in the incubation mixture of sialyl lactose, a substrate of Hemagglutinin-Neuraminidase. Incubation with PLP of the water-soluble enzymatic domain of the neuraminidase has no effect on enzymatic activity, while the allosteric inhibition (Dallocchio et al. (1991) Biochem. Int. 25, 663–668) disappears. Both virus-bound and solubilized neuraminidase are selectively modified by PLP at the lysine-553. Our data suggest that PLP inactivates a previously undetected inhibitory site on the viral neuraminidase, and that a physiological effector is present on the viral envelope.

Tiziana Bellini - One of the best experts on this subject based on the ideXlab platform.

  • Anomeric specificity and protein-substrate interactions support the 3D model for the Hemagglutinin-Neuraminidase from sendai virus.
    Biochemical and biophysical research communications, 1999
    Co-Authors: Tiziana Bellini, Claudia Pasti, Maria Cristina Manfrinato, Maurizio Tomasi, Franco Dallocchio
    Abstract:

    The 3D structure of paramyxovirus Hemagglutinin-Neuraminidase has not yet been resolved; however, a theoretical model has been built by using influenza virus and bacterial neuraminidases as template [V. C. Epa (1997) Proteins Struct. Funct. Gen. 29, 264-281]. Two common features of the catalytic mechanism of the neuraminidases of known 3D structure are the anomeric specificity and the involvement of a tyrosine residue in the stabilization of the transition state. These key features have been investigated on the water-soluble ectodomain of the Hemagglutinin-Neuraminidase from Sendai virus (cHN). The anomeric specificity of the hydrolysis of the substrate by cHN has been investigated by NMR spectroscopy. The immediate product of the reaction was the alpha-anomer, meaning that cHN belongs between glycohydrolases retaining anomeric configuration like influenza virus neuraminidase. Measurements of the UV difference spectrum upon binding of the substrate analogue 2,3-dehydro 2-deossi N-acetyl neuraminic acid indicate the ionization of a tyrosine residue and decreased polarity in the environment of a tryptophan residue. Functional significance of the spectral data was derived from the known structure of influenza neuraminidase, where a tyrosinate ion is involved in the stabilization of the transition-state carbonium ion, and a tryptophan residue is involved in the binding of the acetyl moiety of the substrate. The data give experimental support to the 3D model of paramyxovirus neuraminidase.

  • Inhibition of Sendai virus hemagglutinin neuraminidase by the fusion protein.
    Biochemical and biophysical research communications, 1994
    Co-Authors: Franco Dallocchio, Maurizio Tomasi, Tiziana Bellini
    Abstract:

    Abstract The Sendai virus envelope contains two glycoproteins: the fusion (F) protein and the Hemagglutinin-Neuraminidase (HN). Inactivation of F causes the loss of fusogenic activity and an increase of the neuraminidase activity of HN. After inactivation of F, HN can be inhibited by fetuin or asialofetuin, as already observed on the water-soluble, C-terminal fragment of HN (Dallocchio, F., Bellini, T., Martuscelli, G., Baiocchi, M., and Tomasi, M. (1991) Biochem. Int. 25, 663-668). Disruption of viral envelopes by detergents does not affect the neuraminidase activity of virions contaning inactive F, while it causes an increase of the neuraminidase activity in native virions. Reconstitution of HN into liposomes is accompanied by a decrease of enzymatic activity, due to the random inside-outside distribution of the protein. However, the decrease of the neuraminidase activity is higher in liposomes contianig both HN and F. These data suggest that F inhibits the neuraminidase activity of HN.

  • Selective modification of Sendai virus hemagglutinin neuraminidase by pyridoxal 5'-phosphate: evidence for an allosteric modulation of neuraminidase activity.
    Biochimica et biophysica acta, 1993
    Co-Authors: Tiziana Bellini, Maurizio Tomasi, Franco Dallocchio
    Abstract:

    Abstract Incubation of Sendai virus with pyridoxal 5′-phosphate (PLP) causes inhibition of hemolytic activity, a slight reduction of hemagglutinating activity, and an increase in neuraminidase activity. The effects on hemagglutination and neuraminidase are prevented by the presence in the incubation mixture of sialyl lactose, a substrate of Hemagglutinin-Neuraminidase. Incubation with PLP of the water-soluble enzymatic domain of the neuraminidase has no effect on enzymatic activity, while the allosteric inhibition (Dallocchio et al. (1991) Biochem. Int. 25, 663–668) disappears. Both virus-bound and solubilized neuraminidase are selectively modified by PLP at the lysine-553. Our data suggest that PLP inactivates a previously undetected inhibitory site on the viral neuraminidase, and that a physiological effector is present on the viral envelope.